Undercover Police Agent Provocateurs Caught Initiating Looting and Rioting

Agent Provocateur Jacob Pedersen?

Agent Provocateur Jacob Pedersen

Remember guys – question everything. Especially anything passed on by the media that insists you should only trust information from sources the powers that be labels “verified“.

Unlike the mainstream press, I have been telling you to verify, double verify and trust no one but your own gut since long before the ZOG arrested me for informing the public, but after 12 years of awakening the world if you were to only take one thing for your own, let it be that you need to question everything. Including whatever I say.

I have been exposing the lies that were declared as official truths for years, and got to be well familiar with TPTB’s modus operandi. And my gut is telling me – COVID-19 was just a test to see how well the plebs will respond to severe restrictions on their universal rights, with the real plague yet to be released upon the world. They’ve been spraying us with chemtrails for decades, which is a well documented and proven fact, so this method could well be used to disperse an actual seriously dangerous agent all over the world.

COVID-19 has been around for a few months, so we now have months worth of data which clearly shows the coronavirus is not any worse than many previous virus outbreaks that didn’t require lockdowns. But TPTB now know that fear mongering over nothing got the plebs to worship their lockdown and be so scared of their fellow men, they would rat them out to the Cheka type enforcers, so the test run was a success. Thus, the real virus can be released with expected reaction from the public, who will believe it is the second wave of COVID-19.

Still, because the COVID-19 overload is clearly wearing off, a false flag was needed to step up the divide and conquer objectives. The racial divide has been playing out the best for the TPTB. So here comes a white cop allegedly killing an unarmed black man in Minneapolis.

Except, the alleged killer and the alleged victim knew each other well, and spent a year working together as bouncers for a night club. The alleged killer cop’s neighbors claim they didn’t know he was a cop. The vehicle plate shown in the video has no numbers just the letters POLICE. EMT didn’t check vitals nor try to resuscitate the victim. The cops took off the cuffs, helped EMT flip him unto the gurney and off they went. But the best part – none other but Dr. Michael Baden, the go-to doctor for legitimizing dubious deaths, will perform autopsy on George Floyd. The whole fiasco stinks of George Floyd getting a new identity to hang out with the new and improved Jeffrey Epstein in Tel Aviv.

But a divide and conquer false flag would not be complete without undercover police agent provocateurs initiating looting and rioting in order to make the legitimate protesters who respond to the oppression appear violent. We’ve seen it play out by the book in police states, so once you get to understand their modus operandi, everything really makes sense.

It figures that Dear Leader Trump, who demonstrably hates the American people, would openly call for deaths of the American citizens who dare voice their disdain and protest, even though when it comes to people of Hong Kong who dare voice their disdain and protest, he’s vehemently against the Chinese government quelling the protests. Donald Trump simply is the most anti American American president in history of America. He doesn’t give a shit about the real looting going on, whereby over the past 30 years the top 1% gained $21 trillion in wealth, while the bottom 50% lost $900 billion in wealth. He just wants to see the average American suffer.

Here’s the video of a man who started the looting and rioting during protests in Minneapolis. He was destroying property and was being told to stop by local folks who were angered by this and chased him off. He was identified by the good folks on teh interweb as Jacob Pedersen, a cop from St. Paul. Jacob Pedersen’s ex wife also confirmed it was him. It sure seems as though the police had him infiltrate and catalyze the destruction. He wears a sophisticated police issue Honeywell 760008A gas mask, padded gloves for protection from broken glass and no fingerprints, and an umbrella for fellow cops to identify him. Plants need a way to coordinate and be distinguishable so they can be followed, including from the air:

And here’s a video of an agent provocateur caught leaving undercover police vehicle in DC. An example of great citizen reporting and journalism:

Pics:

Author: Vincit Omnia Veritas

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131 thoughts on “Undercover Police Agent Provocateurs Caught Initiating Looting and Rioting”

    1. Preaching to the Converted.

      Just have to keep getting the word out there, wherever one can. To anyone, that’s more than the brain dead conformist do.

      But people are not going to slide so easily back into self imposed house arrest a second time, slowly people ARE waking up, a world wide resistance movement is what needs forming but that’s idealistic wishful thinking on my part.

      Every Day I have this in Mind.

      .“Humanity would sink into eternal darkness, it would fall into a dull and primitive state, were the Jews to win this war” Joseph Goebbels.”
      ++

    2. I don’t know why you guys dislike “CHINA!”
      Let me show you the first sentence of the chinese national anthem :
      Stand up! The people who don’t want to be a slave!

      It time to stand up my friend.
      Fuck the damn politics.
      Fuck the damn consortium.
      Fuck all of goddamn unfair.
      Burn the Wall Street
      To get what should belong to you.
      Let the democracy be purity.
      Let the gore be righteousness.
      God bless you because when you stand up
      You are the GOD

      1. Maybe you will ask me
        Why I don’t speak this to the Hongkong protestors.
        Because they are just some crowd and never stand up.
        They just want to change their ID from UK-slave to US-slave.
        Fake democracy make me sick.
        But now ,I feel the light of real democracy .
        That is what Americans doing.

        1. I don’t agree with the rioters but I also don’t agree with your communist bullshit. Chinese don’t know anything even a peaceful protest will have them kidnapped and disappear with their organs harvest.

          1. Oh man!
            Am I disappear?
            Who is talking to you about the fact of China.
            You’re right my friend.
            When the education and economic have not reached appropriate level. Communist is a goddanm bullshit.
            All of the “peaceful” protest your media show you is fake .They just want to get a green card of other country and leave away from the life which is excluding them .
            They are COWARD,
            They are shame of China!
            They should be disappear and killed.
            Fucking betrayaler.
            The real protesters have gotten into the organization for fighting against the wicked power.
            China is changing. I saw it and you

            will see it.

      2. COMMUNIST China is controlled by Jews as well. They funded Mao and it’s not as though they ever walk away from such a huge investment (and they certainly weren’t expelled…again). They have also transferred all of our wealth to China along with our military secrets, while simultaneously weakening the West from within. Nevertheless, even most Jew-wise right-wingers are still blind to the obvious fact that China has the same (((problem))) that the rest of us have, and once their usefulness has been worn out they will get the same treatment too.

        https://web.archive.org/web/20160407230351/http://theforbiddentruth.net/viewtopic.php?f=8&t=92&sid=a5c381a96b2f309e328beda5cc2ed4aa

        1. Thank you for tell me a nice story.
          Maybe something is the fact about history.But most of the information you show me is from another mouth of unknowing person. If you change Jews to E.T, the story can be reasonable too .Sorry my friend.
          You guys hate Jews just because Jews make the Wall Street and president of America kneel down in front of them.
          You don’t know China .You should come here live for a while .
          In the fact.We have always stand your sit and support you guys fight against those totalitarian which is exploiting your peaceful life.
          You should get right target. Not just look at the Twitter of D.Trump :CHINA!

      3. Ha ha. I was there during the Dalian protest. They quelled that quickly. I didn’t see much “standing up ” but some beatings to the citizens. It was gone by the next morning. Chinese know how to stay in their place. And the police there are pretty nice as long as you don’t do stupid shit or pay them off. I got to respect the patience of the Chinese despite their obnoxious CCP.

        1. Are you chinese?
          If you aren’t,I would like to tell you ,you don’t have to respect chinese for that.
          Some of CCP are obnoxious.Not all of them.At least my girlfriend isn’t.
          If CCP deal CO-19 like Trump during the epidemic time. They will be burned by us.
          We don’t have patience, but we have patience when we are geting RICH.
          Every chinese people know a simple fact that hunman right is a fucking bullshit
          when you have no money.
          Just like the “poor” Floyd ,he is poor, so he have no right to breathe .Even though he is a American.
          POOR guys are POOR.
          Poor people are animal.

          I bet that you don’t know how rich you will be when you have gotten close with the CCP. Now I know you’re not chinese.
          That’s why we don’t choose to do protest. Protest is useless and we love to be quelled, that makes us get money.

          Easy to understand.
          Welcome to China.

          1. 倍思特鸡儿 >”I bet that you don’t know how rich you will be when you have gotten close with the CCP. Now I know you’re not chinese.
            That’s why we don’t choose to do protest. ”

            So you’re telling me that most Chinese citizens have high hopes on getting close connections to government or government officials? You and I both know that Chinese are smart enough to know it happens only to a very small percentage of the average Chinese population. And for the average Chinese that does indeed have Guanxi, it doesn’t reap huge personal wealth for them.

          1. 90 million CCP members? Are they all rich? Aren’t some of them just owning a membership card and only paying their yearly dues with little or no benefit?

        2. My friend.
          I’m a chinese !
          I know that how many CCP in my life . My teacher my boss my doctor. All of them are CCP.Even though my parents are not one of them.But they are doing business with CCP.
          The whole country is under CCP’s control.We want them be happy, just like you want to make your boss be happy. For what?For freedom? For human right? For money my friend.
          The same with africa-Americans ,We are lower-class people .
          But different is ,we have chance to get close with the high-class.But the black peoples in America ,never have the chance

          1. Well, my wife was also a member of the CCP and decided the payments were not worth the benefits. She built wealth and independence without their help and basically said “Why should I keep paying for this, it’s just club membership.”

          2. I know you’re Chinese. “My friend” is the indicator. Anyway, my time limit to edit my post got cut off prematurely. I just want to add, that my wife also built her wealth and independence without any help from The CCP, and dumped their club membership. She sees the Chinese media reporting, and is disgusted on what they report to their citizens when the story is entirely different outside of China. She’s had arguments with her mother back in China about what CCTV told her mother, and what the mother believes to be factual while the rest of the world is reporting something entirely different. Her sister living in Germany 20 years also has the same views. Don’t get me wrong, I loved Dalian. Best 2 years of my life. Amazing and warm people, but the biggest daily barrage of nightly news propaganda I’ve ever seen in my life. I was surprised at some of the stuff they were reporting on Japan back then. And what you say is untrue about black people having no chance to be close to upper class in America. Absolutely untrue unless you decide to continue to live the “Ghetto/Thug Life.”

        3. Thank you for the communication.
          But I’m not your wife’s mother.
          The same with all of young chinese ,I hate to watch the CCTV.Compared with the interesting YouTube, CCP’s media is so boring. I join searching some political comment of Taiwan. I would like to see those people wagging their tail in front of the West.

          If you think or infer that CCP is just a simple club,
          You’re totally wrong.

          Don’t laugh, I gonna tell a viewpoint you will never approve in your whole life.

          The CCP is the greatest development of chinese people.
          (Hahaha…Sorry ,I seemingly saw your puzzled expression )

          Today’s CCP is the product of China’s culture of 5000 years. That culture is “fusion”. CCP is a fusion of politics ,religion, patriotism,nationalism, imperialism,democracy and freedom,possibly others as well.
          And the catalyst of the fusion are 100 years of war,poor,faithless,torment,suffering and sacrifice. Cultural Revolution and “6.4” are catalyst .The most recent are Hongkong, Xinjiang and co-19.
          The fusion,Individualistic western people will never understand it.Culture is different,blood is different.

          You take your wife out to prove your point.
          But you don’t know how sad she was when she debated with her mom, and how sad she was when she had left her oath to CCP and chinese. Smiling ,she said, “I never regret”,Just because she loves you.

          The tearing like what happened in your wife’s family is also one of catalyst of the fusion. Thousands of examples right here in China. They have their right to choose their life, be easy ,be simple .But the real warriors had jumped into the chaotic fusion for helping 1.4 billion to seek the light.
          Not me ,my friend. After I ended my study in Japan and back to China up to now. I feel so weak and dying .
          “I can’t breathe”
          The fucking CCP,The fucking fusion.lol

          I know you will negate me.But before the CCP get fucked, your negation is useless to me.

    3. Wow these fuckin retard life matters people blowing up like 7 cars in my city. I hope that they r launching cans of carona
      Black lives…. Sicka hearing black people fuckin powting fuckin cry baby bitches

  1. None of this shit is truly real. It’s all a show, a big show. Designed and orchestrated. Paid for. There are things that are already being planned and scripted for later in the year. And next year. And the year after that.

  2. There are NO limits to their Depravity.
    They will not stop at, or stoop to any levels
    COVID-19 is a Myth it DOES NOT EXIST.
    I’ve been safe with this knowledge for months, and spreading the word wherever possible.
    It’s just WTF will they try next, trying to second guess them is again NOT enough but, put the work in and do your BEST…!!

    1. If COVID-19 is a Myth then how come millions of people are dying from it. Mostly in “your” country. Maybe you’re safe just because you’re going by the Rules. But don’t say it’s ain’t Real.

      1. Not ONLY is it a Myth, it’s a scam, confidence trick, smoke screen cover for something else, do your own research if you are capable of it, I could tell you alot more but obviously you believe the narrative and MSM.
        If you had been more respectful I could have enlightened you but you are ignorant so FUCK OFF.

        People die all the time their massaging figures to have people such as yourself blindly swallow the Shit.
        I’m not hear to educate you with the attitude you have.

        My country, you haven’t a clue where I live, I’m following no rules, unless there my own. I’ve NOT been shiting myself under house arrest in my own home like you.
        I’ll say what ever I feel, don’t care what you think, you ARE a Sheep following Bo Peep.
        Bhaa baa baa bleat bleat.
        IT’S NOT REAL AND I CAN PROVE THAT..!
        Put your head firmly back in your Ass where it belongs.
        There’s obviously NO hope for you. Bye bye. That all you get. Fucking ignoramus..!

        1. And I’m Sorry for my Attitude. Because some of my friends are suffering or suffered from it. I just lost my mind. There is a Lockdown going on here too. But I don’t give a shit, I go out everyday to see the green in the woods. Also I’ve been safe for months.

        2. @jxk777
          Who would I believe the infectiologists, virologists, pulmonologists, microbiologists, epidemiologists ? The ten studies, memoirs and theses that I have read on the subject made by people who dedicate their lives to science or a poor negro like you?

          You’re just another moron who believes in all this nonsense conspiracy theory that makes no sense.

          …..and the earth is still flat isn’t it ?

          1. Crawl back into YOUR hole Troll.
            You spout NOTHING but pure Shyte. FFS..!
            Nigger this jew that white Nigger. You self hating prick.
            FUCK OFF with your projections, presumptions, and worthless diatribe.
            No one is listening.

            Read all you can on the subjects, only get the correct books to READ.
            Conspiracy theories exist because their ARE Conspiracies. I subscribe to NONE.
            FACTS AND TRUTH is what it’s about, hadn’t you noticed..?
            Every time you are here, which thankfully isn’t that often you’re blowing your mouth off, and NEVER produce or verify anything, other than Just having a tantrum. And attacking what doesn’t suit your tunnel visions, combined with your limited intellect which makes you no better than a pavement apes droppings.

            I’ve researched this from day 1. Keeping up on said almost every FUCKING DAY..!
            Also certainly not going to start pasting scientists and scientific papers medical and doctors assessments in the Freaking comments section. Just especially for you, when all you are doing is running off with your bad mouth. Even the inventor of the test being used has stated that it cannot test for causation and viral load. It will detect corona but there are many types.
            White Americans are armed, why do they not use them to defend themselves that is all I want to know.

            Put yourself in a Box go back into hibernation.
            Utter Fucking Fool, listen to the hollow noise you make.
            What age are you Mann, 10..?
            Was Mammies CUNT to dry for you to FUCK, and your having a period out of your mouth again.
            Defense and justification I’ve plenty but not to waste on imbeciles such as you.
            Go find some traffic to play with…
            @kasper88

        3. @jxk777

          Insanity, stupidity and insults,
          not even an argument and just insults, but I don’t really expect better from a nigga like you.

          seriously you convince me, you got me when you said
          ” I’ve researched this from day 1.

          So you research this from day 1 ? Coronavirus exist for more than 10 years moron so you research this for more than 10 years?

          ” Even the inventor of the test being used has stated that it cannot test for causation and viral load. It will detect corona but there are many types. ”

          LMFAO
          WHO INVENTED THIS TEST FUCKING IDIOT ??????????????

          NO ONE INVENTED THE TEST
          There are several tests in more than 218 countries, poor invertebrate slug. If you know nothing about the matter shut your poor ignorant mouth, these ten times better than saying bullshit

          The Coronavirus has been around for years poor asshole, the novel coronavirus called SARS Cov 2 has more than 11 (strain) different and not ” type ” as you say.

          If you are really interested in this subject, read well and then we will discuss

          Study real studies written by eminent doctors, researchers who spent their lifetime studying virology, infectiology, microbiology, epidemiology, pneumology, immunology, biology….

          Have you studied its patogen?
          Do you know the genetic sequencing of SARS-CoV-2?
          What do you know about an infectious agent or biological agent?
          What do you know about pathophysiology in infectious disease?
          What is the viral component of a microbiome?
          We characterize a virus by its inability to reproduce by mitosis, by scissiparity or by meiosis do you know that?

        4. @jxk777

          So your guy invent all these tests liar ?

          1copy COVID-19 QPCR Kit 1DROP INC. (imported by Luminarie Canada Inc.)
          (South Korea) Nucleic acid technology Lab-based test 2020-03-30

          Abbott Realtime SARS-COV-2 Abbott Molecular Inc.
          (United States) Nucleic acid Technology Lab-based test 2020-03-25

          Allplex 2019-NCoV Assay Seegene Inc.
          (Korea) Nucleic Acid Technology Lab-based test 2020-04-09

          BD SARS-CoV-2 Reagents For BD Max System Becton Dickinson and Company (Canada) Nucleic acid technology Lab-based test 2020-04-19

          Biofire COVID – 19 Test & Biofire COVID – 19 External Control Test Kit (+)Table 1 – Footnote 2 Biofire Defense LLC (United States) Nucleic Acid Technology Lab-based test 2020-05-04

          Biofire Respiratory Panel 2.1 (RP2.1) Biofire Diagnostics, LLC. (United States) Nucleic acid technology Lab-based test 2020-05-26

          cobas SARS-CoV-2 Roche (United States) Nucleic acid technology Lab-based test Initial authorization – 2020-03-18
          Amended authorization 2020-04-09

          DiaPlexQ Novel Coronavirus (2019-nCoV) Detection Kit Life Sciences Research Institute (South Korea) Nucleic acid technology Lab-based test 2020-04- 05

          DiaSorin Simplexa COVID-19 Direct Molecular Assay on the LIAISON MDX Instrument DiaSorin Molecular LLC

          (United States) Nucleic Acid technology Lab-based test 2020-04-09
          Genefinder CoVid-19 Plus Realamp Kit Osang Healthcare Co., Ltd. (South Korea) Nucleic Acid technology Lab-based test 2020-04-21

          Liaison Sars-Cov-2 S1/S2 IgG, Liaison Control Sars-Cov-2 S1/S2 IgG Diasorin Inc.
          (United States) Serological technology Lab-based test 2020-05-12

          LYRA SARS-COV-2 ASSAY Diagnostic Hybrids, Inc. – Also trading as Quidel Corporation (United States) Nucleic acid technology Lab-based test 2020-03-25

          NxTAG COV Extended Panel Luminex Molecular Diagnostics, Inc.
          (Canada) Nucleic acid technology Lab-based test 2020-03-26
          PerkinElmer New Coronavirus Nucleic Acid Detection Kit PerkinElmer, Inc. (United States) Nucleic Acid

          Technology Lab-based test 2020-04-06
          Qiastat-DX Respiratory SARS-CoV-2 Panel Qiagen Gmbh (Germany) Nucleic acid technology Lab-based test 2020-05-27

          Real-time Fluorescent RT-PCR Kit For Detecting SARS-CoV-2 BGI Americas Corp. (China) Nucleic Acid Technology Lab-based test 2020-05-04

          SARS-CoV-2 Assay (Panther Fusion System) Hologic (United States) Nucleic acid technology Lab-based test 2020-03-25

          SARS-CoV-2 IgG Assay Abbott Laboratories , Diagnostics Division (United States) Serological technology Lab-based test 2020-05-14

          Spartan Cube COVID-19 System Spartan Bioscience Inc. (Canada) Nucleic acid technology Point of care test Research Use Only
          2020-05-02
          TaqPathTM COVID-19 Combo Kit Thermo Fisher (United States) Nucleic acid technology Lab-based test 2020-03-18

          Xpert Xpress SARS-CoV-2 Cepheid (United States) Nucleic acid technology Lab-based test
          Point of care test 2020-03-24

        5. @jxk777
          The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell–cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examined, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.
          Subject terms: Membrane fusion, Electron microscopy
          Go to:
          Introduction

          In April of 2018, the World Health Organization (WHO) established a priority list of pathogens, including Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS) and Disease X, a disease with an epidemic or pandemic potential caused by an unknown pathogen1,2 (Fig. ​(Fig.1a1a).
          An external file that holds a picture, illustration, etc. Object name is 41422_2020_305_Fig1_HTML.jpg
          Open in a separate window
          Fig. 1
          Establishment of SARS-CoV-2 S protein-mediated cell–cell fusion system.

          a The emerging timeline for highly pathogenic viruses and the proposed Disease X. b Schematic representation of SARS-CoV-2 S protein. Its S1 subunit contains NTD (14–305 aa), RBD (319–541 aa), and RBM (437–508 aa). Its S2 subunit contains FP (788–806 aa), HR1 (912–984 aa), HR2 (1163–1213 aa), TM (1214–1237 aa) and CP (1238–1273 aa). c The formation of syncytium in Huh-7 cells 24 h after SARS-CoV-2 infection, with scale bar of 200 µm. d Images of SARS-CoV and SARS-CoV-2 S-mediated cell–cell fusion on 293T/ACE2 cells at 2 h (left) and 24 h (right). e SARS-CoV (I–II) and SARS-CoV-2 (III–IV) S-mediated syncytium formation on 293T/ACE2 cells at 48 h. f SARS-CoV (I–II) and SARS-CoV-2 (III–IV) S-mediated syncytium formation on Huh-7 cells at 48 h. Scale bar equals 400 µm in d–f.

          In late December 2019, an outbreak of pneumonia with an unknown etiology in Wuhan, China was considered as the first Disease X following the announcement by WHO. Shortly thereafter, a novel coronavirus, 2019-nCoV, as denoted by WHO,3 was identified as the pathogen causing the coronavirus disease COVID-19.4,5 2019-nCoV with 79.5 and 96% sequence identity to SARS-CoV and a bat coronavirus, SL-CoV-RaTG13, respectively,6 was renamed SARS-CoV-2 by the Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses (ICTV),7 while, in the interim, it was renamed HCoV-19, as a common virus name, by a group of virologists in China.8–10

          As of 24 February 2020, a total of 79,331 confirmed cases of COVID-19, including 2618 deaths, were reported in China and 27 other countries,11 posing a serious threat to global public health and thus calling for the prompt development of specific anti-coronavirus therapeutics and prophylactics for treatment and prevention of COVID-19.

          Coronaviruses (CoVs), the largest RNA viruses identified so far, belonging to the Coronaviridae family, are divided into 4 genera, α-, β-, δ- and γ-coronaviruses, while the β-coronaviruses are further divided into A, B, C, and D lineages. The seven CoVs that can infect humans (HCoVs) include HCoV-229E and HCoV-NL63 in the α-coronaviruses, HCoV-OC43 and HCoV-HKU1 in the β-coronaviruses lineage A, SARS-CoV and SARS-CoV-2 in the β-coronaviruses lineage B (β-B coronaviruses), and MERS-CoV in the β-coronaviruses lineage C.6 To develop specific SARS-CoV-2 fusion inhibitors, it is essential to study the fusion capacity of SARS-CoV-2 compared to that of SARS-CoV. Particularly, SARS-CoV and SARS-CoV-2 have 89.8% sequence identity in their spike (S) proteins S2 subunits, which mediate the membrane fusion process, and both of their S1 subunits utilize human angiotensin-converting enzyme 2 (hACE2) as the receptor to infect human cells.6 Most importantly, the ACE2-binding affinity of the receptor-binding domain (RBD) in S1 subunit of S protein of SARS-CoV-2 is 10- to 20-fold higher than that of SARS-CoV,12 which may contribute to the higher infectivity and transmissibility of SARS-CoV-2 compared to SARS-CoV. However, it is unclear whether SARS-CoV-2 can mediate membrane fusion in a manner that exceeds the capacity of SARS-CoV.

          After binding of RBD in S1 subunit of S protein on the virion to the ACE2 receptor on the target cell, the heptad repeat 1 (HR1) and 2 (HR2) domains in its S2 subunit of S protein interact with each other to form a six-helix bundle (6-HB) fusion core, bringing viral and cellular membranes into close proximity for fusion and infection.13 Therefore, the 6-HB fusion core structure of SARS-CoV-2 and SARS-CoV S proteins should also be compared in order to investigate the structural basis for membrane fusion mediated by their S proteins and thus set the stage for the rational design of coronavirus fusion inhibitors.

          In our previous studies, we designed a pan-coronavirus fusion inhibitor, EK1, targeting the HR1 domains of HCoV S proteins, which proved to be effective in inhibiting infection of 5 HCoVs, including SARS-CoV and MERS-CoV, and 3 SARS-related CoVs (SARSr-CoVs). By intranasal application of this peptide, either pre- or post-challenge with coronavirus, the treated mice were protected from HCoV-OC43 or MERS-CoV infection, suggesting that this peptide has prophylactic and therapeutic potential against SARS-CoV-2 infection.14 Indeed, our recent studies have shown that EK1 peptide is effective against SARS-CoV-2 S protein-mediated membrane fusion and PsV infection in a dose-dependent manner.15

          In this study, we have shown that SARS-CoV-2 exhibits much higher capacity of membrane fusion than SARS-CoV, suggesting that the fusion machinery of SARS-CoV-2 is an important target for development of coronavirus fusion inhibitors. We have solved the X-ray crystal structure of SARS-CoV-2’s 6-HB core and identified several mutated amino acid residues in HR1 domain responsible for its enhanced interactions with HR2 domain. By conjugating the cholesterol molecule to the EK1 peptide, we found that one of the lipopeptides, EK1C4, exhibited highly potent inhibitory activity against SARS-CoV-2 S-mediated membrane fusion and PsV infection, about 240- and 150-fold more potent than EK1 peptide, respectively. EK1C4 is also highly effective against in vitro and in vivo infection of some live HCoVs, such as SARS-CoV-2, HCoV-OC43 and MERS-CoV, suggesting potential for further development as pan-CoV fusion inhibitor-based therapeutics and prophylactics for treatment and prevention of infection by the currently circulating SARS-CoV-2 and MERS-CoV, as well as future reemerging SARS-CoV and emerging SARSr-CoVs.
          Go to:
          Results
          The capacity of SARS-CoV-2 S protein-mediated membrane fusion

          From the GISAID Platform (https://platform.gisaid.org), we obtained the full-length amino-acid sequence of SARS-CoV-2 (BetaCoV 2019–2020) S protein (GenBank: QHD43416). Through alignment with SARS-CoV and SL-CoVs S proteins, we located the functional domains in SARS-CoV-2 S protein, which contains S1 subunit and S2 subunit with the cleavage site at R685/S686.15 S1 subunit is located within the N-terminal 14–685 amino acids of S protein, containing N-terminal domain (NTD), receptor binding domain (RBD), and receptor binding motif (RBM). S2 subunit contains fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2), transmembrane domain (TM) and cytoplasmic domain (CP) (Fig. 1b).

          Recent biophysical and structural evidence showed that SARS-CoV-2 S protein binds hACE2 with 10-fold to 20-fold higher affinity than SARS-CoV S protein, suggesting the higher infectivity of the new virus.12 Unlike other β-B coronaviruses, S protein of SARS-CoV-2 harbors a special S1/S2 furin-recognizable site, indicating that its S protein might possess some unique infectious properties. Indeed, in live SARS-CoV-2 infection, we found a typical syncytium phenomenon naturally formed by infected cells, which is rarely reported in SARS-CoV infection (Fig. 1c). To further explore the special characteristic of SARS-CoV-2 infection, we cloned the S gene into PAAV-IRES-GFP vector and established the S-mediated cell–cell fusion system, using 293T cells that express SARS-CoV-2 S protein and EGFP (293T/SARS-CoV-2/EGFP) as the effector cells, and ACE2/293T cells expressing human ACE2 receptor as the target cells (Fig. 1d and Supplementary information, Fig. S1a). After effector cells and target cells were cocultured at 37 °C for 2 h, the fused cells showed at least 2-fold larger size than normal cells and multiple nuclei, and these cells were observed in the SARS-CoV-2 group, but not the SARS-CoV group. After coincubation for 24 h, hundreds of target cells fused together as one big syncytium, containing multiple nuclei (Fig. 1d). Another 24 h later, the syncytium grew bigger and could be easily observed under both light and fluorescence microscopy (Fig. 1e). Similar results were observed in the fusion between 293T/SARS-CoV-2/EGFP cells and Huh-7 cells, which naturally express human ACE2 receptor on the cell surface. Their syncytium was obviously formed after coincubation for 48 h, similar to the syncytium formed by live SARS-CoV-2-infected Huh-7 cells (Fig. 1c, f). On the contrary, SARS-CoV S protein lacked the ability to mediate the cell–cell fusion under the same conditions (Fig. 1d) based on the required presence of exogenous trypsin to complete membrane fusion in our previous studies. Therefore, compared to SARS-CoV, SARS-CoV-2 S protein showed much more efficiency in mediating viral surface-fusion and entry into target cells.14 Meanwhile, no fusion was observed for 293T/EGFP cells without S-expression or 293T cells without ACE2-expression (Fig. 1d and Supplementary information, Fig. S1b), confirming that S-receptor engagement is necessary for the S-mediated viral fusion and entry.
          X-ray crystallographic analysis of the 6-HB fusion core formed by HR1 and HR2 domains in S2 subunit of SARS-CoV-2 S protein

          Previously, we identified that the 6-HB formed by HR1 and HR2 domains of the S2 subunit plays a very important role in the membrane fusion process mediated by MERS-CoV or SARS-CoV S protein.16,17 Similarly, our recent study suggested that HR1 and HR2 in subunit S2 of SARS-CoV-2 also interacted to form coiled-coil complex to support membrane fusion and viral infection15 (Fig. 2a and Supplementary information, Fig. S2). However, the specific binding characteristics of SARS-CoV-2 6-HB remained to be explored.
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          Fig. 2
          Overall structure of post-fusion 6-HB in SARS-CoV-2.

          a Sequence alignment of HR1 and HR2 domains in SARS-CoV and SARS-CoV-2. b Structure of SARS-CoV-2 6-HB is shown in cartoon representation with HR1 colored in green and HR2 in cyan. The structural dimensions are indicated in angstroms. c HR1 trimer of SARS-CoV-2 6-HB is shown in electrostatic surface, and HR2 domain is shown in cartoon representation, the important binding residues of which are shown in sticks and labeled. d The superposition of 6-HB structure of SARS-CoV (PDB entry 1WYY), MERS-CoV (PDB entry 4NJL) and SARS-CoV-2 is shown in ribbon. The RMSD between structures is indicated. e The sequence comparison of 6-HB structure of different HCoVs is shown in cartoon representation with different colors for HR1 and HR2. The helical fusion core regions are indicated.

          To understand the structural basis of the interactions between HR1 and HR2 regions of SARS-CoV-2, a fusion protein containing the major parts of HR1 (residues 910–988) and HR2 (residues 1162–1206) with a flexible linker (L6, SGGRGG) in between was constructed for crystallographic study. The crystal structure of HR1-L6-HR2 shows a canonical 6-HB structure with a rod-like shape 115 Å in length and 25 Å in diameter (Fig. 2b). The three HR1 domains form a parallel trimeric coiled-coil center, around which three HR2 domains are entwined in an antiparallel manner. The interaction between these two domains is predominantly a hydrophobic force. Each pair of two adjacent HR1 helices forms a deep hydrophobic groove, providing the binding site for hydrophobic residues of the HR2 domain, including V1164, L1166, I1169, I1172, A1174, V1176, V1177, I1179, I1183, L1186, V1189, L1193, L1197 and I1198 (Fig. 2c). The hydrophobic interactions between HR1 and HR2 are mainly located in the helical fusion core region, which will be discussed later.

          The overall 6-HB structure of SARS-CoV-2 is similar to that of other HCoVs with root-mean-square deviation (RMSD) of 0.36 Å to SARS-CoV 6-HB and 0.66 Å to MERS-CoV 6-HB for all the Cα atoms (Fig. 2d). This finding suggested that the overall 6-HB conformation is an important and highly conserved component for these dangerous coronaviruses. When comparing with the 6-HB of other common coronaviruses causing mild respiratory disease, such as 229E and NL63, the SARS-CoV-2 6-HB has a similar overall structure, except for the different length of HR2 helix in the 6-HB. The HR2 domain of 229E or NL63 forms a longer and bending helix to interact with trimeric HR1 core (Fig. 2e). The relationship between the structural difference and the pathogenicity of these HCoVs remains to be elucidated.

          According to sequence alignment, the S2 subunits of SARS-CoV-2 and SARS-CoV are highly conserved, with 92.6% and 100% overall homology in HR1 and HR2 domains, respectively. Inside the fusion core region of HR1 domain, there are 8 different residues (Fig. 3a), which may contribute the enhanced interactions between HR1 and HR2 and stabilize 6-HB conformation of SARS-CoV-2 as revealed by crystallographic analysis, compared with those of SARS-CoV. This significant difference has not been observed in other SARS-like viruses, such as WIV1, Rs3367, and RsSHC014. As shown in Fig. 3b, the K911 in SARS-CoV HR1 could bind to E1176 in HR2 through a salt bridge 2.9 Å in distance. However, with the Lys-Ser replacement, S929 in SARS-CoV-2 binds to S1196 through a strong hydrogen bond 2.4 Å in distance. In SARS-CoV, Q915 in the HR1 domain does not bind to the HR2 domain. However, with Q-K replacement in the new virus, K933 in the HR1 domain binds to carbonyl oxygen of N1172 in HR2 through a salt bridge 2.7 Å in distance (Fig. 3b). In SARS-CoV, E918 in the HR1 domain binds to R1166 in the HR2 domain through a weak salt bridge 3.7 Å in distance. In SARS-CoV-2, E918 is mutated to D936 and binds to R1185 in the HR2 domain through a salt bridge 2.7 Å in distance (Fig. 3c). In SARS-CoV, K929 in HR1 binds to E1163 in HR2 through a salt bridge 3.2 Å in distance, while T925 is not involved in the interaction. However, when T925 was mutated to S943, it could bind to E1182 in the HR2 domain with a hydrogen bond 2.6 Å in distance, and K947 could also bind to E1182 through a salt bridge 3.0 Å in distance (Fig. 3d). These results suggested that the multiple replacements in the HR1 domain of emerging SARS-CoV-2 virus could enhance the interactions between HR1 and HR2 domain to further stabilize the 6-HB structure, which may lead to increased infectivity of the virus.
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          Fig. 3
          Interaction between HR1 and HR2 of SARS-CoV-2 and SARS-CoV.

          a–d The 6-HB structure of SARS-CoV-2 and SARS-CoV is shown in cartoon representation. The HR1 domain is shown in green for SARS-CoV-2 and forest for SARS-CoV, while the HR2 domain is shown in cyan for SARS-CoV-2 and orange for SARS-CoV. Important residues are shown in sticks and labeled.
          Design and structure-activity relationship (SAR) analysis of lipopeptides with remarkably improved fusion inhibitory activity

          Previously, we found that peptide EK1 could disturb viral 6-HB formation and effectively inhibit SARS-CoV-2 PsV infection. However, the potent stability of SARS-CoV-2 6-HB structure might reduce the antiviral efficacy of EK1. Recently, numerous reports have shown that the lipidation strategy can effectively improve the antiviral activity of fusion inhibitory peptides, such as the ant-HIV-1 peptide LP-19,18 and the anti-Nipah virus lipopeptides.19 In order to improve the inhibitory activity of EK1, cholesterol (Chol) and palmitic acid (Palm) were covalently attached to the C-terminus of EK1 sequence under the help of a flexible polyethylene glycol (PEG) spacer, and the corresponding lipopeptides EK1C and EK1P were constructed, respectively (Fig. 4a). Both of them could completely inhibit SARS-CoV-2 mediated cell–cell fusion at the concentration of 2.5 μM (Fig. 4b). The inhibitory activity with mean 50% inhibitory concentration (IC50) values is 48.1 nM for EK1C and 69.2 nM for EK1P, respectively (Fig. 4c). Meanwhile, the EK1-scrambled peptide showed no inhibitory activity with the concentration up to 5 μM (Fig. 4c). These results strongly suggest that lipidation of EK1 is a promising strategy to improve its fusion-inhibitory activity against SARS-CoV-2 infection, especially, cholesterol-modification.
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          Fig. 4
          EK1-Lipopeptides showed potent inhibitory activity against SARS-CoV-2 infection.

          a Amino acid sequences of the designed peptides EK1, EK1P and EK1C. The dotted lines represent E–K salt-bridge with i to i + 3, or i + 4 arrangement. b SARS-CoV-2 S protein-mediated cell–cell fusion in the presence of EK1-scramble (I), EK1 (II), EK1C (III), and EK1P (IV) at 2.5 μM (scale bar: 400 µm). c Inhibitory activity of EK1-scramble, EK1, EK1C and EK1P against SARS-CoV-2 S-mediated cell–cell fusion. d Design diagram of EK1-lipopeptides with cholesterol modification, including EK1C1-EK1C7. e Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 S-mediated cell–cell fusion. f Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 PsV infection. Experiments were repeated twice, and the data are expressed as means ± SD (error bar).

          On the basis of the structure of EK1C, series of cholesteryl EK1 with multiple linkers were constructed, where the glycine/serine-based linker, i.e., GSG, or PEG-based spacer was employed between EK1 and the cholesterol moiety (Fig. 4d). Compared with EK1C1, EK1C2 and EK1C showed similar inhibitory activities. Strikingly, EK1C3 peptide with both the 3-amino acid linker “GSG” and the PEG4-based spacer, exhibited 4-fold more potency than EK1C1. It is noteworthy that changing “GSG” in EK1C3 to a longer 5-amino acid linker “GSGSG” significantly increased the inhibitory potency of the hybrid molecule, and EK1C4 had IC50 value of 1.3 nM, which was 43-fold more potent than EK1C1. These findings indicate that the linker length has a significant effect on the overall activity of lipopeptides. Comparison of increasing PEG-based arm lengths in EK1C4 shows that inhibitors potency slightly decreased in the cell–cell fusion assay (Fig. 4e). The data suggest that “GSGSG-PEG4” linker was optimal to bridge both parts of the conjugates. Similarly, EK1C4 showed the most potent inhibitory activity against SARS-CoV-2 PsV infection, with IC50 value of 15.8 nM, providing 149-fold stronger anti-SARS-CoV-2 activity than that of EK1 (IC50 = 2,375 nM) (Fig. 4f).
          The lipopeptide EK1C4 exhibits the most potent inhibitory activity against membrane fusion mediated by S proteins and entry of pseudotyped coronaviruses

          We have previously demonstrated that EK1 could effectively inhibit divergent HCoV infection by targeting the HR1 domains, including α-HCoV and β-HCoV. Here, we further systematically evaluated the broad-spectrum surface-fusion inhibitory activity of EK1C4 on cell–cell fusion mediated by S proteins of divergent coronaviruses, including SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63 and HCoV-229E. Among them, SARS-CoV has the closest relative to SARS-CoV-2, and its S protein-mediated cell–cell fusion could be effectively inhibited by EK1C4 with IC50 of 4.3 nM, which is about 94-fold more active than that of EK1 (IC50 = 409.3 nM) (Fig. 5a). Similarly, EK1C4 showed extremely potent fusion-inhibitory activity on MERS-S- and OC43-S-mediated cell–cell fusion with IC50 of 2.5 nM and 7.7 nM, which were 95- and 101-fold more potent when compared to EK1, respectively, indicating that EK1C4 could potently and broadly inhibit S protein-mediated cell–cell fusion of various β-HCoVs (Fig. 5b, c). For α-HCoVs, EK1C4 also effectively blocked the fusion process mediated by the S protein of HCoV-229E and HCoV-NL63 with IC50 of 5.2 nM and 21.4 nM, respectively, while EK1 showed inhibitory activity of IC50 ranging from 207.4 to 751.0 nM (Fig. 5d, e). Moreover, with their potential for human infection, SL-CoVs, including WIV1, Rs3367 and RsSHC014, the fusion process of which is mediated by S protein, could also be significantly prevented by EK1C4 with IC50 ranging from 4.3 to 8.1 nM, as well as EK1 with IC50 ranging from 237.0 to 279.6 nM (Fig. 5f–h). As control, the EK1-scrambled peptide showed no inhibitory activity with concentration up to 5 μM in all those coronavirus cell–cell fusion assays (Fig. 5a–h).
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          Fig. 5
          EK1C4 broadly and potently inhibited cell–cell fusion and PsV infection mediated by S protein of divergent HCoVs.

          a–h Inhibitory activity of EK1C4 in cell–cell fusion mediated by the S proteins of SARS-CoV (a), MERS-CoV (b), HCoV-OC43 (c), HCoV-229E (d), HCoV-NL63 (e), WIV1 (f), Rs3367 (g) and SHC014 (h). i–o Inhibitory activity of EK1C4 in PsV infection assays against SARS-CoV (i), MERS-CoV (j), HCoV-OC43 (k), HCoV-229E (l), NL63 (m), WIV1 (n) and Rs3367 (o). Experiments were repeated twice, and the data are expressed as means ± SD.

          We also assessed the antiviral activity of EK1C4 on PsV infection by divergent coronaviruses. As expected, EK1C4 showed much more potent activity than EK1 (IC50 ranging from 631.8 to 3,237 nM) against SARS-CoV, MERS-CoV, and HCoV-OC43 infection with IC50 of 11.7 nM, 11.1 nM and 37.7 nM, respectively (Fig. 5i-k). EK1C4 also effectively blocked PsV infection of α-HCoVs, including HCoV-229E and HCoV-NL63, with IC50 of 12.4 nM and 76.6 nM, respectively, which was about 319- and 99-fold more active than EK1 (IC50 ranging from 3,963 to 7,666 nM) (Fig. 5l, m). Similarly, by cholesteryl modification with “GSGSG-PEG4” linker, the inhibitory activity of EK1 could be significantly increased on PsV infection from SL-CoVs, including WIV1 and Rs3367, where EK1C4 showed potent inhibitory activity with IC50 of 30.8 nM and 66.9 nM, respectively, which is 175-fold to 89-fold more potent than that of EK1 (Fig. 5n, o).
          EK1C4 possesses the most potent inhibitory activity against in vitro infection by live coronaviruses

          We further assessed the inhibitory activity of EK1C4 against live HCoVs infection, including SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-229E, and HCoV-NL63. Importantly, EK1C4 effectively blocked SARS-CoV-2 infection at the cellular level in a dose-dependent manner with IC50 of 36.5 nM, being 67-fold more active than that of EK1 (IC50 = 2,468 nM) (Fig. 6a), which is consistent to the results of cell–cell fusion assay and PsV infection assay mediated by SARS-CoV-2 S protein. Similarly, EK1C4 also showed more potent antiviral activity than EK1 against MERS-CoV, HCoV-OC43, HCoV-229E, and HCoV-NL63 infection with IC50 of 4.2 nM, 24.8 nM, 101.5 nM and 187.6 nM, respectively, which are 190-, 62-, 42- and 19-fold more potent than those of EK1, respectively (Fig. 6b–e). We next assessed the cytotoxicity of EK1C4 on various target cells and found that the half cytotoxic concentration (CC50) was beyond 5 μM, which is the highest detection concentration of EK1C4 (Supplementary information, Fig. S3). Therefore, the selectivity index (SI = CC50/IC50) of EK1C4 is >136, suggesting that EK1C4 is a promising SARS-CoV-2 fusion inhibitor with little, or even no, toxic effect in vitro. Further, we explored the potent antiviral mechanism of EK1C4 and found that the complexes of EK1C4/SARS-HR1, EK1C4/MERS-HR1, and EK1C4/SARS-2-HR1 harbor higher stability and increased Tm values than those of the complexes formed by EK1 and HR1s (Supplementary information, Fig. S4). These results suggested that increased antiviral activity of EK1C4 should be related to its increased binding affinity with HR1, but their detailed interactions require further studies.
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          Fig. 6
          EK1C4 effectively inhibited live-CoVs infection in vitro and in vivo.

          a–e Inhibitory activity of EK1C4 on live HCoV replication for SARS-CoV-2 (a), MERS-CoV (b), HCoV-OC43 (c), HCoV-229E (d), and HCoV-NL63 (e). f–g In vivo prophylactic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (f) and survival curves (g) of mice challenged with HCoV-OC43. h–i In vivo therapeutic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (h) and survival curves (i) of mice challenged with HCoV-OC43. Experiments were repeated twice, and the data are expressed as means ± SD.
          Intranasally applied EK1C4 showed strong protection of mice against HCoV-OC43 infection

          Recently, SARS-CoV-2 rapidly spread in humans by transmitting through the respiratory tract. Here, we used an HCoV-OC43 infection mouse model to further investigate the potential prophylactic effect of EK1C4 in clinical applications via the intranasal administration route (Fig. 6f, g). In the OC43-infected mouse model, we treated newborn mice with EK1C4 at a single dose of 0.5 mg/kg 0.5 h (Pre-0.5), 2 h (Pre-2), 4 h (Pre-4), 12 (Pre-12) and 24 h (Pre-24) before challenging with HCoV-OC43 at 100 TCID50 (50% tissue culture infectious dose). Starting from 4 days’ post-infection (dpi), the body weight of mice in the viral control group decreased significantly along with 100% mortality (Fig. 6f, g). The final survival rates of mice in Pre-0.5, Pre-2, Pre-4, Pre-12 and Pre-24 groups were 100%, 100%, 100%, 83 and 0%, respectively (Fig. 6f, g). In contrast, EK1 with a single dose of 20 mg/kg via nasal administration exhibited very promising prophylactic effect in the Pre-0.5 h and Pre-1 h groups, whereas all mice in the EK1-Pre-2 h group eventually died similarly to the mice in the viral control group (Supplementary information, Fig. S5). These results suggested that EK1C4 has better stability, antiviral activity, and prolonged half-life in the airway environment when compared with EK1.

          We then tested the therapeutic effect of EK1C4 0.5 h (Post-0.5 group) and 2 h (Post-2 group) after HCoV-OC43 infection (Fig. 6h, i). The Post-0.5 group and Post-2 group mice showed 100% and 16.7% survival rate, respectively, suggesting that EK1C4 harbors good therapeutic effect after a short period of HCoV-OC43 infection, possibly resulting from the establishment of HCoV-OC43 infection in mouse brain where EK1C4 cannot get through the blood brain barrier via nasal administration.14 As shown in Supplementary information, Fig. S6, high viral titer was detected in brains of all 5 mice in Pre-24 group and 4 out of 5 mice in Post-2 group, but was not detected in brain tissues of all mice in Pre-0.5, Pre-2, Pre-4, and Post-0.5 groups, while only moderate level of viral titer was detected in brain tissue in one of the 5 mice in Pre-12 group (Supplementary information, Fig. S6a, b). Similar to those in the viral control mice, mice in Pre-24 and Post-2 groups exhibited similar histopathological changes in brain tissues, including vacuolation, degeneration, and infiltration. However, the brain tissues of mice in Pre-0.5, Pre-2, Pre-4, Pre-12 and Post-0.5 group as well as the normal control group showed no apparent histopathological changes (Supplementary information, Fig. S6c).
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          Discussion

          Over the past 20 years, highly infectious pathogens have been emerging increasingly, such as SARS-CoV in 2003 and MERS-CoV in 2012.20–22 In 2018, WHO proposed “Disease X” in the blueprint priority diseases for any new unknown pathogen that may cause an epidemic or pandemic in the future, calling for the development of effective and safe vaccines and antivirals to prevent and treat such Disease X. Indeed, at the end of 2019, the outbreak of Wuhan pneumonia with an unknown etiological agent, the first Disease X following WHO’s announcement was reported to WHO. Shortly thereafter, a novel coronavirus, SARS-CoV-2 (also known as 2019-nCoV or HCoV-19), was identified to be the etiology of the Wuhan pneumonia, i.e., COVID-19 as designated by WHO.

          Unlike SARS-CoV, live SARS-CoV-2-infected cells were found to form typical syncytium, suggesting that SARS-CoV-2 may mainly utilize the plasma membrane fusion pathway to enter and replicate inside host cells. Consistently, in the cell–cell fusion system, SARS-CoV-2 S protein could effectively mediate the formation of syncytium between the effector cell and the target cell in the absence of an exogenous proteolytic enzyme, e.g., trypsin, while SARS-CoV S protein could not. Actually, the plasma membrane fusion pathway is more efficient than the endosomal membrane fusion pathway for most viruses because the latter is more prone to activating the host cell antiviral immunity.23,24 Generally, β-B coronaviruses lack the S1/S2 furin-recognition site, and their S proteins are uncleaved in the native state. For example, SARS-CoV enters into the cell mainly via the endosomal membrane fusion pathway where its S protein is cleaved by endosomal cathepsin L and activated.25 Inducing the S1/S2 furin-recognition site could significantly increase the capacity of SARS-CoV S protein to mediate cellular membrane surface infection.26 Interestingly, SARS-CoV-2 harbors the S1/S2 cleavage site in its S protein, but its specific role in S protein-mediated membrane fusion and viral life-cycle remains to be further explored (Supplementary information, Fig. S7). A recent report suggested that SARS-CoV-2 mainly used TMPRSS2 for plasma membrane fusion; this means that the TMPRSS2 inhibitor might constitute an option for blocking SARS-CoV-2 fusion with and entry into the host cell.27

          The 6-HB structure formed by HR1 and HR2 regions in the S2 subunit of HCoVs plays a key role during the viral membrane fusion process, which makes it one of the most important targets for drug design. In previous studies, we have found that HR1 and HR2 of SARS-CoV-2 could form a stable coiled-coil complex, but the detailed conformations remain unknown. According to the X-ray crystallographic analysis of the complex formed by HR1 and HR2 of SARS-CoV-2 (Fig. 2b), it is a typical 6-HB fusion core structure similar to those of SARS-CoV and MERS-CoV. Although the amino acid sequences of HR2 domain from SARS-CoV and SARS-CoV-2 are fully identical, multiple residue differences occur in the HR1 domain of SARS-CoV-2. However, instead of weakening the interaction between HR1 and HR2, such unilateral difference seems to form new interactions in some regions and enhance the existing ones in other regions (Fig. 3). When K991 in SARS-CoV HR1 was replaced with S929 in SARS-CoV2 HR1, a new, strong hydrogen bond was formed with a distance of 2.4 Å. K933 forms a new interaction with N1192 in SARS-CoV-2 with a distance of 2.7 Å, whereas the corresponding position in SARS-CoV has no such interaction. In the other two regions, E918 binds to R1166 and K929 binds to E1163 in SARS-CoV, both of which were enhanced in SARS-CoV-2. These results suggest that this new HCoV has evolved with improved binding affinity between HR1 and HR2 domains, which may accelerate the viral membrane fusion process and enhance viral infectivity or transmissibility. A recent study also found that the binding affinity between ACE2 receptor on the host cell and RBD in S protein of SARS-CoV-2 is more than 10-fold higher than that of SARS-CoV, which may also be associated with the increased infectivity and transmissibility of SARS-CoV-2.12

          The conjugation of cholesterol to viral entry inhibitor has been proved to be an effective strategy to enhance the antiviral activity, such as C34 peptide for HIV-1.28 However, the mechanism of this enhancement, especially the role of cholesterol group in the C-terminal tail of entry inhibitor, is still unclear. There is a possibility that the cholesterol group could anchor to the target membrane to facilitate the binding of inhibitor to the HR1 targets. However, we noticed that binding affinity between EK1C4 and SARS-CoV-2-HR1P is significantly enhanced than EK1 peptide alone, which suggested that cholesterol group may be involved in binding to HR1P directly (Supplementary information, Fig. S4). Therefore, using structural simulation and docking method, we predicted a possible model of EK1C4 in binding with SARS-CoV-2 HR1P (Supplementary information, Fig. S8). In this model, the EK1C4 peptide anchors to one of the three hydrophobic grooves of HR1 trimer via its EK1 moiety, and also anchors to another adjacent hydrophobic groove of HR1 trimer via its cholesterol moiety. The cholesterol group of EK1C4 may bind to HR1P through hydrophobic interactions, while several hydrogen bonds may form between HR1 and helical region of EK1C4. The intermediated GSGSG-PEG4 linker of EK1C4 peptide is just enough to connect these two moieties on the two binding targets. Admittedly, the exact mechanism and structure of EK1C4 need more studies in the future.

          In the past few decades, the viral HR1 domain has been proved to be an important target for the development of viral fusion and entry inhibitors. In the early outbreak of MERS, we quickly solved the 6-HB fusion core structure formed by MERS-CoV S protein HR1 and HR2 domains and designed the fusion inhibitory peptide HR2P-M2 which proved to be highly effective in blocking its spike protein-mediated membrane fusion and inhibit in vitro MERS-CoV infection.16 The results from animal experiments showed that intranasal application of HR2P-M2 peptide could effectively protect mice from MERS-CoV infection with reduction of virus titers in the lung more than 1000-fold.29 However, the MERS-CoV HR2P-M2 peptide could not inhibit SARS-CoV infection, suggesting that this peptide lacks cross-inhibitory activity against other β-CoVs, such as SARS-CoV and bat SARSr-CoVs. To be well prepared for combating the emerging coronaviruses with epidemic or pandemic potential, we designed and synthesized the first pan-coronavirus fusion inhibitor, EK1, and found that EK1 exhibited potent inhibitory activity against all HCoVs that we tested, including SARS-CoV and MARS-CoV, as well as bat SARSr-CoVs. As expected, we recently have shown that EK1 is also effective in inhibiting infection of the novel β-CoV, SARS-CoV-2.15 We then optimized EK1 peptide in hopes of improving its fusion inhibitory activity. Indeed, we found that one of the modified EK1 peptides, EK1C4, was 226-fold and 149-fold more potent against SARS-CoV-2 S protein-mediated membrane fusion and PsV infection, respectively, than EK1. EK1C4 also showed broad-spectrum inhibitory activity against infection by SARS-CoV, MERS-CoV and other HCoVs. EK1C4 showed prolonged and significant prophylactic effect against HCoV-OC43 infection in mouse model, suggesting that EK1C4 may also be used as an inhibitor against SARS-CoV-2 infection in vivo. Consistent with other studies,30 HCoV-OC43 was shown as a typical neurotropic virus in the mouse model, and quickly entered and established infection in mouse brain tissue, leading to the relatively weak therapeutic effect of EK1C4 via intranasal administration. However, SARS-CoV-2 mainly infected and caused severe pathological changes in human lung tissue.4 Therefore, EK1C4 administered intranasally is expected to have good therapeutic potential against SARS-CoV-2 infection.

          Currently, no specific anti-CoV therapeutics or prophylactics have been used in clinics for treatment or prevention of SARS-CoV-2 infection. A number of nonspecific antiviral drugs, including IFN, lopinavir-ritonavir (HIV protease inhibitors), chloroquine, favipiravir (T-705) and remdesivir (GS-5734), have been used in clinics in China to treat SARS-CoV-2 infection.31 Their in vivo efficacies still require further confirmation. Their potential use for treatment of infection by other coronaviruses and emerging coronaviruses in the future is unclear. Compared with these clinically used nonspecific antiviral drugs, EK1C4 has more advantages for treatment and prevention of SARS-CoV-2 infection. First, the sequence of its target, the HR1 domain in S2 subunit of S protein, is highly conserved. Therefore, EK1C4 possesses a high genetic barrier to resistance and cannot easily induce drug-resistant mutations. Second, EK1C4 can be used in an intranasal formulation to prevent coronavirus infection. The small bottles can be carried easily by persons who will have close contact with infected patients or high-risk populations. Third, EK1C4 can be used in inhalation formulation for treatment of patients to reduce the viral loads in their lungs, thus attenuating the acute lung injury caused by viral infection and reducing the chance to spread the virions to the closely contacted persons. The inhalation equipment can be used in home or hotel room, reducing the expense of staying in hospitals. Fourth, EK1C4 is expected to be safe to humans because it will be used locally, not systemically, and peptide drugs are generally safer than chemical drugs. Fifth, because of its broad-spectrum anti-coronavirus activity, EK1C4 can be used for treatment and prevention of infection by not only SARS-CoV-2, but also other HCoVs. Sixth, recently 103 SARS-CoV-2 genomes have been identified,32 but we found that both HR1 and HR2 domains among those reported genomes show 100% identity (Supplementary information, Fig. S9), indicating the high conservation of EK1C4 target. In the meantime, the HR2 derived peptides have much larger interface on HR1 domain, making it more resistant to the viral mutations. Therefore, EK1C4 shows exceptional promise to be developed as the first pan-CoV fusion inhibitor-based antiviral therapeutic or prophylactic for treatment or prevention of infection by the currently circulating SARS-CoV-2 and MERS-CoV and the future reemerging SARS-CoV and emerging SARSr-CoVs.
          Go to:
          Materials and methods
          Cell Lines, viruses and peptides

          The human primary embryonic kidney cell line (293T) (CRL-3216™), Vero E6 (CRL-1586™), RD (CCL-136™), and LLC-MK2 Original (CCL-7™) cells were obtained from the American Type Culture Collection (ATCC). Human hepatoma Huh-7 cells were from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and 293T cells stably expressing human ACE2 (293T/ACE2) cells were kindly provided by L.D. All of these cell lines were maintained and grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Carlsbad, CA, USA) containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated fetal calf serum (FCS) (Gibco).

          Patient-derived COVID-19 (BetaCoV/Wuhan/WIV04/2019) was isolated by the Wuhan Institute of Virology.6 MERS-CoV-EMC/2012 was originally provided by Chuan Qin (Beijing Key Laboratory for Animal Models of Emerging and Re-emerging Infectious Diseases). ATCC strain of Human coronavirus 229E (VR-740), as well as Human coronavirus OC43 (VR-1558) and HCoV-NL63 (Amsterdam strain) strains were amplified in Huh-7, HCT-8 and LLC-MK2 cells, respectively.

          Peptides were synthesized by Chao Wang (Beijing Institute of Pharmacology and Toxicology). The sequences of EK1 (SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL) and EK1-scrambled (LKVLLYEEFKLLESLIMEILEYQKDSDIKENAEDTK) have been reported in our previous study.14
          Plasmids

          The envelope-expressing plasmids of SARS-2-S (pcDNA3.1-SARS-2-S), SARS-S (pcDNA3.1-SARS-S), MERS-S (pcDNA3.1-MERS-S), OC43-S (pcDNA3.1-OC43-S), NL63-S (pcDNA3.1-NL63-S), 229E-S (pcDNA3.1-229E-S), and bat SARS-like CoV-S (pcDNA3.1-WIV1-S, pcDNA3.1-Rs3367-S and pcDNA3.1-SHC014-S), and the plasmids pAAV-IRES-EGFP that encode EGFP as well as the luciferase reporter vector (pNL4-3.Luc.R-E-) were maintained in our laboratory.
          Cell–cell fusion assay

          The establishment and detection of several cell–cell fusion assays are as previously described.14,16 In brief, Huh-7 cells (for testing all coronaviruses) or 293T/ACE2 cells (for testing SARS-CoV-2) were used as target cells. For preparing effector cells expressing S protein a coronavirus, 293T cells were transfected with one of the S protein expression vectors, including 293T/SARS-CoV-2/GFP, 293T/MERS-CoV/GFP, 293T/HCoV-229E/GFP, 293T/SARS-CoV/GFP, or 293T/SL-CoV/GFP, 293T/HCoV-OC43/GFP, 293T/HCoV-NL63/GFP or empty plasmid pAAV-IRES-EGFP. For SARS-CoV S-, SL-CoV S-, OC43 S- or NL63 S-mediated cell–cell fusion assays, effector cells and target cells were cocultured in DMEM containing trypsin (80 ng/mL) for 4 h, while for SARS-CoV-2 and MERS-CoV S-mediated cell–cell fusion assays, effector cells and target cells were cocultured in DMEM without trypsin but 10% FBS for 2 h. After incubation, five fields were randomly selected in each well to count the number of fused and unfused cells under an inverted fluorescence microscope (Nikon Eclipse Ti-S).
          Inhibition of HCoV S-mediated cell–cell fusion

          The inhibitory activity of a peptide on a HCoV S-mediated cell–cell fusion was assessed as previously described.14,16 Briefly, a total of 2 × 104 cells/well target cells (Huh-7) were incubated for 5 h. Afterwards, 104 cells/well effector cells (293T/S/GFP) were added in the presence or absence of a peptide at the indicated concentrations at 37 °C for 2 h. 293T/EGFP cells with phosphate-buffered saline (PBS) were used as a negative control. The fusion rate was calculated by observing the fused and unfused cells using fluorescence microscopy.
          Inhibition of pseudotyped HCoV infection

          293T cells were cotransfected with pNL4–3.luc.RE (the luciferase reporter-expressing HIV-1 backbone) and pcDNA3.1-SARS-CoV-2-S (encoding for CoVs S protein) using VigoFect (Vigorous Biotechnology, Beijing, China).16,33,34 Pseudotyped particles were efficiently released in the supernatant. The supernatant was harvested at 72 h post-transfection, centrifuged at 3000 × g for 10 min, and frozen to −80 °C. To detect the inhibitory activity of a peptide on infection of coronavirus PsV, target cells (293T/ACE2 for SARS-CoV-2, SARS-CoV and SL-CoVs; RD cells for HCoV-OC43; Huh-7 for other CoVs) were plated at a density of 104 cells per well in a 96-well plate one day prior to infection.14 PsV was mixed with an equal volume of a peptide which was series diluted with PBS at 37 °C for 30 min. The mixture was transferred to the Huh-7 cells. Medium was changed after 12 h and incubation continued for 48 h. Luciferase activity was analyzed by the Luciferase Assay System (Promega, Madison, WI, USA).
          Inhibition of live HCoV replication

          The inhibition assay for live SARS-CoV-2 and MERS-CoV was performed in a biosafety level 3 (BSL3) facility at the Wuhan Research Institute and Beijing Key Laboratory for Animal Models of Emerging and Re-emerging Infectious Diseases, respectively.6 Inhibition activity of peptides on SARS-CoV-2 and MERS-CoV was determined by plaque reduction assay. Peptides with different dilution concentrations were mixed with SARS-CoV-2 (100 TCID50) for 30 min and then added to monolayer VERO-E6 cells. After adsorption at 37 °C, the supernatant was removed, and 0.9% methyl cellulose was overlaid on the cells. After 72 h, the plates were fixed and stained. Plaques were counted by fixing with 4% paraformaldehyde and staining with 0.1% crystal violet. To test the effect of peptide on HCoV-OC43, HCoV-229E and HCoV-NL63 replication, 50 μL of 100 TCID50 virus were mixed with an equal volume of peptide and incubated at 37 °C for 1 h. Afterwards, the mixture was added to RD, Huh-7 and LLC-MK2 cells, respectively. Cell Counting Kit-8 (CCK8, Dojindo, Kumamoto, Kyushu, Japan) assay was applied to determine cytopathic effect.
          Circular dichroism spectroscopy

          The peptides or peptide mixtures were dissolved in PBS to prepare a solution with a final concentration of 10 μM at 37 °C for 30 min and then measured on a Jasco-815- circular dichroism spectrometer.35 The scanning wavelength range was 198–260 nm. Thermal denaturation detection starts at 222 nm with a 5 °C/min thermal gradient detection.
          Mouse infection studies

          Newborn mice were bred from pregnant mice purchased from the Animal Center of Fudan University, and all the related experiments were carried out in strict accordance with institutional regulations (approval number 20190221-070, approval date 21 February 2019). Each group had 12 3-day-old mice. To test the protective effect of peptides on HCoV-infected mice, EK1C4 (0.5 mg/kg), EK1 (20 mg/kg) in 2 µl 28% Hydroxypropyl-β-Cyclodextrin (HBC), or phosphate-buffered saline (PBS) solution, were administered intranasally 0.5, 1, 2, 4, 12, and 24 h before challenge, or 0.5 and 2 h after challenge. Then mice were challenged intranasally with HCoV at a dose of 102 TCID50. For the viral control group, the same volume of 28% HBC or PBS was administered intranasally. In each group, six mice were randomly selected for euthanasia on day 5 after infection, then five mice for collecting and assessing the viral titer in mouse brain, one mouse for brain histological examination. Body weight and survival of the remaining six mice in each group were monitored for 14 days.30
          Cytotoxicity assay

          Cytotoxicity of the peptides to the cells (Vero-E6, Huh-7, LLC-MK2 and RD cells) was tested by using the Cell Counting Kit-8 (CCK-8). Briefly, each cell type was seeded into the wells of a 96-well microtiter plate (10,000 per well) and incubated at 37 °C for 12–15 h, replacing medium with DMED containing EK1C4 at graded concentrations to culture at 37 °C for 2 days; CCK-8 solution (10 μL per well) was added, followed by an additional incubation for 4 h. The absorbance was measured at 450 nm.
          Expression and purification of fusion protein HR1-L6-HR2 of SARS-CoV-2

          The coding sequences of HR1 (residues 910–988) and HR2 (residues 1162–1206) domains of SARS-CoV-2 S2 subunits were tandem linked though a 6-residue linker (L6: SGGRGG). The resulting sequences encoding the fused HR1-L6-HR2 protein were then cloned into a modified pET-28a vector containing a His6-SUMO tag upstream of the multiple cloning site. The recombinant construct was expressed in Escherichia coli BL21 (DE3). Cells were grown in lysogeny broth (LB) media supplemented with 50 μg/mL kanamycin at 37 °C and were induced with 1 mM IPTG for 12 h at 16 °C overnight. Cells were harvested by centrifugation at 4500 g for 10 min at 4 °C and were lysed by high-pressure homogenizer twice after resuspension in buffer containing 25 mM Tris–HCl, pH 8.0, and 200 mM NaCl. The fusion proteins were isolated by Ni-affinity chromatography, and the SUMO tag was removed by Ulp1 enzyme (1:100 w/w) cleavage. HR1-L6-HR2 protein was concentrated and gel-filtered on a 10/300 Superdex 75 (GE Healthcare) column. Peak fractions containing HR1-L6-HR2 trimer were pooled and concentrated to 20 mg/ml through centrifugation (EMD Millipore).
          Crystallization and structure determination

          Crystals were obtained at 16 °C for 7 days using the hanging drop vapor diffusion method by mixing equal volume of protein solution (HR1-L6-HR2, 10 mg/mL) and reservoir solution (10% PEG8000, 200 mM zinc acetate, 0.1 M MES, pH 6.0). Then crystals were flash-frozen and transferred to liquid nitrogen for data collection. On the in-house (Institute of Biophysics, Chinese Academy of Sciences) X-ray source (MicroMax 007 generator (Rigaku, Japan)) combined with Varimax HR optics (Rigaku, Japan), HR1-L6-HR2 crystals at 100 K were diffracted to 2.9-Å resolution at a wavelength of 1.5418 Å. A native set of X-ray diffraction data was collected with the R-AXIS IV++ detector (Rigaku, Japan) with an exposure time of 3 min per image and was indexed and processed using iMosflm.36 The space group of the collected dataset is P21. Molecular replacement was performed with PHENIX.phaser37 to solve the phasing problem, using the SARS-CoV S protein core structure (PDB code 1WYY) as a search model. The final model was manually adjusted in COOT and refined with Refmac.38 Data collection statistics and refinement statistics are given in Table 1. Coordinates were deposited in the RCSB Protein Data Bank (PDB code: 6LXT). The interaction model of EK1C4 peptide and HR1 domains of SARS-nCoV-2 was predicted by SWISS-MODEL sever39 using 6XLT as reference for EK1 moiety, and by Autodock 4 software40 for cholesterol moiety (Supplementary information, Fig. S8).

        6. @jxk777

          An outbreak of atypical pneumonia, referred to as severe acute respiratory syndrome (SARS) and first identified in Guangdong Province, China, has spread to several countries. The severity of this disease is such that the mortality rate appears to be ∼3 to 6%, although a recent report suggests this rate can be as high as 43 to 55% in people older than 60 years (1). A number of laboratories worldwide have undertaken the identification of the causative agent (2, 3). The National Microbiology Laboratory in Canada obtained the Tor2 isolate from a patient in Toronto and succeeded in growing a coronavirus-like agent in African green monkey kidney (Vero E6) cells. This coronavirus was named publicly by the World Health Organization and member laboratories as the “SARS virus” (WHO press release, 16 April 2003) after tests of causation according to Koch’s postulates, including monkey inoculation (4). This virus, which we refer to as SARS-HCoV, was purified, and its RNA genome was extracted and sent to the British Columbia Centre for Disease Control in Vancouver for genome sequencing by the BCCA Genome Sciences Centre.

          The coronaviruses are members of a family of enveloped viruses that replicate in the cytoplasm of animal host cells (5). They are distinguished by the presence of a single-stranded plus-sense RNA genome about 30 kb in length that has a 5′ cap structure and 3′ polyadenylation tract. Upon infection of an appropriate host cell, the 5′-most open reading frame (ORF) of the viral genome is translated into a large polyprotein that is cleaved by viral-encoded proteases to release several nonstructural proteins, including an RNA-dependent RNA polymerase (Rep) and an adenosine triphosphatase (ATPase) helicase (Hel). These proteins, in turn, are responsible for replicating the viral genome as well as generating nested transcripts that are used in the synthesis of the viral proteins. The mechanism by which these subgenomic mRNAs are made is not fully understood. However, recent evidence indicates that transcription-regulating sequences (TRSs) at the 5′ end of each gene represent signals that regulate the discontinuous transcription of subgenomic mRNAs. The TRSs include a partially conserved core sequence (CS) that in some coronaviruses is 5′-CUAAAC-3′. Two major models have been proposed to explain the discontinuous transcription in coronaviruses and arterioviruses (6, 7). The discovery of transcriptionally active, subgenomic-size minus strands containing the antileader sequence and of transcription intermediates active in the synthesis of mRNAs (8–11) favors the model of discontinuous transcription during the minus strand synthesis (7).

          The viral membrane proteins, including the major proteins S (Spike) and M (membrane), are inserted into the endoplasmic reticulum (ER) Golgi intermediate compartment while full-length replicated RNA plus strands assemble with the N (nucleocapsid) protein. This RNA-protein complex then associates with the M protein embedded in the membranes of the ER, and virus particles form as the nucleocapsid complex buds into the lumen of the ER. The virus then migrates through the Golgi complex and eventually exits the cell, likely by exocytosis (5). The site of viral attachment to the host cell resides within the S protein.

          The coronaviruses include a large number of viruses that infect different animal species. The predominant diseases associated with these viruses are respiratory and enteric infections, although hepatic and neurological diseases also occur. Human coronaviruses identified in the 1960s (including the prototype viruses HCoV-OC43 and HCoV-229E) are responsible for up to 30% of respiratory infections (12). Coronaviruses are divided into three serotypes: groups 1, 2, and 3 (13). Phylogenetic analysis of coronavirus sequences also identifies three main classes of these viruses, corresponding to each of the three serotypes. Group 2 coronaviruses contain a gene encoding hemagglutinin esterase (HE) that is homologous to that of influenza C virus. It is presumed that the precursor of the group 2 coronaviruses acquired HE as a result of a recombination event within a doubly infected host cell. We note that the Tor2 genome sequence appears to lack an HE gene.

          Purification of viral particles and RNA, and DNA sequencing. Virus isolation was performed on a bronchoalveolar lavage specimen of a fatal SARS case belonging to the original case cluster from Toronto, Canada. Viral particles from this Tor2 isolate were purified, and the genetic material (RNA) was extracted (14) from the Tor2 isolate (15). The RNA was converted to cDNA by means of a combined random-priming and oligo(dT) priming strategy (14). Size-selected cDNA products were cloned, and single sequence reads were generated from each end of the insert from randomly chosen clones. Sequences were assembled and the assembly was edited to produce a draft sequence of the viral genome on 12 April 2003 (14). Rapid amplification of cDNA ends [RACE (14)] was performed to capture the 5′ end of the viral genome. The SARS genomic sequence has been deposited into GenBank (accession number AY274119.3). The final sequence we produced (also available as Release 3; http://www.bcgsc.bc.ca) is essentially identical to that released independently by the U.S. Centers for Disease Control (CDC) (16). We report additional bases in the Tor2 sequence that correspond to the 3′ (encoded) polyadenylation tail. Eight base differences between the two sequences could represent sequencing errors, polymerase chain reaction (PCR) artifacts, or mutable sites in the genome. The differences we detect between our sequence and that of the CDC are summarized in Table 1.
          Table 1.

          Nucleotide base differences between the Tor2 sequence and the Urbani sequence [(16), http://www.cdc.gov/ncidod/sars/sequence.htm%5D. Boldface indicates a base difference resulting in an amino acid change (32); X indicates a nonconservative amino acid substitution.
          PositionView inline Tor2 Urbani Frame Protein
          Base Amino acid Base Amino acid
          7,919 C A T V 1 Replicase 1A
          16,622 C A T A 3 Replicase 1B
          19,064 A E G E 3 Replicase 1B
          19,183 T V C A 3 Replicase 1B
          23,220 G A T S X 3 S (Spike) glycoprotein
          24,872 T L C L 3 S (Spike) glycoprotein
          25,298 A R G G X 2 ORF 3
          26,857 T S C P X 1 M protein

          View inline* GenBank AY274119.3.

          Non–protein-coding features of the Tor2 SARS-CoV genome sequence. At the 5′ end of the genome, we detected a putative 5′ leader sequence with similarity to the conserved coronavirus core leader sequence, 5′-CUAAAC-3′ (6, 7). Putative TRS sequences were determined through manual alignment of sequences upstream of potential initiating methionine codons (see below) with the region of the coronavirus genome sequence containing the leader sequence (Table 2). Candidate TRS sequences were scored as strong, weak, or absent on the basis of inspection of the alignments.
          Table 2.

          Nucleotide position, associated ORF, and putative transcription regulatory sequences (see text for details). Numbers in parentheses within the alignment indicate distance to the putative initiating codon. The conserved core sequence is indicated in boldface in the putative leader sequence. Contiguous sequences identical to region of the leader sequence containing the core sequence are underlined. No putative TRSs were detected for ORFs 4, 13, and 14, although ORF 13 could share the TRS associated with the N protein.
          Base ORF TRS sequence
          60 Leader UCUCUAAACGAACUUUAAAAUCUGUG
          21,479 S (Spike) CAACUAAACGAACAUG
          25,252 ORF 3 CACAUAAACGAACUUAUG
          26,104 Envelope UGAGUACGAACUUAUG
          26,341 M GGUCUAAACGAACUAACU (40) AUG
          27,001 ORF 7 AACUAUAAAUU (62) AUG
          27,259 ORF 8 UCCAUAAAACGAACAUG
          27,590 ORF 9 UGCUCUA—GUAUUUUUAAUACUUUG (24) AUG
          27,766 ORF 10 AGUCUAAACGAACAUG
          27,852 ORF 11 CUAAUAAACCUCAUG
          28,099 Nucleocapsid UAAAUAAACGAACAAAUUAAAAUG

          The 3′ untranslated region (3′UTR) sequence contains a 32–base pair region corresponding to the conserved s2m motif (17). The s2m motif is believed to be a universal feature of astroviruses that has also been identified in avian infectious bronchitis virus (avian IBV) and the ERV-2 equine rhinovirus. The high degree of conservation between the s2m motifs in these different viruses and their evolutionary distance suggests that the avian IBV and ERV-2 have acquired the s2m motif through separate horizontal RNA transfer events (17). The inferred distance of the SARS coronavirus to IBV from our phylogenetic analysis (Fig. 1) would also suggest that the SARS coronavirus has obtained its s2m motif through a horizontal transfer event.

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          Fig. 1.

          Phylogenetic analysis of SARS proteins. Unrooted phylogenetic trees were generated by clustalw 1.74 (33) using the BLOSUM comparison matrix and a bootstrap analysis of 1000 iterations. Numbers indicate bootstrap replicates supporting each node. Phylogenetic trees were drawn with the Phylip Drawtree program 3.6a3 (34). Branch lengths indicate the number of substitutions per residue. GenBank accession numbers for protein sequences are as follows: (A) Replicase 1A: BCoV (bovine coronavirus), AAL40396; HCoV-229E (human coronavirus), NP_073555; MHV (mouse hepatitis virus), NP_045298; IBV (avian infectious bronchitis virus), CAC39113; TGEV (porcine transmissible gastroenteritis virus), NP_058423. (B) Membrane glycoprotein: PHEV (porcine hemagglutinating encephalomyelitis virus), AAL80035; BCoV, NP_150082; IBV 1, AAF35863; IBV 2, AAK83027; MHV, AAF36439; TGEV, NP_058427; HCoV-OC43, AAA45462; FCoV (feline coronavirus), BAC01160. (C) Nucleocapsid: MHV, P18446; BCoV, NP_150083; IBV 1, AAK27162; IBV 2, NP_040838; FCoV, CAA74230; PTGV (porcine transmissible gastroenteritis virus), AAM97563; HCoV-229E, NP_073556; HCoV-OC43, P33469; PHEV, AAL80036; TCV (turkey coronavirus), AAF23873. (D) S (Spike) protein: BCoV, AAL40400; MHV, P11225; HCoV-OC43, S44241; HCoV-229E, AAK32191; PHEV, AAL80031; PRCoV (porcine respiratory coronavirus), AAA46905; PEDV (porcine epidemic diarrhea virus), CAA80971; CCoV (canine coronavirus), S41453; FIPV (feline infectious peritonitis virus), BAA06805; IBV, AAO34396.

          Predicted protein coding features of the Tor2 SARS-CoV genome sequence. ORFs were determined initially through sequence similarity to known coronavirus proteins. This approach identified replicases 1a and 1b, the S protein, the small envelope (E) protein, the M protein, and the N protein. ORFs that did not match database sequences were identified if they were larger than 40 amino acids, unless a strong match to the TRS consensus was found close to and upstream of the potential initiating methionine residue. We note that Rota et al. (16) did not identify potential proteins of less than 50 amino acids. We attempted to identify putative TRSs upstream of all ORFs, both known and predicted (Tables 2 and 3). However, TRSs are not required for transcription of all coronavirus genes, because internal initiation from larger RNA transcripts is also able to facilitate translation (18, 19). Certain ORFs overlap (ORFs 10 and 11, by 12 amino acids; Fig. 2), and some are contained entirely within another ORF or ORFs (ORF 4 and ORFs 13 and 14; Fig. 2). The biological relevance of these ORF predictions remains to be established, but in the cases of ORFs 10 and 11, we detect strong matches to the TRS consensus in close proximity to their respective initiating methionine codons (Table 2). Construction of unrooted phylogenetic trees using the set of known proteins and representatives of the three known coronaviral groups reveals that the proteins encoded by the SARS virus do not readily cluster more closely with any one group (Fig. 1). Hence, we propose that this isolate be considered the first representative of “group 4” coronaviruses.

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          Fig. 2.

          Map of the predicted ORFs and s2m motif in the Tor2 SARS virus genome sequence.
          Table 3.

          Features of the Tor2 genome sequence.
          Feature Start—EndView inline No. of amino acids No. of bases Frame Candidate TRSView inline Rota et al. ORFView inline
          ORF 1a 265-13,398 4,382 13,149 +1 N/AView inline 1a
          ORF 1b 13,398-21,485 2,628 7,887 +3 N/A 1b
          S protein 21,492-25,259 1,255 3,768 +3 Strong S
          ORF 3 25,268-26,092 274 825 +2 Strong X1
          ORF 4 25,689-26,153 154 465 +3 AbsentView inline X2
          E protein 26,117-26,347 76 231 +2 Weak E
          M protein 26,398-27,063 221 666 +1 Strong M
          ORF 7 27,074-27,265 63 192 +2 Weak X3
          ORF 8 27,273-27,641 122 369 +3 Strong X4
          ORF 9 27,638-27,772 44 135 +2 Weak N/R
          ORF 10 27,779-27,898 39 120 +2 Strong N/R
          ORF 11 27,864-28,118 84 255 +3 Weak X5
          N protein 28,120-29,388 422 1,269 +1 Strong N
          ORF 13 28,130-28,426 98 297 +2 AbsentView inline N/R
          ORF 14 28,583-28,795 70 213 +2 AbsentView inline N/R
          s2m motif 29,590-29,621 N/A 30 N/A N/A N/R

          View inline* End coordinates include the stop codon, except for ORF 1a and s2m. The right coordinate of ORF 1a is the end position of the ribosome slippage site (UUUAAC). The most likely frameshift site, based on alignment with other replicase proteins, is 13,392.

          View inline† See text for details.

          View inline‡ Corresponding ORFs from Rota et al. (16). N/R indicates the feature was not reported.

          View inline§ These ORFs overlap substantially or completely with others and may share TRSs.

          View inline∥ N/A, not applicable.

          The coding potential of the 29,751-base genome is depicted in Fig. 2. Recognizable ORFs include the replicase 1a and 1b translation products, the S glycoprotein, the E protein, the M protein, and the N protein. We have, in addition, conducted a preliminary analysis of the nine novel ORFs in an attempt to ascribe to them a possible functional role. These analyses are summarized below.

          The replicase 1a ORF (base pairs 265 to 13,398) and replicase 1b ORF (base pairs 13,398 to 21,485) occupy 21.2 kb of the SARS virus genome (Fig. 2). Conserved in both length and amino acid sequence to other coronavirus replicase proteins, the genes encode a number of proteins that are produced by proteolytic cleavage of a large polyprotein (20). As seen in other coronaviruses and as anticipated, a frame shift interrupts the protein-coding region and separates the 1a and 1b reading frames.

          The Spike (S) glycoprotein (Fig. 2; base pairs 21,492 to 25,259) encodes a surface projection glycoprotein precursor predicted to be 1255 amino acids in length. Mutations in the gene encoding the Spike protein have previously been correlated with altered pathogenesis and virulence in other coronaviruses (5). In some coronaviruses, the mature Spike protein is inserted in the viral envelope, with most of the protein exposed on the surface of the viral particles. It is believed that three molecules of the Spike protein form the characteristic peplomers or corona-like structures of this virus family. Our analysis of the Spike glycoprotein with SignalP (21) reveals a high probability of a signal peptide (probability 0.996) with cleavage between residues 13 and 14. TMHMM (22) reveals a strong transmembrane domain near the C-terminal end. Together these data predict a type I membrane protein with the N terminus and the majority of the protein (residues 14 to 1195) on the outside of the cell surface or virus particle, in agreement with other coronavirus Spike protein data. Supporting this conclusion, it has recently been shown that for HCoV-229E virions, residues 417 to 546 are required for binding to the cellular receptor, aminopeptidase N (23). However, it is known that various coronaviruses use different receptors, and hence it is likely that different receptor binding sites are also used.

          ORF 3 (Fig. 2; base pairs 25,268 to 26,092) encodes a predicted protein of 274 amino acids that lacks significant BLAST (24), FASTA (25), or PFAM (26) similarities to any known protein. Analysis of the N-terminal 70 amino acids with SignalP provides weak evidence for the existence of a signal peptide and a cleavage site (probability 0.540). Both TMpred (27) and TMHMM predict the existence of three transmembrane regions spanning approximately residues 34 to 56, 77 to 99, and 103 to 125. The most likely model from these analyses is that the C terminus and a large 149–amino acid N-terminal domain would be located inside the viral or cellular membrane. The C-terminal (interior) region of the protein may encode a protein domain with ATP-binding properties (ProDom ID PD037277).

          ORF 4 (Fig. 2; base pairs 25,689 to 26,153) encodes a predicted protein of 154 amino acids. This ORF overlaps entirely with ORF 3 and the E protein. Our analysis failed to locate a potential TRS sequence at the 5′ end of this putative ORF. However, it is possible that this protein is expressed from the ORF 3 mRNA using an internal ribosomal entry site. BLAST analyses fail to identify matching sequences. Analysis with TMpred weakly predicts a single transmembrane helix.

          The gene encoding the small envelope (E) protein (Fig. 2; base pairs 26,117 to 26,347) yields a predicted protein of 76 amino acids. BLAST and FASTA comparisons indicate that the predicted protein exhibits significant matches to multiple envelope (alternatively known as small membrane) proteins from several coronaviruses. PFAM analysis of the protein reveals that the predicted protein is a member of the well-characterized NS3_EnvE protein family (26). InterProScan (28, 29) analysis reveals that the protein is a component of the viral envelope, and conserved sequences are also found in other viruses, including gastroenteritis virus and murine hepatitis virus. SignalP analysis predicts the presence of a transmembrane anchor (probability 0.939). TMpred analysis of the predicted protein reveals a similar transmembrane domain at positions 17 to 34, consistent with the known association of this protein with the viral envelope. TMHMM predicts a type II membrane protein with most of the hydrophilic domain (46 residues) and the C terminus located on the surface of the viral particle. In some coronaviruses such as porcine transmissible gastroenteritis virus (TGEV), the E protein is essential for virus replication (30). In contrast, in mouse hepatitis virus (MHV), although deletion of the gene encoding the E protein reduces virus replication by more than four orders of magnitude, the virus still can replicate (31).

          The gene encoding the membrane (M) glycoprotein (Fig. 2; base pairs 26,398 to 27,063) yields a predicted protein of 221 amino acids. BLAST and FASTA analyses of the protein reveal significant matches to a large number of coronaviral matrix glycoproteins. The association of the Spike glycoprotein (S) with the matrix glycoprotein (M) is an essential step in the formation of the viral envelope and in the accumulation of both proteins at the site of virus assembly (5). Analysis of the amino acid sequence with SignalP predicts a signal sequence (probability 0.932) that is not likely cleaved. TMHMM and TMpred analyses indicate the presence of three transmembrane helices, located at approximately residues 15 to 37, 50 to 72, and 77 to 99, with the 121–amino acid hydrophilic domain on the inside of the virus particle, where it is believed to interact with the nucleocapsid. PFAM analysis reveals a match to PFAM domain PF01635 and alignments to 85 other sequences in the PFAM database bearing this domain, which is indicative of the coronavirus matrix glycoprotein.

          ORF 7 (Fig. 2; base pairs 27,074 to 27,265) encodes a predicted protein of 63 amino acids. BLAST and FASTA searches yield no significant matches indicative of function. TMHMM and SignalP predict no transmembrane region; however, TMpred analysis predicts a likely transmembrane helix located between residues 3 and 22, with the N terminus located outside the viral particle. Similarly, ORF 8 (Fig. 2; base pairs 27,273 to 27,641), encoding a predicted protein of 122 amino acids, has no significant BLAST or FASTA matches to known proteins. Analysis of this sequence with SignalP indicates a cleaved signal sequence (probability 0.995) with the predicted cleavage site located between residues 15 and 16. TMpred and TMHMM analyses also predict a transmembrane helix located approximately at residues 99 to 117. Together these data indicate that ORF 8 is likely to be a type I membrane protein, with the major hydrophilic domain of the protein (residues 16 to 98) and the N terminus oriented inside the lumen of the ER/Golgi or on the surface of the cell membrane or virus particle, depending on the membrane localization of the protein.

          ORF 9 (Fig. 2; base pairs 27,638 to 27,772) encodes a predicted protein of 44 amino acids. FASTA analysis of this sequence reveals some weak similarities (37% identity over a 35–amino acid overlap) to Swiss-Prot accession Q9M883, annotated as a putative sterol-C5 desaturase. A similarly weak match to a hypothetical Clostridium perfringens protein (Swiss-Prot accession CPE2366) is also detected. The functional implications, if any, of these matches are unknown. TMpred predicts the existence of a single strong transmembrane helix, with little preference for alternate models in which the N terminus is located inside or outside the particle. Similarly, ORF 10 (Fig. 2; base pairs 27,779 to 27,898), encoding a predicted protein of 39 amino acids, exhibits no significant matches in BLAST and FASTA searches but is predicted to encode a transmembrane helix by TMpred, with the N terminus located within the viral particle. The region immediately upstream of ORF 10 exhibits a strong match to the TRS consensus (Table 2), providing support for the notion that a transcript initiates from this site. ORF 11 (Fig. 2; base pairs 27,864 to 28,118), encoding a predicted protein of 84 amino acids, exhibits only very short (9 or 10 residues) matches to a region of the human coronavirus S glycoprotein precursor (starting at residue 801). Analyses by SignalP and TMHMM predict a soluble protein. As was the case for ORF 10, a detectable alignment to the TRS consensus sequence was found (Table 2).

          The gene encoding the nucleocapsid protein (Fig. 2; base pairs 28,120 to 29,388) yields a predicted protein of 422 amino acids. This protein aligns well with nucleocapsid proteins from other representative coronaviruses, although a short lysine-rich region (KTFPPTEPKKDKKKKTDEAQ) (32) appears to be unique to SARS. This region is suggestive of a nuclear localization signal, and although it contains a hit to InterProDomain IPR001472 (bipartite nuclear localization signal), the function of this insertion remains unknown. It is possible that the SARS virus nucleocapsid protein has a novel nuclear function, which could play a role in pathogenesis. In addition, the basic nature of this peptide suggests that it may assist in RNA binding.

          ORF 13 (Fig. 2; base pairs 28,130 to 28,426) encodes a predicted protein of 98 amino acids. BLAST analysis fails to identify similar sequences, and no transmembrane helices are predicted. ORF 14 (Fig. 2; base pairs 28,583 to 28,795) encodes a predicted protein of 70 amino acids. BLAST analysis fails to identify similar sequences. TMpred weakly predicts a single transmembrane helix.

          Conclusions. We used genome sequencing to determine that the virus named by the WHO as causally associated with SARS is a novel coronavirus. This has been confirmed by the sequence of two independent isolates: the Tor2 isolate, reported here, and the Urbani isolate, reported by the CDC (16). Although morphologically a coronavirus (3), this SARS virus is not more closely related to any of the three known classes of coronavirus, and we propose that it defines a fourth class of coronavirus (group 4) and that it be referred to as SARS-CoV. Our sequence data do not support a recent interviral recombination event between the known coronavirus groups as the origin of this virus, but this may be due to the limited number of known coronavirus genome sequences. Apart from the s2m motif located in the 3′UTR, there is also no evidence of any exchange of genetic material between the SARS virus and non-Coronaviridae. These data are consistent with the hypothesis that an animal virus for which the normal host is currently unknown recently mutated and developed the ability to productively infect humans. There also remains the possibility that the SARS virus evolved from a previously harmless human coronavirus. However, preliminary evidence suggests that antibodies to this virus are absent in people not infected with SARS-CoV (3), which implies that a benign virus closely related to the Tor2 isolate is not resident in humans. Identification of the normal host of this coronavirus and comparison of the sequences of the ancestral and SARS forms will further elucidate the process by which this virus arose.

          The availability of the SARS virus genome sequence is important from a public health perspective. It will allow the rapid development of PCR-based assays for this virus that capitalize on novel sequence features, enabling discrimination between this and other circulating coronaviruses. Such assays will allow the diagnosis of SARS virus infection in humans and, critically, will consolidate the association of this virus with SARS. If the association is further borne out, SARS virus genome–based PCR assays may form an important part of a public health strategy to control the spread of this syndrome. In the longer term, this information will assist in the development of antiviral treatments, including neutralizing antibodies and development of a vaccine to treat this emerging and deadly disease.

        7. …..The software used was the KRUNCH suite illustrated in the appendix to Ref. [48], using a model of “pseudoatoms” (artificial interaction centers) representing first shell of water. The approach, described in more detail in Section 4.8, is one of molecular mechanics using energy minimization capable of exploring multiple minima, boosted by various algorithms to help explore the conformational energy space as a whole. However, many conformers of comparable low energy are found, simply supporting the idea that these candidate peptides in Section 4.6 are conformationally flexible. Starting from an extended structure, essentially e for each residue but specifically backbone dihedral angles Φ = −90°, Ψ = +150°, there is a preferred overall secondary structure in reasonable accord with the predictions made on the 2019-nCoV S protein made above using GOR IV for the segment PSKRSFIEDLLFNKVT, and the d-amino acid sequence in reverse is consistent with the mirror image conformation of the reverse sequence. In effect, that simply means that an α-helical conformation (and its mirror image in the d-amino acid peptide case) did not appear. There was departure from local deep minima. Notably EDLLFNK tended occasionally to adopt angles around Φ = −60° Ψ = −50° characteristic of α-helix, but they returned to the extended conformer. Very preliminary binding studies similar to those of Barr’e et al., but using 2019-nCoV spike protein and the retorinverso peptide suggest various modes of binding varying between circa −11 and −16 kcal/mol but these numbers should not be taken too seriously. Only relative values are meaningful but they have been scaled (“adjusted”) by correction factors established in previous higher grade molecular dynamics calculations [48]. In any event the above calculations do not include a potentially strong entropy contribution for each conformer (and ideally for the overall solute-solvent system).
          4.9. Preliminary studies on the proposed peptides and smaller ligands using a model pharmacophore for 2019-nCoV antagonism

          The system used to explore 2019-nCoV to host cell binding, activation and cell entry, i.e. the “binding model”, was chosen on the basis of inhibitors common to, or similar between, (a) SARS binding, activation and entry and (b) and ligands including inhibitors of an enzyme of medical importance, as discussed here below. This model system was initially and in part simply a practice “set up” for relevant studies, suggested by certain relationships between the actions of small molecules revealed by auto-surfing as described below. The choice is undoubtedly likely to be of concern because it involves a human enzyme unlikely to be involved in 2019-nCoV entry. Nonetheless, it proved insightful, and consider that, until relatively recently in the history of pharmacology, ignorance of the atomic details of a protein to which a ligand (as the proposed bioactive small molecule) binds, has been the norm. Pharmacologists have used evidence and hunches to deduce a pharmacophore, i.e. an abstract description of molecular features that are necessary for molecular recognition of the ligand by the protein. The rational for the strategy used in the present study is that one can make model binding sites and draw some useful conclusions, but using the actual experimental structure of a protein known to bind similar ligands is more likely to give realistic insight. A justification is that if the pharmacophore in the target choice has no clear features to refute that choice, which the present author calls the “target refutation principle”, then it is at least a worthy first choice as a “straw man”, i.e. a conjecture for criticism and further debate. After all, any preliminary pharmacophore model might be later refined by adding an “except when” condition to its description.

          The choice of the target protein as model was governed by several considerations discussed below, mostly revealed by the auto-surfing approach, and motivated by the above problems of conformational flexibility for peptide ligands. Other investigations by the author and collaborators have focused on rigid, steroid-like scaffolds for binding groups, and also on molecules roughly resembling steroid-like “pieces”. In one such study [48], using as the lead a known inhibitor and a steroid-like plant derivative called carbenoxolone, a list of such “pieces” and related molecules screened for binding more at least a superficial resemblance to emodin (1,3,8-trihydroxy-6-methylanthraquinone). This compound came to attention as important in the present study because the “auto-surfing” found that emodin has already been shown to be an inhibitor of SARS-CoV entry [57,58]. See Fig. 3. Thus by analogy, it is a putative antagonist for 2019-nCoV entry. Even more importantly, it was found that several studies (e.g. Ref. [60] have shown that emodin, carbenoxelone (again, see Fig. 3), and other molecules with some similarity can inhibit 11-beta-hydroxysteroid dehydrogenase type 1 in rodents and humans. The experimental structure of this enzyme is available with bound carbenoxolone (Protein Data Bank entry 2BEL; the structure also includes the NADP cofactor). In a series of anthraquinone compounds shown to inhibit the steroid-processing enzyme 11-beta-hydroxysteroid dehydrogenase type 1 of both humans and mice, emodin was identified as the most potent selected [60].
          Fig. 3

          Download : Download high-res image (112KB)Download : Download full-size image

          Fig. 3. Emodin, found elsewhere to be an inhibitor of SARS-CoV entry, has some binding features involving the ketone group that are akin to carbenoxelone and to a number of molecules that may be considered as substructures of carbenoxelone’s steroid-like ring.

          The KRUNCH method of molecular modeling used [48,62,63] is not commercially available although the molecular dynamics, docking and other parts of the suit as a whole are essentially the same as standard available versions, and have in the past sometimes been replaced by the standard available versions [48]. The core approach, which is of less usual character, is predominantly concerned with conformational space, not phase space based on molecular dynamics with a dimension of simulated time. This core approach is a descendant of earlier developments in protein and peptide modeling [11] that did not simulate Newtonian dynamics but rather they applied energy minimization techniques, and in particular those called Simplex methods [11]. Because they do not depend on continuous derivatives these methods can navigate a potential energy surface pitted with multiple small energy minima [11,63]. This approach is then extended in pursuit of identifying the global minimum or minima by a Globex method that looks for trends in deep minima on the larger scale [11], and then finally embellished by a variety of techniques that explore conformational space efficiently [63]. Roughly speaking, however, history of a simulation gives a similar impression to molecular dynamics, albeit run at high temperatures with periods of cooling or annealing.

          Initial binding studies using 11-beta-hydroxysteroid dehydrogenase type 1 as “receptor” and activator of the virus are similar to those used in Ref. [48]. Early alternative choices of binding protein as model included growth hormone secretagogue receptor type 1 GenPept accession NP_796304 because of some evidence in the literature of the action of anthraquinone, emoghrelin derivatives, and indanthrone, amongst others, which have some of the features of emodin, and because of an initial suspicion that a sequence segment in growth hormone receptor might contribute to binding and had some features relating to the current peptide of interest, but none of the alternatives were ultimately seen as persuasive as the steroid dehydrogenase choice. Preliminary studies in the present project also suggested possible similarities of binding to the active site of steroid dehydrogenase between (a) the peptide epitope proposed above, (b) the retroinverso peptidomimetic, (c) a compound carbenoxelone already studied somewhat intensively by the author and collaborators [48], and (d) emodin. See below. Although initial results for binding of the peptides −8 to −10 kcal/mol initially seemed promising, emodin and carbenoxelone bound significantly lower as discussed below, and the peptide binding differed by showing multiple similar energy binding modes.

          Although the dehydrogenase is a very different model system to any directly relating to 2019-nCoV, there were some interesting observations regarding emodin binding that might be extensible to more relevant simulations in a manner consistent with the above “target refutation principle”. Using the software described in Ref. [62] and the appendix to Ref. [48], initial studies comprised simply superimposing the smaller analogues into the steroid core of carbenoxolone using the position of the analogous ketone O11 atom on C11 of the steroid-like framework of carbenoxelone and serine 170 side chain oxygen atom as a pivot, removing the carbenoxolone, and initially locally minimizing the energy of the new enzyme-ligand system. Conformational changes of the enzyme on replacing by emodin depended critically on choice of dialectric constant, values less than 20 gave less than 1.9 Å rms. In practice, one conventional rule of thumb is that each aromatic ring can contribute up to about −1.7 kcal/mol in going from an aqueous to a non-polar environment. But although carbenoxolone is steroid-like, and so expected to fit in very hydrophobic pockets, there is ample evidence that there is often hydrogen bonding occurring between steroids and their receptors [61]. In the present system, however, despite significant and differing conformational adjustments by the protein to accommodate these two ligands, there is a negative electrostatic tension in the region of the ketone group because due to close approach of the serine 170 side chain oxygen atom the phenolic oxygen of Tyr 183 and also by oxygen O7N in the NADP cofactor. These electrostatic tensions would seem to provide evidence against the choice of the target protein according to the above target refutation principle. However, it is to be recalled that there is catalysis to consider (including a serine protease type of mechanism) for the proteolysis that is believed to be required to activate the virus for entry. Also, this is at a cut at the lysine residue following an arginine, both carrying a positively charged sidechain. Consequently, relevance cannot be dismissed. Also of course, binding can be favorable overall. Indeed, despite the electrostatic tension, binding energies of −11 and −16 kcal/mol (again adjusted against previous high grade calculations as in Section 4.7) were obtained for emodin and carbenoxelone respectively which seem reasonable indicators of significant binding. Again, however, such values should not be taken as predictions of experimental binding free energy because the above comments on relative values and entropy still apply for the overall solute-solvent system even though the ligands themselves are much more rigid. Other compounds containing one or few steroid-like rings such as (2,3)-dithio-6-hydroxy-8-carboxy-(1,7,9)-azanaphthalene showed as binding similarly in some binding simulation methods but not in others, and when binding showed multiple binding modes.
          5. Discussion and conclusions
          5.1. The proposed L-peptide

          Recall that it is the l-amino sequence GPSKRSFIEDLLFNKVTLAC that was proposed as an B-epitope to be synthesized with L amino acids and attached to a carrier, for purposes of raising antibodies as a diagnostics, or as a potential vaccine. In the letter case it might require an additional peptide as a T-epitope (for immune system memory), which might come from anywhere in the S protein or even elsewhere in virus, and perhaps other agents such as molecular adjuvant. These are known considerations in the state of the art [50,51], although vaccines based on this synthetic approach are still relatively disfavored for human use despite being in increased use in veterinary medicine. The reason may in part be the historical difficulty in synthesizing longer peptides without side reactions and accumulative errors in the insertions of the amino acids, but now whole proteins can be synthesized, even out of d-amino acids (in which case they fold in mirror image of the native form) [43]. It is increasingly held that the synthetic peptide vaccine approach can sometimes be more effective than traditional vaccine methods [50], and it has been steadily increasing in popularity [51]. The main problem to overcome for vaccine and diagnostic design, with some analogous issues for therapeutic peptidomimetics, seems to be that in many pathogens, the B epitope antigenic determinants for antibody production are discontinuous sections of sequence, and using just one of them typically delivers poor results [51]. However, since sections can be linked chemically with runs of connecting amino acids such as glycine, this is, strictly speaking, just a further layer of design, especially if the three dimensional structure of the protein is known. For the immediate future, that will probably still require a significant amount of experimental trial and error to refine the design, but it is certainly facilitated if one can first obtain activity from studies on different continuous epitopes, ultimately linking the peptides together as above.
          5.2. The proposed D-peptide

          Recall also that is was d-amino acid sequence GNFLLDEIFSRKSRKSPC that was proposed for synthesis from d-amino acids to act as an antagonist to viral entry. The reason for the retroinverso approach is that an L-peptide would be susceptible to proteolysis from a variety of human proteins. In silico (computer simulations) make it possible to use both an L-peptide and D-peptide, as standard molecular mechanics or dynamics will not emulate catalysis (and it would take a remarkably sophisticated and fast quantum mechanical calculation to do so), and the L-peptide results may also be helpful in the design process. In vivo, a D-peptide would have a much longer half-life to work either at the human protein required for binding, most often believed to be the angiotensin converting enzyme type 2 (ACE2) as responsible for binding SARS-CoV to lung cells, or as an inhibitor preventing proteolysis required for activation of the S spike protein. At the time of writing, it is the type II transmembrane serine protease TMPRSS2 that is favored as responsible for the activation cleavage. The author favors the inhibition of proteolysis. If the peptide is correct, at least we know form the SARS spike protein experiments that the lysine (K) in the terminal segment PKSPC corresponds to the proteolytic cleavage point in the segment PSKRS. There must be a sufficient degree of binding of an inhibitor to the active (i.e. catalytic) site and many competitive inhibitors work by binding reversibly to it. It is a common view even to the point of view of some authors defining competitive inhibition that way. However, the main binding and recognition site may not be the same thing, and there are also mechanisms by which an enzyme may bind either the inhibitor or the substrate but never both at the same time. Allosteric interactions allow competitive, non-competitive, or uncompetitive inhibition, and the peptide binding at another site could act to enhance enzyme activity (and so act as an agonist to viral entry). Certainly a practical advantage of focusing on the protease in the laboratory is that its inhibition can initially be tested without having to use virus or virus protein. Strictly speaking it is the relative strengths of binding virus and peptide that matter, but this is not generally seen as a serious obstacle in practice.

          The original proposal for the retroinverso compound [2] suggested a proline (P) to substitute for arginine (R) in the end section RKSPC. This modification had somewhat the status of a “hunch” and is at best a “weak rule” in the sense of Section 5.3 below. Having been demoted in the present paper it would seem hardly worthy of mention here except that proline has played a recurrent high profile role in the history of design of peptidomimetics, and the modification might still be worth considering if binding of the recommended retroinverso peptide is negligible or weak. Briefly, the various ideas used in the president study involved considering the advantages of a proline residue lacking an NH group that in the retroinverso peptide was suspected to involve an unfavorable N–H.H–N interaction, and a different flexibility at that critical point in which peptide bond cis-trans isomerization replaced extensive N-Cα rotation. There was also a concern that residual L or specific D proteolysis or racemization to L can still occur at some points in a d-amino acid residue sequence. However, the former stereochemical considerations apply to the steroid processing enzyme as the binding and inhibitor model, not necessarily to the lung proteases, and even in considering the former model there are also some objections, discussion of which is beyond current scope.
          5.3. Use of bioinformatics and selection of sequences for synthesis

          There has been an argument amongst pharmaceutical scientists that, especially at early stages of a study, bioinformatics combined with hunches of the experienced pharmaceutical synthetic chemistry can often provide more reliable guidelines than, for example computational ligand-protein binding simulations that are hampered by the complexity of conformation space [63]. At least, for some decades investigation by bioinformatics has been considered as an important early step, certainly in the views of the present author and collaborators [[64], [65], [66]]. The main role of the project described in the present paper was initially to describe bioinformatics strategies, rather than binding studies, that have worked in other cases as discussed briefly shortly below, and to explore automation. Compared with the tasks of automating protein modeling and drug design in which the present author has been involved (e.g. Refs. [[21], [22], [23], [24], [25], [26], [27],48]) it seems clear that the above relatively simple series of bioinformatics steps can readily be automated. The main tasks are for this kind of work are (a) detecting those sequences that are conserved in a surface protein across a viral (and potentially bacterial or parasitic) group of organisms, strain and likely to be exposed to the exterior (prediction of coil C helps here as discussed in the text), and (b) programming the set of rules for the detailed choice of amino acid residue sequence and changes to it. Section 4.6 gave a set of rules for the present study that are readily automatable, and those rules are essentially the ones that have stood the test of time and appear logically justified. For design of synthetic vaccines, such automation has already been extensively implemented as prediction software by many workers [49]. All these are essentially tools for predictions, and they are rules-of-thumb particularly in the sense that they may not work in very case, and may needed to be adjusted on a trial-and-error basis. There are also “weak” rules for which evidence for general applicability is questionable, but which could work in a few special cases. The only “rule” in the original specification of Section 4.6 that on further consideration lacked generality, is that for proline discussed above in Section 5.3.
          5.4. Computational calculations and binding simulations

          Currently, computational chemistry, protein modeling, and binding simulations are popular tools. In practice, however, they must be used with caution because of complexity of the conformational space and, related to that, strong entropic contributions, including of course from the surrounding solvent. This is probably the main reason why IBM’s very high performance computer Blue Gene, originally developed on the premise by some workers (including the present author) that it might be able to predict at least the three dimensional structure of some smaller proteins by folding simulation lasting for about a year of computer time [63], did not succeed. It did however achieve many insights into protein modeling as discussed in Ref. [63], and went on to have other applications, including drug design based on reading all US patents at the time, and evolving designs by DOCK and molecular dynamics simulations [48]. The scaling “adjustment” factors use in the molecular mechanics KRUNCH approach in Section 4.8 also depended on these more sophisticated, high grade simulations [48]. Other sources of error includes also (typically) include neglect of quantum mechanical contributions, neglecting quantization of vibrations and with modeling based primarily with atoms representing centers of interaction, essentially meaning limitations in potential functions, which in turn may largely mean neglect of changes in interactions and interaction centers in the environment of other electrical fields.

          From the preliminary conformation and binding studies carried out in the present study, one may conclude that these approaches are, for the proposed peptides, challenging by involving many degrees of freedom, both in the conformational and general sense. Simulation methods need to be enhanced by methods that can assess protein and protein-ligand interaction model (e.g. Ref. [62]), and heuristics for overcoming the multiple minimum problem (e.g. Ref. [63]), but so far these particular cited approaches have not provided a quick solution on a standard personal computer..For pharmaceutical reasons too, moving toward smaller non-peptide analogues may be the best direction. It is probably best to await detailed experimental structures of viral and human proteins involved in activation, and their interaction. The electrostatic tension that applied in the above choice of pharmacophore may not make it an optimal model system, but “computer experiments” and incidental observations can provide useful clues and insights. In earlier studies on related compounds including carbenoxelone, converting the ketone group = O to a thioketone = S derivative in most of the compounds studied appeared to enhance binding at first by relaxing the electrostatic tension despite increasing increased the van der Waal’s repulsion, but it was subsequently appreciated that a stronger partial charge is required for the thioketone sulfur on a benzene ring because chemically this is strongly electronegative, carrying closer to a full electron unit of charge. The CS bond length of thiobenzophenone is 1.63 Å thioformaldehyde, but due to steric interactions, the phenyl groups are not coplanar and the dihedral angle SC-CC is 36°. Nonetheless, the binding energy of the emodin thioketone fell by 2 kcal/mol, and this is being investigated. However, the thioketone group is not an optimal choice to explore and might well cause oligomerization in practice, but studies like that above do support the idea…

          that other replacements to the corresponding ketone group in multiple ring substructures of the steroid-like structure might reasonably be explored in pursuit of potent analogues. Of course, the appropriate pharmacophore could be one with hydrogen bonding requirements not met with the above model.
          5.5. Effectiveness of peptide based approaches in general

          Many principles similar to those of epitope selection apply to therapeutic pepidomimetic design, at least for the relatively simple step of proposing a retroinverso candidate. It is a worthy early strategy, if only on the grounds that it shows suggestive wanted activity, or it does not, and one might well consider it unfortunate if a great deal of computational medicinal chemistry and laboratory synthesis and testing is done, only to find later that using the retroinverso approach would have created a successful first candidate at the outset. A limitation of the approach is that one must be aware of coincidental matches that are not truly significant unless one can see that the proteins being compared are essentially of the same function or family, with the order of similar sections preserved, and that the correspondences make sense in the light of background biological knowledge. Even armed with such knowledge, one must be wary of making too big a jump. A curious example from the present study is as follows. Because in the author’s experience a simple reversal of the peptide sequence (i.e. using l-amino acids) can sometimes have required activity, it was interesting to also perform a BLAST search on that reverse sequence. One of the closest matches is with a close match of the above reverse sequence with a sequence in a bacterial enzyme that interconverts D and L alanine: bifunctional UDP-N-acetylmuramoyl-tripeptide:D-alanyl-d-alanine ligase, also known as alanine racemase. More specifically, NFLLDEIFSRKSPKS finds a similar section NFLLNEEFSVIKSPKS in the racemases ID WP_090409885.1 and WP_158209963.1. This is an interesting coincidence, but it does not (as yet) stand up to investigation. This is primarily because amino acid residues from a variety of parts of the racemase appear to interact with itssubstrate, and because the sequence is not significantly preserved in a large number of both related and diverse racemases.
          5.6. Future therapeutic aspects and studies

          In the ongoing fight against infectious disease there is accelerating progress in the identification of relevant peptides and, where appropriate, design of smaller drug molecules based on them should be applied (e.g. Refs. [[67], [68], [69], [70], [71], [72]]) Such progress provides many additional tools and strategies that could be used. In some cases, one might find that repurposing existing drugs or herbal extracts with suspected genuine efficacy may help in the challenge. As a kind of “stop press”, note that at the time of this study it seemed plausible that angiotensin converting enzyme inhibitors could do the same job as a petidomimetic. This was based on certain molecular similarities, but recent studies have highlighted that customary ACE inhibitors are likely to have a counterproductive effect, although an ACE 2 inhibitor might well work usefully. It is likely that the concentrations required may have an excessive effect on blood pressure, but an aerosol preparation might be useful. However, much research on this is needed. However, a peptidomimetic based on the above motif perhaps also delivered as an aerosol, could perhaps be more specific and have less side effects. Design or discovery of more rigid molecules with similar van der Waals’s and electrostatic surfaces would be the more traditional pharmaceutical choice.

          In the 2011 fictional movie “Contagion” [73] a herbal plant supplement claimed to cure a fictional viral infection played the role of the villain compared with established mainstream methods of vaccine and drug design; however, the villainy was not in the herbal origins but rather the fraudulent experiment in the movie that claimed to support its efficacy. For example, medicinal plants are often found to have an ACE inhibitor action (e.g. ref. [74]). Many compounds studied in works referenced in Section 4.7 were plant extracts, derivatives, or analogues. Carbenoxelone is a derivative of a product from the licorice plant already proven in clinical trials and once marketed as an anti-inflammatory treatment for stomach ulcers, and is steroid-like. Emodin (6-methyl-1,3,8-trihydroxyanthraquinone) can be isolated from rhubarb, buckthorn, and Japanese knotweed and is produced by many species of fungi. The preliminary comparisons with the retroinverso peptide, carbenoxelone and emodin could, of course, emerge as a coincidental similarity of the same kind as that between the reverse sequence used in the retroinverso petidomimetic and a sequence in alanine racemease discussed above.

          A future Thinking Web WW4 beyond the current World Wide Web and emerging Semantic Web [28] remains an important goal to facilitate the response to old and emerging diseases. It seems clear, nonetheless, that rapid access to the emerging literature and the bioinformatics tools available on the Internet, guided by a human researcher, will for some time yet remain important weapons in the battle against viruses and other pathogens. Q-UEL tools, or other similar approaches, can help there.
          Declaration of competing interest

          This paper is provided to the community to promote the more general applications of the thinking of Professor Paul A. M. Dirac in human and animal medicine in accordance with the charter of The Dirac Foundation, to emphasize the advantages and simplicity of the basic form of the Hyperbolic Dirac Net, to encourage its use, and to propose at least some of the principles of the associated Q-UEL, a universal exchange language for medicine, as a basis for a standard for interoperability. These mathematical and engineering principles are used, amongst many others in an integrated way, in the algorithms and internal architectural features of the BioIngine.com, a distributed system developed by Ingine Inc. Cleveland, Ohio, for the mining of, and inference from, Very Big Data for commercial purposes.

        8. @jxk777

          I have only one account buddy and btw your right i start the insult but when you post some stupid comments on coronavirus and about police officers who kill this FUCKING NIGGER, this is only help nigger.

          Now The cops are the only people who fight these animals in this RACE WAR !!!

          If your are not a nigga or a Jew just prove it by your action and your comment please.

          Police are fucking pigs and I HATE THEM MORE THAN YOU but now they don’t rape people, burning house, buisness, police station, beat innocent people, they don’t loot etc….

          Now our ennemys are Niggers and Jews who start this race war, not pigs

          Cops are the only one who protect people who are too stupid to have firearms, fron rape, beating, looting, they protect their homes and buisness from burning.

          They should kill all theses fucking niggers and not the reverse. If you help Niggers to kill police officers your are an ennemy for me.

          You can make mistake thats happen to everybody but please stop posting comments like thats who ONLY help Niggers and jews.

          Thanks

          1. You need to switch off mainstream news for a minute. Once the programming of your mind stops, then you may notice that it’s primarily the cops who rape people, burn houses, businesses, police stations, beat innocent people, loot, etc. Most of those are done exclusively by them, though in some instances they manage to trick well meaning citizens to act as their useful idiot. Much as you act as their useful idiot by shilling for their divide and conquer agenda here.

          2. “I have only one account buddy and btw your right i start the insult but when you post some stupid comments on coronavirus and about police officers who kill this FUCKING NIGGER, this is only help nigger.”

            Firstly let us deal with this shall we;
            (Remember that there is no I in WE).
            What I was trying to convey was and is; what is spewing from MSM is total lies and disinformation/misinformation for the maintenance of the fear factor and irrational panic status for the herd, keep the pot simmering so to speak.
            When ‘sheeple’ then start looking in here (the site BG ) for possible clues and/or answer(s) as to what is REALLY happening, I’m floored, They have an internet connection, the Wire I call it, and that’s what I’ll refer to it from here on in/out ok.

            Now, I’ve NEVER possessed a TV. Television (Tell-You-Your-vision). Why are not these then capable of making searches for factual/truthful information, when it really is there to be found. Their looking for someone to join dots for them, rather than do it themselves, and come across as helpless.
            So to provoke them into doing and thinking for themselves I will throw in a wild card, as an attempt to trigger their own thought. Example;
            (Checked the link, still active)
            http://whiterabbitradio.net/audio/FTWR-17.mp3

            This can teach you humiliation tactics in order to deprogram liberals and other Anti-Whites in your area. Make sure that you “humiliate” and “threaten” liberals regarding the implications of their destructive views in order to DEPROGRAM them. Make sure that your INTENT is never “Hate” and don’t be motivated by it , only by what is RIGHT and WRONG. “Hating” will not solve our problems. Only doing what’s RIGHT will.

            Now, it’s apparent that you are thinking but you are angry, hateful, and resenting, somewhat confused.
            Sorry that I can’t fix that for you, you have to do that by yourself.

            NEXT; as far as I am aware of I’ve posted NOTHING about the nigga being killed (assuming Floyd’s death). You mean, and certainly would NOT.
            There is an example of your confusion.

            Also there really is no point in cluttering up the comments section with text from text.
            Alot of which I would have disseminated, and digested already and reached my own conclusions.
            Your NOT impressing anyone by that.
            Certainly not myself anyway but you’ve put it there maybe others might get something from it.

            Don’t refer, infer, or call me again Jew or Nigger.
            Stop the angry hateful projections towards my good self.
            Otherwise I will NOT continue with what I know that I can impart to you. Ok.
            Then if you are willing and drop the Attitude.
            WE could progress.
            That is now entirely up to YOU.
            Bare in mind also I’m not getting any sort of remuneration for this…
            @kasper88

        9. @jxk777

          RT-PCR has been around for a long time in nuclear medicine
          It is not too reliable if you do not use another method to confirm the diagnosis of
          of covid 19 but some strain of sars cov 2 cannot be identified correctly with this method
          To minimize the risk of diagnostic errors, encompassing
          improving diagnostic accuracy by combining clinical evidence with
          the results of the thoracic computed tomography (CT) and the RT-PCR,
          interpretation of RT-PCR results according to epidemiological data,
          clinical and radiological factors and testing of upper respiratory samples
          (or lower) in patients with negative RT-PCR test results and
          suspicion or high probability of infection, issuing clear instructions
          for the collection, management and storage of samples (in particular the swab),
          as well as refinement of molecular targets and strict compliance with
          analytical procedures, including quality assurance.

          ▪ Among the 273 COVID-19 confirmed specimens:
          – 43.6% (119/273) were positive for RdRp / Hel,
          ▪ Among these, 28.2% (77/273) were positive for RdRp-P2
          (p value <0.001);
          – Average viral load for RdRp-Hel samples
          positive = 3.2×104 copies / mL.
          ▪ There is no cross-reactivity for trials targeting genes
          RdRp / Hel, S and N.

          1. @happy

            Ho yeah now police burn house, make riot, rape and kill white people and the Niggers protect those whites people ????? Yeah sure it’s very believable. Hahahaha !!

            My gosh I see that jewish propaganda did some big damage to your brain my friend but you know thats ok, i totally expect that from weak people.

            At least you made me smile today, thanks dude (:

            I am a big fan btw

      2. The real virus has yet to be unleashed, but it’s coming. As for this Covid 19 crap you simply cannot trust ZOG ever when it comes to their statistics, polls, or anything else.

        Some old lady has a heart attack = “Covid”
        Some guy gets his throat slit = “Covid”
        All the blacks shooting each other are really just using “Covid” bullets.

        Point is, they can throw out any number they wanted to and the sheep would believe them. And even if people didn’t believe them it would still give them the narrative they need to keep doubling down.

  3. With this possibly being staged you can bet the murders will be found not guilty of all charges and we get another massive riot perhaps in all major states with trump pushing more police state laws. yup the brain dead NPC are digging they’re graves and the zombified bootlickers will welcome it like it’s the next best thing after trump and his Zionist buddies taking all of America wealth. after the re-election of trump we’ll likely see America invading Syria in the coming years after of course they stage another false flag attack in US soil which they pin the blame on Iran and use it as an excuse to invade Syria just like they did with Iraqi using 9/11 attacks as an excuse.

  4. Yeah the JEWS are controlling everything in the USA they do the 9-11 in that day mossad jews rats agents was dancing in front the two towers celebrating the consolidation of the new jew order, and they do the Communism first and second war vietnam, etc FUCK! If you see a jew in the street show him the finger.

  5. I agree with pretty much everything said, particularly the use of agent provocateurs. They have been using them for years.

    Niggers however don’t need much persuading to commit acts of theft and violence. Its just in them. If they see a chance to rape and rob they will. Every single time.

    Niggers are also jealous of everyone and everything because they are life’s perpetual losers and so they get angry. They get angry a lot. They then lash out at society, putting all of their own failings onto others to bare responsibility for so as to avoid it themselves.

    They talk about ‘da racist wite supremacist society keepin dem down’. A white society I add that is so racist and supremacist that Asians and Indians etc tend to do better than white people in them, lol.

    So yeah. I agree with what you say Mark. However. These dumb fuck pavement apes were not in on the gag. They were not aiding a grander scheme by will. They were just being niggers. Full stop.

  6. I’m confused. Many of you on here contradict yourselves. You want niggers dead, but hate the white cops that kill them. You want Jews dead, but hate the Muslims that want them dead also. Ya’ll need to pick a side and stand for something, rather than hate everyone for everything.

    1. There is no contradiction. Contradiction only exists for those unable to think beyond a subjects individual example at hand.

      Those who hate niggers but who also take umbrage with the cops who kill them do so because they know cops don’t stop at niggers when it comes to using excessive force. They do not want to be the Turkey who votes for Christmas.

      That said, a lot of cop on nigger murders are justified. This particular one wasn’t though.

      Those who hate Jews and Muslims simultaneously do so because they know that both groups are insidious and controlling towards any society they infect. To say that one cannot hate both they must pick one is to make someone choose between dick cancer and testicle cancer.

      There is no contradiction then because life isn’t so black and white.

  7. Its always been this way and it seems it always will be..

    You can ‘wake’ the ‘sheep’ all you want, when is it going to make a difference? When are we going to stop being played in our day to day existince…how can we fight and take back the power FOR REAL in FULL FORCE I just wish something would fucking happen that actually makes a difference and change!

      1. @desp Well well, You were a much nicer cunt a few years ago, guess you get bitter with age. Nice to see you again too! Thought we were friends before but guess not.

        You and the other “long timers” arnt cooler than anyone else. So stop being a bitch, you dont like something move the fuck on already…I’m not seeing you everywhere so if you are noticing ME that’s YOUR problem. 😉

      2. You @desp and @illegalsmile55 youre both just fake old ass bitches. Because we were friends and you act like I cant see how quick you turned on me and THEN kept talking about it. You know I fucking see you right? Like all the shit you are STILL saying. Like if im so dead to you, just dont comment to me, go back to ignoring me cause who the fuck cares. You all dont know wtf happened and funny how 3 years go by and you still talking about it. I called mark a homosexual women hater….I was trying to get my profile deleted which at the time was NOT an option like it is now. I may have been going through my own issues with how mark addresed certain women here at the time, playing favorites ETC which was like I said years ago.

        I grew up, did you?

        I’m sorry @happy! I was going through shit but who the fuck doesn’t. Thank you for not deleting me again…or yet..cause I’m not trying to fuck around but then here you two chicks are being bitches like I have to apologize to you too for something….I thought we could be civil but guess not.

        1. Fake old bitches?! BAHAHAHA That’s some funny shit coming from the attention seeker smeared with makeup. What the fuck did I say to piss you off? ‘Cause I’m friends with despy? Why don’t you show me where I “dissed” you? That’s right, I didn’t…. Why don’t YOU fuck back off to whatever shithole you’ve been lurking from? You’re the one lying, telling someone here that an old moderator “who didn’t like me” kicked you off here…MARK gave you the boot…justifiably. Acneska thanked MARK for kicking your attention seeking ass out of here. There were more members happy to see you go than weren’t…You shoulda stayed gone Bella, because here you are starting shit already. Don’t worry, I’m sure you’ll have a cuntpig-fit again and that will be the last of your fat ass. I would’ve just ignored you, but you had to drag me into YOUR shit…not mine…Ms. Six-Monther….

          1. Check again, I ignored you both shit talking fucks it was @desp who decided to talk her shit. And hunny I read the forums okay ive seen all the shit said by anyone the last 3 years because tags dont seem to die here. You do YOU and ill do ME because there are far worse people on here yet you guys wont get off my dick! Let me fuck off here like everyone else without all your damn menopause in my buinsness

          2. @desp
            https://gph.is/2d9obMH

            I checked again, you are WRONG bella, I did not ‘shit talk’ about you, but even if I did, you would’ve deserved it. You threw a bitch fit and got the boot. PERIOD. But I do like your excuse of why YOU shit talked Mark….really? Because you couldn’t delete your account you called Mark a faggot so HE would delete your account?! Imaginative! Why not just quit commenting and go away if you disagree with his stance on women? Like I said to dre on Acneska’s profile, I had nothing against you and did not talk shit about you. I wouldn’t have a problem admitting it because that’s how I am and as you said, it’s all here to see… except all your comments got deleted from the forums. Have to say, never seen that before. Tell you what, you find where I was a big old meany to poor little bella…if you can prove it, I’ll apologize for saying I didn’t say shit about you. Admit it, you had to know you would catch some shit if you came back, RIGHT? This is Best Gore where no shits are given and not one person here has escaped others ‘shit talking’.

          3. @illegalsmile55

            I don’t care to look back ahgggain because shits not easy to find. Ill just go ahead and agree with you and apologize for dragging your name into what was just @Desp saying shit for no reason.

            I did take a break and leave, remember, 3 years worth. If I couldnt handle anyone with something to say to me I would have used a different name. I came here as me and thats just how it is. People like or dont like me and no one cares. There are people here just to say dumb ass shit and retarted ass comments that make ZERO sense, so I really don’t see why ANYONE needs to get all worked up over anything I say. I’m done with whatever this back and forth is, I’m not fighting over something from 3 years ago, I was calm as a cucumber then, I knew what I wanted and how to get the attention I needed to get myself erased. Could have handled it better sure, but I just feel like the people who didnt like me then or now can just go about their buisness.

  8. I’m not saying you are wrong because there is definitely more to it. With that said the responders did check his neck for a pulse while that guy was still on him. All Minneapolis police cars have a license plate that says police.

    Absolutely spot on about the covid 19 bs.

  9. “agent provocateur” is not a word created without a reasons.
    that technique was used back in the 1700’s and 1800’s, where some governemental or military men where used to infiltrate gangs and criminals, in order to do that, they used to break things and all.

    things then changed and todays is used as every possible excuse to try starting a riot or else , expecting some idiots to join them and then get caught by cops.

    as exempl, they are some cops that used to disguise as “gilet jaune” in france, with the only order to create shit and blame the gilet jaune expecting people to start disliking them and stop supporting their actions.

    (source : a friend police officer / CRS)

  10. At the end of the day, we are all sheeple, everyone of us….its just that some of us are in different fields

    I dont think there is a ‘top person’….. everyone is following someone

    The question is, can you see the other fields from where you are grazing?

    Martin Taylor, 30.05.2020

  11. There was quite a crowd at the moment when Floyd was getting his neck crushed. But now that he’s past and the video is viral suddenly people are outraged and want to react. Its easy to whine, scream, burn and loot but very select few people actually want to make a sacrifice for what they believe in.

    1. Yeah the dude was high with his pizza, saw some dumb shit, then when confronted he smokes his joint.
      “ya wanna go? whats up” *hits joint*
      It doesn’t matter if your pizza is cold when you’re stoned.

  12. Can we create a topic and thread please as to why white countries, and white countries only, must adhere to open borders, diversity and multiculturalism and yet every other race and their countries can have their own place and space and be homogeneous without concern, question or outcry.

  13. This is a message to all women. DON’T DATE COPS! They’re all corrupt, they’re all in secret societies such as Freemasonry. They are secretly using MK Ultra mind control on innocent men and women. All women need to boycott dating cops. Because if you date them and marry them, you are supporting and condoning the crimes they are committing against innocent citizens. Let them pay the consequences of joining a criminal secret society.

    Ladies, turn your back on corrupt cops. Let them jerk off to porn hub and cry. Divorce your cop husband. Leave his crooked ass.

    As a matter of fact, date and marry truthers!!! Have kids with honest men!!! Don’t have children with lying sneaky murdering crooks!

  14. THIS IS FOR EVERYONE. PLEASE READ…

    Have you experienced a sense of unease when witnessing a group of individuals wearing masks and/or keeping strictly two meters apart..? If so, that’s good news, because it means you’re still sufficiently human to be able to respond to life’s deeper survival instincts.

    For those who don’t have an uneasy sense that something is wrong in this situation, I feel a genuine sense of concern and the strong hope that this will change, very soon. Here’s the reason why: you are being indoctrinated to allow a carefully constructed lie to direct your thinking, your behaviour and your individual powers of judgement.

    Coronavirus Covid-19, is most certainly not something to fear as a significant threat to health. And that the mask is a worse than useless piece of so called ‘protection’ which only serves to accentuate the fact that what it is supposed to protect against doesn’t even exist in the form in which it is being described.

    However, there is something about what this mask represents that should give rise to genuine alarm. That something, is the huge effort being made to turn what is essentially a corona virtual reality construction into a real life event. An event whose momentum is created by pressing down on the irrational fear button and keeping it down until the repetition of the preplanned indoctrination exercise has gone on long enough for the majority to believe that any other version of events must be ‘a conspiracy theory, or theorists’.

    This is basically where we are NOW. This moment in time, ladies and gentlemen. Yet, in spite of the best efforts of our oppressors, the truth is coming out and awareness will follow, not so far behind. However, until such awareness forms the bedrock for a critical mass of humanity, we will go on witnessing the manifestation of the lie in all public places and in homes where individuals stick doggedly to the scripted formula they have been fed.

    Here is where the uneasy feeling rises into something considerably more than just ‘uneasy’. Because what is on exhibit is an open admission of slavery to the unquestioned commands of ‘the leader of the day’; and what better symbol of such (typically unconscious) subservience than the wearing of the mask of the beast..?

    What better conformism to ‘the system’ is there than maintaining belief in the validity of ‘social distancing’ at two military paces from our collective brothers and sisters..? What starker statement can one make concerning one’s loss of ability to recognise one is being deceived, than not to question being segmented into anti-humanitarian social isolation through a modern day act of apartheid..?

    If there is a pervasive sickness connected with the ‘grand covid pandemic’ it is this: the willingness of tens of millions of individuals to adopt that which is so far removed from the truth that not even those who are in charge of purveying it can make it sound like sense.

    The British Prime Minister, recently attempting to explain the inexplicable rules he expects the population of Britain to conform to, sounded and looked like a man suffering from ‘cognitive dissonance’. INSANITY..!

    Think carefully about what this mask actually is. If it doesn’t have anything to do with health, what does it have something to do with..? If keeping two metres apart from fellow human beings also has nothing to do with health, and my research endorses that is indeed the case, then what does ‘keeping distance’ actually stand for..?

    We know the answer, but perhaps as yet, not deeply enough to bring about the change that should result from such knowledge. So I am going to say it again, in the hopes that those who have not recognised the reality of the trick being perpetrated on us hold onto this truth: Do not waver or fall due to the insidious levels of brainwashing being directed at dominating our psyches.

    The mask is a statement. A statement of conformity. Conformity with a plan to destroy humanity and civilization and by extension, the living environment and the overall aspiration to spiritual evolution which is innate in all life forms. Symbolically, traditionally, and actually, the covering of the nose and mouth with a mask is an expression of ‘secrecy’, ‘subversion’, ‘disguise’ and ‘undercover operations’. SUBTERFUGE..!

    The mass wearing of such masks (with the exception of doctors/medical practitioners) becomes a conformist identity symbol “I see, he/she is also wearing a mask, so they are one of us, we who agree to follow the regulations, to obey the rules and to behave in a politically correct manner. In this way we will be seen to be upstanding members of the community and normal individuals.”

    To be ‘normal’ is a treasured status within 21st century urban/suburban communities and beyond. There is a terrible fear of being seen to be ‘different’ and thereby ‘not normal’. DARE to be DIFFERENT..!
    Because you already know I am and I do. But to fear being different more than to fear being a slave, is a truly terrible sickness. Far worse than that being framed as Coronavirus. One for which the only cure may be a bang on the door by the NWO police who have come to remove such a ‘normal individual’ from his/her home, with worse to follow if that person still fails to raise a finger of self defence for fear of ‘disobeying the law’.

    We must break the chains of such paralysing conformity. A conformity historically epitomised by mass obedience to the communist regime in post WWII Russia. Stand up, anyone who fails to recognise that such a time bears a sinister similarity to the in-your face rampant top down usurpation of power happening today, under the excuse that it is ‘necessary’ to prevent the spread of some sort of fake pandemic.

    At a time like this, we must make visible statements of our belief in fundamental life values. Where there are occasions being advertised in which peaceful collective assembly in stated places/locations is the objective, be at those events to demonstrate ‘en masse’ that it is WE, the people who rightfully hold the destiny of this planet in our hands and NOT the control system which has usurped those powers. Then go home and continue to act on your freshly resurrected sense of self belief.

    Anyone still passively allowing the state/corporate alliance to manufacture their lives for them at this point in time, is not worthy of the title HUMAN.

    Dear friends, let us rebel from our own susceptibility to fall prey to indoctrination, of any kind. Let us refuse to allow ourselves to take the easy way out..! Let us disabuse ourselves of obeisance to the absurd Anti Social Distancing commandments emanating from The World Health Organisation and passed down to us by a cohort of puppet dictators styling themselves as bona fide representatives of the Ministry of Truth.

    Anyone who has warmth in their heart knows instinctively that people need each other and need to be close to each other, especially in times of stress and hardship. They know that to impose ‘distancing’ has nothing to do with preserving the health of the nation, and everything to do with preserving the top down divide and conquer programme of the NWO fascist dream.

    This is where civil disobedience becomes our primary and most effective tool of resistance. Let us not delay putting it into effect.

    Over the next few weeks, months and years, we are going participate in one of the most moving uprisings this world has ever known. Literally millions of us are going to step out of our psychological imprisonment and declare ourselves sovereign independent human beings who do not consent to a life of squalid serfdom. SLAVERY..!

    So uplifting will this great rising be that deluded enforcement bodies attempting to exercise some form of ILLEGAL arrest or restraining coercion upon us all, they will then shrink back into the night from whence they came and quite simply fail to achieve their sick devious mission.

    This is the future I clearly see ahead, and because I see it I believe you do too, and if you do, I do and we all do, it will happen. All it takes is a sprinkling of courage and a pinch of passion, stirred well into the natural heart led instinct that favours life over death. Then we’re away, unstoppable forces for the great emancipation of humanity..!

    Get off your knees, join the fight because this is WAR on a totally different scale to which you have ever known, people are going to Die, people are going to be Killed. Wouldn’t you much rather die or be Killed while standing on your own two feet..?

    Than cowering in a corner on your knees waiting for the liquidators coming thru your door..?

    As you stand now, you are standing in the calm before the storm. Don’t wait for the storm to reach you, get out, face it, meet it head on..!

    Thank You…

  15. Chemtrails are bullshit it’s just water like what comes out of your car exhaust in winter…. the earth is not flat if you blow bubbles there not fucking flat like a plate there the shape of the

      1. Was it recently, because contrails existed for a while. Even if you live in a city. If its not just water vapor then whatever particles would be blown off into the bumfuck desert or ocean tens of hundreds of miles away. And even if then you’d have to accommodate for weather and meteorological patterns in a given place on a weekly basis. There’s way too much calculation to be involved for the govt when people just want to get their ass across the country.

        1. I’ve noticed it for the last couple of years above Vancouver BC. I’ve flown over a hundred times. I know what regular contrails look like. I was born in the 70’s. I’ve seen regular contrails for decades. These chemtrails are not contrails. I am 100 percent sure of it. I have watched them spraying them. Massive grid patterns across the sky, then the slowly spread out and cover the whole city.

  16. All of this shit is to help collapse the economy. Keep filling your house with food preps. Ignore all the crazy bullshit going on, get prepared for the collapse of the economy. GET FOOD AND WATER AND A WEAPON TO DEFEND IT. Buy gold and silver so you don’t lose your wealth.

  17. Who would of thought? The sheeple don’t know and won’t ever know the truth about their overseers. Even us chosen few are powerless against the zionist run goverments. Until something is exposed all we can do is live our small little lives and hope to die before being enslaved.

  18. I was watching the riots last night and connected the dots quickly. This shit confirmed my suspicions. It’s actually funny though. They’re gonna reduce the population or sum shit which I hope they get rid of all the annoying niggers.

  19. Fucking wild man. Destroying shit for the CNN Camera Crews to record, & Then Blame on the Good Citizens of Minneapolis. Fucking *Disgracefully-Shameful* in every Possible way is what this is.
    🙁 JEW-FUCKS! 🙁

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