Furin cleavage site and molecular features

This is the site that is analogous to the furin cleavage site between gp120 and gp41 of HIV.

Fauci Intelligence Community Release, p.28 · original PDF · in context

Zheng-Li Shi at the University of Wuhan has been working for years in GOF in coronaviruses to allow adaptation of the spike protein to bind to the human ACE2 receptor, we could not let this go.

Tony's Diary Package, p.14 · original PDF · in context

Speaking of furin - Bob, your old buddy is on the Virological again and it's a pretty nice theory for how the furin site was acquired in bats - just need to add a little extra divergence and there you have it:http://virological.org/t/tackling-rumors-of-a-suspicious-origin-of-ncov2019/384/3

Slack / Private Message Drop, p.7 · original PDF · in context

Typically SARS-like CoVs are cultured in the presence of trypsin (to allow for cleavage of S1/S2), but if trypsin isn't added, there's a *huge* selection pressure for gaining a furin site.

Slack / Private Message Drop, p.23 · original PDF · in context

The example from bovine CoVs (post #6 on the thread on virological) is exactly a 12bp insertion seen in tissue culture creating a furin cleavage site.3.

Slack / Private Message Drop, p.23 · original PDF · in context

It's obvious, but the only bits I'm really interested in are the RBD and the cleavage site of the progenitor of SARS-CoV-2.

Slack / Private Message Drop, p.34 · original PDF · in context

The furin site in SARS2 is exactly the same as it is in some of the HKU1 sequences.

Slack / Private Message Drop, p.69 · original PDF · in context

I was reading Mike's paper to suggest that D614G was under selection to increase the infectivity of SARS-CoV-2, because it was directly related to the furin cleavage insertion, such that it not perfectly adapted to humans on emergence.

Slack / Private Message Drop, p.69 · original PDF · in context

HKU1 actually does have an optimal furin site - some strains have a insert or deletion of SSS - yes some of these Shave predicted O-glycans [shared file(s): HKU-1 Spike.pdf]

Slack / Private Message Drop, p.69 · original PDF · in context

People have put furin sites into CoVs previously - here's one example from SARS: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111780/

Slack / Private Message Drop, p.70 · original PDF · in context

My take is that furin cleavage sites are *everywhere* in natural viruses e.g.

Slack / Private Message Drop, p.70 · original PDF · in context

I also think that focusing on these genetic details - which are not conclusive - is in danger of missing the bigger picture: that bats (and like other wildlife) carry a huge diversity of coronaviruses some of which are closely related toSARS-CoV-2; that coronaviruses link species boundaries all the time; that 4 other CoVs have emerged in humans inthe last 20 years; that furin cleavage sites are common in viruses and come and go all the time; that people have been warning about emerging CoVs for years.

Slack / Private Message Drop, p.71 · original PDF · in context

The little details about the furin site - e.g., that it's exactly in the spot where experiments had previously inserted it,the fact that the exact sequence exist in a cat CoV, Shi herself being sketchy with details and considering lab accident, etc.

Slack / Private Message Drop, p.72 · original PDF · in context

Notable that HKU1 does have a furin cleavage site but NL63 doesn't.

Slack / Private Message Drop, p.74 · original PDF · in context

Sorry to keep going, but HKU1 is really interesting in this context: (i) has an furin cleavage site (RRKRR) in the right spot; (ii) causes human and has spread (it turned up in Australia; (iii) betacoronavirus; (iv) closest relative is MHV but that is way distant.

Slack / Private Message Drop, p.74 · original PDF · in context

Had a quick look...if Alina is suggesting that the pangolin RBD came from humans she's totally mad (look at the synonymous diversity). 100% clear that that RBD is out there in nature.

Slack / Private Message Drop, p.85 · original PDF · in context

I've just emailed Tommy if he can assemble the same RBD without the Liu et al. data and/or if it has been Sanger sequenced.

Slack / Private Message Drop, p.86 · original PDF · in context

I've also asked if they can assemble the same RBD without the Liu et al. data and/or if it has been Sanger sequenced.

Slack / Private Message Drop, p.86 · original PDF · in context

These studies are presented each as independent evidence of the pangolin virus RBD, but if all those papers are relying on a third data set - and don't actually have _any_ data from this very critical CoV RBD, then that's an issue.

Slack / Private Message Drop, p.86 · original PDF · in context

But if the acquisition of the furin site is the point at which it becomes epidemic then once again we are back to the common ancestor of the current diversity being in late 2019.

Slack / Private Message Drop, p.109 · original PDF · in context

I talked to Christian at the WHO meeting and we had an interesting chat - he thought it had been circulating inhumans for a while before the furin site appeared.

Slack / Private Message Drop, p.110 · original PDF · in context

True, but I don't see how a passage in a mouse model would produce a virus so well adapted to humans and with that RBD.

Slack / Private Message Drop, p.149 · original PDF · in context

That's the key here - that mouse model is using the human ACE2 receptor, so it'd be a perfect fit - plus the acquisition of furin sites during rapid passage as we discussed in the paper (as is seen for flu).

Slack / Private Message Drop, p.149 · original PDF · in context

Fair point about ACE2, but the RBD is very different to SARS-CoV.

Slack / Private Message Drop, p.149 · original PDF · in context

However, the issue of insertions and particularly deletions (including deletions near the furin site) is a real thing - becoming a problem with the animal models according to a discussion I was having with respiratory pathogens branch at CDC on Friday - if the stocksare not properly grown [ie by passing a low MOI} the furin site gets screwed and the viruses are no longer pathogenic in animals.

Slack / Private Message Drop, p.154 · original PDF · in context

I've been looking at some of the spikes of non-CoV Nidoviruses - many have furin sites -some have YUGE mucin-like domains.

Slack / Private Message Drop, p.162 · original PDF · in context

They put a restriction site INSIDE the furin site so they can follow the viruses over time - it's all very clever.

Slack / Private Message Drop, p.168 · original PDF · in context

It's another data point that I believe shows that the furin site in SARS2 could evolve naturally.

Slack / Private Message Drop, p.185 · original PDF · in context

Those furin cleavage sites are definitely out there in nature.

Slack / Private Message Drop, p.186 · original PDF · in context

E.g., they mention that SARS1 has a furin cleavage site in the spike - that's clearly not true and suggestive of them not looking very closely at the literature.

Slack / Private Message Drop, p.189 · original PDF · in context

Notable that Rhinolophus sinicus bats are in Hubei and have a range of ACE2 receptors. [shared file(s): Journal of Virology-2020-Guo-e00902-20.full.pdf]

Slack / Private Message Drop, p.197 · original PDF · in context

That cleavage site paper is really interesting...a key point is that the polybasic insertion is selected against in Vero cells: that journalist from the NYT (Nicholas Wade) was trying to argue with me that passage in Vero cells was a way that the polybasic insertion could occur...clearly the conspiracy theorists have been discussing this.

Slack / Private Message Drop, p.208 · original PDF · in context

Still cleaning up the dataset.*Cross-reactive antibodies to SARS-CoV-2 nucleoprotein, spike and receptor binding domain in pre-COVID-19 plasma and serum samples from Sierra Leonians.* A mystery regarding the COVID-19 pandemic has been the relatively low case numbers and fatality rates in most countries in Sub-Saharan Africa compared to many other countries worldwide (Mbow et al., 2020).

Slack / Private Message Drop, p.214 · original PDF · in context

This important study raises the possibility that a novel betacoronavirus(es) related to SARS-CoV and SARS-CoV-2 is present in African bats.To investigate possible exposure to betacoronavirus proteins in Africans we tested plasma and serum samples collected before the reporting of the first Chinese COVID-19 cases in late 2019 for the presence of antibodies that bind to SARS-CoV-2 nucleoprotein, spike or receptor binding domain (RBD).

Slack / Private Message Drop, p.214 · original PDF · in context

The HexaPro Spike and RBD were further purified by size-exclusionchromatography.

Slack / Private Message Drop, p.214 · original PDF · in context

Significant numbers of positive ELISA reactivities to SARS-CoV-2 N, S and RBD were observed in serum and plasma samples from the Ebola survivor cohort.

Slack / Private Message Drop, p.214 · original PDF · in context

As expected there was a correlation between reactivities to trimericS and RBD (p < .0001).

Slack / Private Message Drop, p.214 · original PDF · in context

Significant correlations were also observed between the reactivities to N and S (P=.003)or N and RBD (p < .0001).

Slack / Private Message Drop, p.214 · original PDF · in context

Using conservative cutoffs, 43% of samples (83/192) were positive for reactivity toeither the SARS-CoV-2 N and/or the RBD. 33/192 were reactive to both N and RBD, 19/192 were reactive to N only and 31/192 were reactive to RBD only.

Slack / Private Message Drop, p.214 · original PDF · in context

Actually, I have some inside information from WHO (please keep confidential): > We have tested a variant with four simultaneous changes in the spike protein (Amino acid changes: 69del, Y453F, I692V, M1229I).

Slack / Private Message Drop, p.249 · original PDF · in context

(1) RmYN02 was not being worked on; (2) the spike protein receptor-binding domain in both these viruses is completely different to SARS-CoV-2; and (3) RmYN02 does not have a polybasic furin cleavage site.

Slack / Private Message Drop, p.250 · original PDF · in context

The N rxty has held up [the Spike/RBDmostly did not], but more complicated.

Slack / Private Message Drop, p.280 · original PDF · in context

Yes indeed - for me the GD pango RBD is game-over/mic drop for natural origin of SC2, No way the pango RBD was selected in cell culture via passage of a bat cov.

Slack / Private Message Drop, p.280 · original PDF · in context

Some of the RBD mutations we have seen recently (eg mink) are actually reversions to the bat residues.

Slack / Private Message Drop, p.282 · original PDF · in context

@Andrew Rambaut But of course that is with a non-ACE2 RBD in bats.

Slack / Private Message Drop, p.282 · original PDF · in context

One think for sure is that there is considerable plasticity in the RBD-ACE2 interactons.

Slack / Private Message Drop, p.327 · original PDF · in context

More importantly it clear that *both* NTD and the RBD need toundergo dramatic shifts that are driven by ACE2 binding to accommodate the S1/S2 and S2' cleavages and ultimately the large scale rearrangement of S2 into the 6 helix bundle.

Slack / Private Message Drop, p.327 · original PDF · in context

Just to revisit Baric https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081895/ "Second, residue 501 in 2019-nCoV RBD (corresponding to residue 487 in SARS-CoV) is an asparagine.

Slack / Private Message Drop, p.333 · original PDF · in context

The pattern of prolines in SC1 and SC2 RBDs are distinct - look at PO1 - which creates a very different interaction with ACE2 - but we're seeing in real time the adaptation of SC2 to receptors in humans, minks and mice pretty muchas you'd draw it up.

Slack / Private Message Drop, p.339 · original PDF · in context

Two RBD residues (residues 479 and 487) had to mutate to enable civet SC1 (the 99+% virus) to jump to humans.

Slack / Private Message Drop, p.340 · original PDF · in context

The two SC1 residues are equivalent in how they interact with ACE2 to to SC2 RBD residues Q493 and N501.

Slack / Private Message Drop, p.340 · original PDF · in context

There are other mutations in the elephants that also involve RBD residues, including a Q493K mutant on the Mass patient SC2 that may be due to the fact that he was treated with Regeneron Mabs.

Slack / Private Message Drop, p.341 · original PDF · in context

Th other RBD residues are either immune escape/enhanced ACE2 binders or both.

Slack / Private Message Drop, p.341 · original PDF · in context

The nucleotide and protein database in PubMed is not working well today :rage: for whatever reason, but of the few cat SC2 sequencesit is said that there are not changes in the RBD of the SC2 in cats.

Slack / Private Message Drop, p.344 · original PDF · in context

According to the authors: "S H655Y isnear the polybasic cleavage site, residin between the receptor binding domain and the fusion peptide, and is thoughtto modulate Spike glycoprotein fusion efficiency 55,56,59 262 .

Slack / Private Message Drop, p.344 · original PDF · in context

I don't have the sequence for that Cambodian virus and I'll see I can get hold of the key bits of the spike protein.

Slack / Private Message Drop, p.345 · original PDF · in context

Yes - will be important to repeat what Jesse has done with plasma from vaccinees and to put some of the mutationsin the RBD in in combination.

Slack / Private Message Drop, p.360 · original PDF · in context

H being a positive charge in the P5 position is an upgrade to the furin site from P. https://journals.sagepub.com/doi/full/10.4137/BCI.S2049

Slack / Private Message Drop, p.369 · original PDF · in context

Once this virus got a furin cleavage site it opened up the possibility for Doug and its 613 twins to destabilzed thetrimers so you could get surface entry.

Slack / Private Message Drop, p.380 · original PDF · in context

Speaking of Pangos/RatG13 the more I look at the SC2/RatG13 alignment the more I realize Boni/Rambaut were totally spot on, No need to evoke Pango recombination to generate the RBD of SC2.

Slack / Private Message Drop, p.394 · original PDF · in context

NL63 - some similar in the RBD which I did not expect - will take a further look -

Slack / Private Message Drop, p.401 · original PDF · in context

Use *naïveté* in a sentence: It is the height of *naïveté* to claim that one can infer the evolutionary origins of the RmYN02 S1/S2 cleavage site by performing a simple nucleotide alignment as advanced by some proponents of the lab origin hypothesis [1]. 1.

Slack / Private Message Drop, p.402 · original PDF · in context

Cambobatcov spike pretty interesting - it has the E484 and 5/6 of the GD Pango/SC2 RBD residues we highlighted in PO1.

Slack / Private Message Drop, p.449 · original PDF · in context

Take homes: Thai Bat spike and RmYN02 spike are nearly identical, including at the S1/S2 cleavage site.

Slack / Private Message Drop, p.455 · original PDF · in context

Cambobatcov RBD is very similar to PangoGD RBD and SARS-CoV-2 RBD. 5/6 pf the PO1 key residues.

Slack / Private Message Drop, p.455 · original PDF · in context

The differences in nucleotide and amino acid trees in spike and RBD seem to have attracted some attention, which is good.

Slack / Private Message Drop, p.464 · original PDF · in context

@Eddie Holmes The HA379 and RmYN02 similarity is after the NTD and into the RBD - INTERESTING!

Slack / Private Message Drop, p.471 · original PDF · in context

And you would introduce the perfect cleavage site and theoptimum RBD.

Slack / Private Message Drop, p.489 · original PDF · in context

Doug and the VoC that have followed show that SC2 and its RBD and FCS are still adapting to us.

Slack / Private Message Drop, p.491 · original PDF · in context

So need to show that the elastase cleavage site is used but it's not out of the question and could make some sense.

Slack / Private Message Drop, p.502 · original PDF · in context

Real COVID-19 is not too hard to spot serologically - but with caveats - high correlation btwn rxty to Nucleoprotein, RBD and S2 PLUS neutralization.

Slack / Private Message Drop, p.512 · original PDF · in context

The RBD itself is a smaller loop within the CTD, which is actually a bit bigger than what we estimated in PO1 - so running about 414 to 506.

Slack / Private Message Drop, p.524 · original PDF · in context

So we have a common set of functional sites/effects for all the new variants: An RdRp mutation, an nsp6 modification, spike 613/614, some NTD mutations/deletions and some RBD mutations.

Slack / Private Message Drop, p.529 · original PDF · in context

it's nicely spotted, since it's the only mutation unique to SC2 in the RBD - we should have picked this up earlier.

Slack / Private Message Drop, p.536 · original PDF · in context

Also HA363 and friends have FOUR predicted O-linked glycans near the S1/S2 cleavage site even thou not a FCS.

Slack / Private Message Drop, p.552 · original PDF · in context

I thought the futon cleavage site was that annoying gap between the cushions on the sofa where your butt sometimes gets stuck. [shared file(s): Screen Shot 2021-03-06 at 1.35.36 pm.png]

Slack / Private Message Drop, p.570 · original PDF · in context

Not a perfectly done alignment , but still you can see that RpYN06 spike is VERY close to GD pango spike and very different from RnYN02 spike until you get to the RBD.

Slack / Private Message Drop, p.579 · original PDF · in context

The RpYN06 RBD is VERY close to RNYN02 spike and very different from GD pango spike After the RBD the three viruses are similar, until you get to the S1/S2 junction where they all differ. [shared file(s): image.png]

Slack / Private Message Drop, p.579 · original PDF · in context

I read this channel and see (1) a rat looking thingie rolled up in a bamboo stick, and (2) socks with a furin cleavage site on them - made in China.We have come a long way since we started this channel, gentlemen.

Slack / Private Message Drop, p.599 · original PDF · in context

One bat virus spike protein into a BAC clone of another bat virus - AND the paper even shows those viruses don't infect cells better thanwildtype.

Slack / Private Message Drop, p.821 · original PDF · in context

@Eddie Holmes Yes, no one would engineer that RBD so it has to be natural.

Slack / Private Message Drop, p.841 · original PDF · in context

I.e.:> The ACE2 binding RBD in SARS-CoV-2 was not inserted or created in a lab [Confidence: high]> ....

Slack / Private Message Drop, p.849 · original PDF · in context

Doesn't take much to think that any particular RBD+FCS combination could arise.

Slack / Private Message Drop, p.854 · original PDF · in context

At least that furin cleavage site tweet is coming in handy for the Aussies :scienceparrot: [shared file(s): Screen Shot2021-05-27 at 9.01.45 PM.png]

Slack / Private Message Drop, p.888 · original PDF · in context

Love it: "Yet Baltimore has effectively disavowed his characterization of the furin cleavage site.

Slack / Private Message Drop, p.936 · original PDF · in context

I'd love to know what a 'human specific furin cleavage site' is.

Slack / Private Message Drop, p.1003 · original PDF · in context

In the RBD there are three N-linked glycans in SC2/BANAL, so two are conserved, one is not.

Slack / Private Message Drop, p.1003 · original PDF · in context

The answer to that is a clear no.BANAL has 23 (predicted...) N-linked glycans, SC2 has 22 - 21 of them are shared with BANAL, so SC1 has one unique N-Glycs (in the NTD), whereas BANAL has two unique N-Glycs (one in RBD, one in NTD).

Slack / Private Message Drop, p.1006 · original PDF · in context

It may have antigenic consequences in humans but that is coincidental because it is the RBD

Slack / Private Message Drop, p.1030 · original PDF · in context

Shortly after the teleconference we learned about the pangolin coronavirus that has a highly similar receptor binding domain- RBD.

Slack / Private Message Drop, p.1053 · original PDF · in context

Now, go find one with a furin site so we can be done with this shit.

Slack / Private Message Drop, p.1070 · original PDF · in context

From the 8th Feb version on GoogleDocs: > Although RaTG13 remains the closest relative to 2019-nCoV across the genome as a whole, the Malayan pangolin CoVs are identical to 2019-nCoV at all six key RBD residues.

Slack / Private Message Drop, p.1079 · original PDF · in context

Analyses of these pangolin viruses are ongoing, although they do not carry the furin cleavage site insertion.

Slack / Private Message Drop, p.1079 · original PDF · in context

And by disproving - I mean, it disproves that the specific RBD mutations seen in SC2 arose via culture passage, but,of course, it doesn't directly disprove passage itself.

Slack / Private Message Drop, p.1080 · original PDF · in context

It has a receptor binding domain that is almost identical, nearly identical to SARS-2, but it does have a couple point mutations in it.

Baric Transcribed Interview (Redacted), p.78 · original PDF · in context

BANAL-52 and BANAL-1-something or another, also had receptor binding domains that were virtually identical to SARS-2.

Baric Transcribed Interview (Redacted), p.79 · original PDF · in context

So there's two bad viruses and pangolin virus within the clade, within that branch of SARS-2 that had identical receptor binding domains, which means that it's not engineered.

Baric Transcribed Interview (Redacted), p.79 · original PDF · in context

But this grant, I mean, especially with all the conversations of the furin cleavage site, I mean, nothing about that grant.

Baric Transcribed Interview (Redacted), p.91 · original PDF · in context

There should be sarbecos out there that have furin cleavage sites, so they were going to look for them.

Baric Transcribed Interview (Redacted), p.92 · original PDF · in context

They would introduce changes into the receptor binding domain and look in the context of those pseudotypes, which is the safe system, to ask questions about what the role of those mutations were in tropism and entry of viruses in receptor usage.

Baric Transcribed Interview (Redacted), p.92 · original PDF · in context