How the replication and transcription complex of SARS-CoV-2 functions in leader-to-body fusion

https://www.biorxiv.org/conten…/2021.02.17.431652v1.full

Abstract

Background Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although unprecedented efforts are underway to develop therapeutic strategies against this disease, scientists have acquired only a little knowledge regarding the structures and functions of the CoV replication and transcription complex (RTC) and 16 non-structural proteins, named NSP1-16.

Results In the present study, we determined the theoretical arrangement of NSP12-16 in the global RTC structure. This arrangement answered how the CoV RTC functions in the “leader-to-body fusion” process. More importantly, our results revealed the associations between multiple functions of the RTC, including RNA synthesis, NSP15 cleavage, RNA methylation, and CoV replication and transcription at the molecular level. As the most important finding, transcription regulatory sequence (TRS) hairpins were reported for the first time to help understand the multiple functions of CoV RTCs and the strong recombination abilities of CoVs.

Conclusions TRS hairpins can be used to identify recombination regions in CoV genomes. We provide a systematic understanding of the structures and functions of the RTC, leading to the eventual determination of the global CoV RTC structure. Our findings enrich fundamental knowledge in the field of gene expression and its regulation, providing a basis for future studies. Future drug design targeting SARS-CoV-2 needs to consider protein-protein and protein-RNA interactions in the RTC, particularly the complex structure of NSP15 and NSP16 with the TRS hairpin.

MEK inhibitors reduce cellular expression of ACE2, pERK, pRb while stimulating NK-mediated cytotoxicity and attenuating inflammatory cytokines relevant to SARS-CoV-2 infection

https://www.oncotarget.com/article/27799/text/

ABSTRACT

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. In serum-deprived and stimulated cells treated with remdesivir and MEKi we observed correlations between pRB, pERK, and ACE2 expression further supporting role of proliferative state and MAPK pathway in ACE2 regulation. We show elevated cytokines in COVID-19-(+) patient plasma (N = 9) versus control (N = 11). TMPRSS2, inflammatory cytokines G-CSF, M-CSF, IL-1α, IL-6 and MCP-1 are suppressed by MEKi alone or with remdesivir. We observed MEKi stimulation of NK-cell killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. Pseudotyped SARS-CoV-2 virus with a lentiviral core and SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope infected human bronchial epithelial cells, small airway epithelial cells, or lung cancer cells and MEKi suppressed infectivity of the pseudovirus. We show a drug class-effect with MEKi to stimulate NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells. MEKi may attenuate SARS-CoV-2 infection to allow immune responses and antiviral agents to control disease progression.

INTRODUCTION

Coronavirus 2 (SARS-CoV-2) infection progresses to a rapidly lethal adult respiratory distress syndrome (ARDS) associated with high mortality especially among the elderly or those with multiple comorbid conditions [1–5]. Patients with cancer are particularly vulnerable in part due to their weakened immune system and are further at risk due to the immune suppressive effects of chemotherapy [6–8]. The lethality of SARS-CoV-2, the causative agent for the COVID-19 disease, involves a fulminant cytokine storm with bilateral lung infiltrates observed on chest X-rays and CT scans [9]. It has become clear that COVID-19 disease involves multiple organ systems including pulmonary, neurological, renal, hematological and gastrointestinal systems, among others [10–15]. The SARS-CoV-2 virus binds to angiotensin converting enzyme 2 (ACE2) receptors and cellular entry is facilitated by TMPRSS2 protease [16]. Current therapeutic approaches include a number of agents such as anti-inflammatory agents that block IL-6, steroids, anti-viral agents, convalescent serum and alpha receptor blockers [17–21]. There are ongoing approaches for drug discovery and drug repurposing [22, 23].

Once SARS-Cov-2 enters into cells it triggers a host immune response that leads to pathogenesis and disease progression [24]. A SARS-CoV-2 SPIKE protein variant (D614G) has emerged as the dominant pandemic form with evidence that it increases infectivity of the COVID-19 virus [25]. The host inflammatory response phase of COVID-19 is the phase where patients become critically ill leading to high patient mortality [26]. We sought to better understand and modulate the host immune response to SARS-CoV-2 in order to prevent or reduce disease severity. This includes strategies to inhibit expression of ACE2, the receptor SARS-CoV-2 uses to enter cells.

It is clear that while the host systemic inflammatory response makes patients critically ill, the host innate immune system including natural killer (NK) cells is involved in fighting and eliminating virally-infected cells [27]. Over the last 25 years we have studied this innate immune system pathway that the immune system uses to eliminate transformed and cancer cells as well as virally-infected cells [28–34]. Natural killer cells secrete TRAIL which is involved in killing virally-infected as well as transformed cells [35–38]. Thus, our goal was to better understand and modulate the host immune response to increase the innate immune system early in SARS-CoV-2 infection while reducing the severe inflammation that occurs late in the disease course. We further wanted to understand the impact of current therapeutics used to treat COVID-19 on SARS-CoV-2 infectivity factors, the innate immune system and the cellular inflammatory response.

Prior work has suggested that coronavirus SPIKE protein can through ACE2 activate the MAPK pathway and downstream inflammatory responses [39]. Other data suggested that MAPK regulates ACE2 [40], and so we investigated the impact of MEK inhibition on ACE2 expression as a strategy to attenuate early SARS-Cov-2 infection. Since remdesivir has been shown to reduce hospitalization [19] and may reduce mortality in patients with severe COVID-19 infection [41], we hypothesized that suppression of viral entry into cells through inhibition of ACE2 and TMPRSS2 would reduce the spread of SARS-CoV-2 infection in a given COVID-19-(+) patient and this would allow the innate immune system and antivirals such as remdesivir to more effectively suppress early infection.

Due to its high pathogenicity and the lack of an effective treatment, live SARS-CoV-2 viruses must be handled under Biosafety Level 3 (BSL-3) conditions, which has hindered the development of vaccines and therapeutics. Pseudotyped viral particles are chimeric virions that consist of a surrogate viral core with a heterologous viral envelope protein at their surface. Such pseudoviruses are routinely used by many investigators for other highly pathogenic coronaviruses including SARS-CoV [42, 43] and MERS-CoV [44] to study viral entry, and develop assays for neutralizing antibodies and drug discoveries. For the current study, we have developed a pseudotyped SARS-CoV-2 virus which has a lentiviral core but with the SARS-CoV-2 spike protein on its envelope. The pseudoviruses infect human lung epithelial cells in an ACE2-dependent manner and confer the expression of a fluorescence protein ZsGreen in infected cells for imaging and quantification. The pseudoviruses can only accomplish a single infection cycle and are replication incompetent, thus require only BSL-2 level containment.

Our results suggest that MEK inhibitors, as a class, suppress host SARS-CoV-2 infectivity factors such as ACE2 and TMPRSS2, and that alone or in combination with remdesivir, there is innate immune system activity along with suppression of inflammatory cytokines and stimulation of Natural Killer cell activity. Our results support the further investigation of MEK inhibitors as a strategy to dampen early SARS-CoV-2 infection to allow host immunity as well as potentially anti-viral agents to be more effective.

RESULTS

MEK inhibitors reduce ACE2 expression in human cell lines

Based on prior literature that SARS coronavirus SPIKE protein through ACE2 can activate MAPK signaling [39], we hypothesized that MEK inhibitors (MEKi) may inhibit SARS-CoV-2 cellular effects. We used human tumor cell lines as well as normal human lung cells as a model to test effects of MEKi on ACE2 expression. We initially observed in H1975 human non-small cell lung cancer (NSCLC) cells that at doses below IC50, three different MEKi’s suppressed ACE2 protein expression (Figure 1A). VS-6766 (5 μM), a small molecule RAF/MEK inhibitor, MEKi Selumetinib (20 μM), or MEKi Trametinib (5 μM) all inhibited expression of ACE2 protein (as detected by PAB13444) with more subtle effects detected by another ACE2 antibody (CS4355) that recognizes glycosylated ACE2. We include results with the two commercially available antibodies we used to demonstrate that these antibodies did not always give concordant results. In this experiment the reduction in ACE2 was clearly demonstrated with the PAB13444 antibody. We observed that the cleaved active SP-domain of TMPRSS2 was increased by chloroquine or hydroxychloroquine and this was potentiated by the MEKi’s (Figure 1A). Inflammatory cytokine IL-6 was reduced by all 3 MEKi’s (RAF/MEKi VS-6766 showed the greatest reduction in this experiment) with no benefit from addition of chloroquine or hydroxychloroquine.

FDA says Covid vaccines that target new variants won't need large clinical trials to win approval

https://news.google.com/articl…=en-US&gl=US&ceid=US%3Aen

The Food and Drug Administration said Monday that modified Covid-19 vaccines against new, emerging variants may be authorized without the need for lengthy clinical trials.

The new guidance, released in a 24-page document on the FDA's website, would clear the new vaccines as an amendment to a company's originally approved emergency use application, according to the FDA. The company would need to submit new data that shows the modified vaccine produces a similar immune response and is safe, similar to the process for annual flu vaccines.

Quote from Fm1

The Food and Drug Administration said Monday that modified Covid-19 vaccines against new, emerging variants may be authorized without the need for lengthy clinical trials.

80% of the income form the FDI board members is from Big pharma. What else should they say? Else they will be kicked out of the golf club...In Switzerland the decision is anyway made at the rotary medicine round table.

Germany ended its silly lockdown of children from school. Germany now has more or less the worst death rate and cheats the figures by low testing or we could say Switzerland cheats by high testing....

A lockdown without FP98 mask for going into the crowed is the true killer of German people staying at home.

Despite vaccination Israel sees no reduction in cases else than caused by the lockdown.

Switzerland has reduced the cases by 8x just with lock down Israel is at 2.

UK: Boris thanks the vaccines for the large decline in cases but his 5x decline is far behind Switzerland where only the very old got it. We still could travel to any place we like (by train) we could do ski and individual outdoor sport. But we know Boris is as bright as a stone.

But it is to late for UK to see any real vaccine effect because the vulnerable old are already dead. Nevertheless the cases among the old here declined > 6x.

Pfizer vaccine only works for 85% in UK (now 89% in Israel) among health care people. This is now a more realistic figure and certainly will go down further.

Buy your personal ivermectin. Early taken it clear your virus within 3 days!

Bepridil is potent against SARS-CoV-2 in vitro

https://www.pnas.org/content/1…12201118/tab-article-info

Significance

Guided by a computational docking analysis, we experimentally characterized about 30 FDA/EMA-approved drugs on their inhibition of essential main protease of SARS-CoV-2, the pathogen of COVID-19. From this study, we discovered that bepridil, an antianginal medication, is potent against SARS-CoV-2. The antiviral analysis of bepridil indicated that it had low micromolar EC50 values in inhibiting SARS-CoV-2 in two highly permissive mammalian cell lines. Due to the urgent matter of COVID-19, our current study encourages further preclinical investigations of bepridil in animal models to clear its path for clinical uses in COVID-19 patients.

People who wear glasses may be up to 3 times less likely to catch COVID, new study suggests

https://www.chicagotribune.com…story.html?outputType=amp

People who wear glasses could be up to three times less likely to get coronavirus, according to a new study conducted in India.

The preliminary study suggests that glass-wearers may have the extra protection because they tend to touch their eyes less frequently than most people.

“Touching and rubbing of the eyes with contaminated hands may be a significant route of infection” for COVID-19, the authors wrote in a report published on medRxiv, a website that compiles medical studies before they are peer-reviewed.

The new study found that the risk of infection was two-to-three times lower among those who wear glasses for “long periods of time,” meaning at least eight hours a day, according to the report.

Indian researchers described the findings as “statistically significant.”

The study was conducted last summer in the northern district of Kanpur Dehat. It involved 304 patients ranging in age from 10 to 80 years old. All of them experienced coronavirus symptoms and about 60 were considered long-time glass-wearers, according to the report.

The study authors noted that COVID-19 infection through the eyes “is extremely rare,” but they said that droplets from the virus can easily go from the eyes to one’s nose or mouth.

The best way to prevent this type of infection is to avoid touching the eyes. Medical workers who treat coronavirus patients should go even further and wear safety goggles for extra protection, according to the study.

COVID-19 Drug Study for Out-Patient Care

Phase 2/3 of RHB-107 (Upamostat), a Serine Protease Inhibitor or Placebo for Treatment of COVID-19 Disease

https://www.uhhospitals.org/re…vid-19-research/upamostat

Purpose of the Study

The purpose of this study is to test the safety of the study drug RHB-107, also referred to as Upamostat, in two different doses to see if it can help people with early COVID-19. Participants in this study will receive either a high or low dosage of Upamostat and/or placebo and will take it by mouth once per day for 14 days.

Who Can Participate

Enrollment is competitive, and researchers hope to enroll a total of 310 participants in several U.S. sites.

Participants must be 18 years or older, started having symptoms or had a positive test for COVID-19 disease within three days from the planned start of study treatment, do not require hospitalization, and can use a smartphone may be eligible for the study. Eligible participants will take the study drug or placebo in a capsule by mouth once a day for up to 14 days. This study only has one in-person clinic visit; then, participants will be monitored closely for the study’s duration, up to 57 days, through phone apps, telehealth, and home health care visits.

China did ‘little’ to hunt for Covid origins in early months, says WHO document

https://news.google.com/articl…=en-US&gl=US&ceid=US%3Aen

Chinese officials did “little” in terms of epidemiological investigations into the origins of the Covid-19 pandemic in Wuhan in the first eight months after the outbreak, according to an internal World Health Organization document seen by the Guardian.

The internal WHO travel report summary, dated 10 August 2020, also said the team who met Chinese counterparts as part of a mission to help find the origins of the virus received scant new information at that time, and were not given any documents or written data during extensive discussions with Chinese officials.

The report from last summer, which was written as global infection rates reached 20m, offers new insights into how WHO scientists appear to have been stymied in their early efforts to study the outbreak in China.

The revelation comes after the Biden administration recently issued a pointed statement about its concerns over Chinese cooperation in studying the disease and the need for the WHO to be held to a high standard and protect its credibility.

Is India currently witnessing a second wave of the Covid-19 pandemic?

https://news.google.com/articl…=en-US&gl=US&ceid=US%3Aen

A spike in the number of new Covid-19 infections threatens to overturn the progress India has made in containing the pandemic.

On Feb. 21, the country recorded over 14,000 new infections of the novel coronavirus. As of Feb. 22, the total reported cases of Covid-19 in India were over 11 million.

With the daily new infections—which had come down to close to 10,000 a day—slowly rising in the past week, several states are now considering localised lockdowns, especially in those regions that are seeing a sudden spike. Five states, according to data from India’s health ministry, account for 86% of the new infections in the country.

Quote from Fm1

The Food and Drug Administration said Monday that modified Covid-19 vaccines against new, emerging variants may be authorized without the need for lengthy clinical trials.

That is a sensible decision under the circumstances. This is an emergency. Normally, I think they would demand clinical trials, but in this case it would take too long, and many people would get sick or die.

Here is an article by a virologist that explains the difference between vaccines that produce immunity and sterilizing immunity. You might still have the virus with the former, and you might infect others with it, but you will not get sick. (Or not very sick.) Whereas sterilizing immunity means there is no virus left in the body.

It is not clear yet whether the two COVID-19 vaccines are sterilizing, but they probably are. Most vaccines that are this effective are. A non-sterilizing vaccine can end a pandemic. The Salk vaccine was not sterilizing but it still eliminated polio.

I do not think this is behind the paywall:

https://www.nytimes.com/2021/0…accines-transmission.html

Yes, the Vaccines Will Stop the Spread

How to understand the difference between vaccination to prevent Covid-19 and shots to halt infection.

. . . Many scientists are reluctant to say with certainty that the vaccines prevent transmission of the virus from one person to another. This can be misinterpreted as an admission that the vaccines do not work. That’s not the case. The limited data available suggests the vaccines will at least partly reduce transmission, and the studies to determine this with more clarity are underway. There should be more data within the next couple of months. . . .

It is true that, according to the clinical trial data, both the Pfizer-BioNTech and Moderna vaccines are highly effective at preventing Covid-19, the disease, but it’s unknown how well they prevent infection with SARS-CoV-2, the virus.

When scientists develop a vaccine against a novel virus, it’s difficult to predict whether vaccination will completely prevent infection — what’s called sterilizing immunity. If the Covid-19 vaccines do not provide sterilizing immunity, it means a vaccinated person can still inhale enough of the SARS-CoV-2 virus to develop an infection, and it will be swiftly cleared from the body before becoming Covid-19, but that person could still pass the infection to another person.

There are many vaccines that do not provide fully sterilizing immunity but nonetheless have huge public health benefits. Every year, the flu vaccine saves lives and keeps people out of the hospital despite the fact that it doesn’t prevent infection altogether.

From everything we know so far, it’s highly unlikely that vaccines that are 95 percent effective at preventing symptomatic disease would have no impact whatsoever on infection. . . .

Historical evidence shows that vaccines that do not prevent virus infection can still stop epidemics in their tracks. The polio vaccine developed by Dr. Jonas Salk, which does not provide sterilizing immunity, resulted in the rapid elimination of polio in the United States beginning in the 1950s.

Quote from Fm1

A spike in the number of new Covid-19 infections threatens to overturn the progress India has made in containing the pandemic.

This is classic bash&fear news. Always look at worldometer first then start to think what the message is about... No spike a slight increase over several week by 10%. Most countries have spikes of +-20%. Just because a new mutations makes a small change. In fact you can see the impact of 1.1.7.1 in most curves thus this is why I know that it has no overall impact at all. The 1.1.7.1 (UK) mututation did delay the decline in average by 4 weeks - + added a small spike - nothing more.

Quote from JedRothwell

It is not clear yet whether the two COVID-19 vaccines are sterilizing, but they probably are.

It is clear that it is not. Also the efficiency is steadily going down now its way below 90% in UK among exposed people. This is what people with a severe infection usually get.

But there is good news for Astra Zeneca very high protection against hospital severity of over 90% after one shot where as for Pfizer this is at best 70%.

Bulgaria also allows Ivermectin treatment: The Domino stones start to fall. Only strict fascist-capitalist nations still block.

Quote from Wyttenbach

This is classic bash&fear news. Always look at worldometer first then start to think what the message is about... No spike a slight increase over several week by 10%. Most countries have spikes of +-20%. Just because a new mutations makes a small change. In fact you can see the impact of 1.1.7.1 in most curves thus this is why I know that it has no overall impact at all. The 1.1.7.1 (UK) mututation did delay the decline in average by 4 weeks - + added a small spike - nothing more.

It is clear that it is not. Also the efficiency is steadily going down now its way below 90% in UK among exposed people. This is what people with a severe infection usually get.

But there is good news for Astra Zeneca very high protection against hospital severity of over 90% after one shot where as for Pfizer this is at best 70%.

Bulgaria also allows Ivermectin treatment: The Domino stones start to fall. Only strict fascist-capitalist nations still block.

I hope you're right on the infections, we should know by he end of he week. Eastern europe should start rising by the end of the week if there is another wave.

I don't hold much hope for ivermectin as the FDA has had all the study's for over 6 weeks and not a peep.

I'm still waiting on vaccine but in no rush

long haulers treatment

Quote from Wyttenbach

It is clear that it is not. Also the efficiency is steadily going down now its way below 90% in UK among exposed people.

Thank God you said that! Again, we can be sure it is not true because it comes from you. We don't even need to Google it -- we can just take it for granted that whatever you say wrong. Of course anyone who does Google that will confirm it is wrong.

(On the other hand, the two are somewhat less effective against the South African strain.)

Quote from JedRothwell

Of course anyone who does Google that will confirm it is wrong.

Google is not God.. it is a modern-day Oracle however

https://greekreporter.com/2016…odern-day-search-engines/

Quote from RobertBryant

it is a modern Oracle however

The oracle of Delphi was on the take too..Verily...
https://thirdworldcentre.org/2…-the-story-of-corruption/

Comment from a Covid patient

Geneva in Canada? Feb 21, 2021 8:46am

"I, too, was exposed, got a high fever, brutal body aches and a headache like no other.

I had Ivermectin on hand as I had been following the FLCCC Alliance. Within 30 hours, all symptoms were gone.

The person who "shared" the virus with me, meanwhile, was hospitalized with pulmonary blood clots for 10 days;

he is still walking around with oxygen a month later.

This is a sad story about MONEY. The FLCCC Alliance had a video of their Senate testimony.

YouTube booted it; they have also been bounced by Facebook.

Facebook, for one example, receives about a billion US $ annually from big pharma.

Google (owns YouTube) has a venture with Glaxo thru its sub,

Verily. Since there is such a huge amount of "investor" and government money riding on the development and success of these vaccines, Ivermectin does not stand a chance

. It is off-patent, readily available and cheap. So there is no profit to be made here, only saving lives.

Congressional members also take a lot of money from big pharma; this is no secret.

The NIH (falls under HHS same as the CDC, BARDA and the FDA),

recently begrudgingly upgraded Ivermectin to the equivalent of "neutral".

But the FDA, in turn, gave no EUA (emergency use authorization) which is what is needs!

By contrast, Eli Lilly's drug, bamlanivimab (monoclonal antibodies therapeutic; brand new) was also "neutral" per the NIH.

But the FDA awarded this drug EUA. Why? BARDA awards to Lilly as follows (all US $): $375 mil 10/27; $812.5 mil 12/2; $625 mil 1/26/21.

As for certain 3rd Wold countries which might be holding back, my guess is that governments there have already taken money from big pharma who will at some point sell those vaccines to them. Meanwhile, people are suffering and dying unnecessarily. I could give you many more instances of folks whom I know were immensely and immediately helped by Ivermectin while under attack by COVID.

We need to push both countries to promote its use, end the pandemic and get back to life as we knew it."

https://www.thesuburban.com/ne…cd-9c7b-bf4c2a615ede.html

Quote from JedRothwell

Thank God you said that!

Free masons know always better!... Go back to your round table buddies! There you can listen instead of read what we link!

Quote from RobertBryant

Google is not God.. it is a modern-day Oracle however

Google will lead you to actual sources of information, instead of made-up crackpot nonsense. Of course it will also lead you to crackpot nonsense. You have to be able to distinguish between them. In some cases that is difficult. But not in this case. There are no reports yet that the two vaccines are significantly less effective against the UK variant. The UK variant is a frightening problem, but not because the two vaccines now in use cannot deal with it.

As I said, there are reports that they are less effective against the South African variant.