1. Revisting this famous section from the DEFUSE grant proposal because I think we will be hearing a lot about it again. And let’s analyze what it actually says and compare it to the mantra of ‘they propsed to insert FCSs in bat CoVs” 
2.Firstly thevirology because even 3 yrs, the authors of high profile books don’t get it (or don’t want to). For cell entry CoV spike proteins undergo at least 2 proteolytic cleavage steps by host cell protease enzymes. One is at the S1/S2 boundary, the other at the S2’ site.
3.These processing events allow the S2 fusion peptide to be exposed and promote fusion of the viral and cellular membranes.
4.These cleavage events can be performed by a range of cell surface and endosomal proteases, with TMPRSSs and Cathepsins being the best known,
5.While in 2018 unknown in the sarbeco family, other CoVs have been found to have polybasic cleavage sites (minimally RXXR) at, or near, either position which extends the range of proteases to include also furin-family members. Note the presence RXXR is suggestive NOT indicative
6.What the authors of this grant had found previously for a number of bat CoVs (not just sarbecos) was that while the sequence of the receptor binding domain of spike might be predicted to use its cognate human receptor…
7.Some didn’t infect human cells efficiently. This could be circumvented by exogenous trypsin or cathepsin treatment – implying a major barrier to human infection by some bat CoVs was the ability of S1/S2 OR S2* to be cleaved by species specific differences in proteases.
8.So elsewhere in the proposal the authors say they will clone spike genes of sequenced viruses, make lentiviral pseudotypes (look it up, non hazardous, non GoF) to test human tropism phenotype, and…
9.Potentially clone into one of 3 recombinant SARS1r bat CoV backbones for viral replication and pathogenesis studies – I’m not going to relitigate whether these are GoF or not, suffice to say SARS-CoV-2 is not one of these.
10.So in order: clone spikes and see if lenti-pseudos infect human cells – if not treat with exogenous trypsin/cathepsin – does that work? If not, deprioritize
11.Look at where the cleavage sites are in the latter – are they poor a consensus for cathepsins or trypsin? If so ‘improve” them. AND IMPORTANTLY do all of this in a lenti. Why? Because it’s cheap, quick and by far the easiest way to do it…and a universal technique.
12.Also note, such spikes that do not get cleaved efficiently are not the most interesting to those proposing this in 2018, who are clearly on the look-out for SARS1-like viruses with high spillover potential.
13.After that, all the functional work to understand whether such spikes are capable of mediating invivo infection will be assessed in the SARS1r backbone – I see people disputing this BUT THIS IS WHAT THE GRANT SAYS. (also to be done at UNC but anyway).
14.There is the potential to make molecular clones of novel viruses discovered, but you aren’t going to do that until you’ve got the data above – simply because it’s a lot of work (it was then) and you want to test one variable at a time.
15.Now to the FCS bit. Other CoVs are known to have them but as of then (and now with the exception of SC2) no other sarbeco has one. So they were alive to the possibility that they might find one or a potential one…
16.But in no part of this grant do they propose to insert one into a live virus that doesn’t have one to see what will happen. This is a fundamental (and in the case of many, deliberate) misrepresentation of what the grant says.
17.Now from a practical PoV, lets imagine some of this work was done and they found a virus like RMYN02. The SC2 FCS is PRRAR*S (*being the cleavage site). RMYN02 is the closest known at this point – PAARV.
18.No-one looking at that in 2018 would say ‘that could be made into an FCS”. But they might say “if we change the V to an S like it is in SARS1 it might get into human cells better”.
19.So the idea that this grant proposes to add unicorn horns to unknown viruses is post-hoc motivated revisionism made largely by people who really have no idea of the biology
20.Now we can talk about the merits of this grant and whether it was any good and of course whether it was justified and contained properly, but there is no blueprint for the artificial engineering of a virus to become SARS2.
21.Lastly the FCS of Wuhan SC2 is actually pretty crap. In the native virus it is not that well cleaved by furin and the cleaved spikes that end up on the virion and not that stable (lentiviruses very misleading in this regard.)
22.The D614G mutation came up pretty fast and that stabilizes the furin cleaved spike as well as resisting human antiviral restriction by GBP5 (@JollyLab).
23.The subsequent VOCs gained infectivity associated with P-to-H or P-to-R changes in the cleavage site. These all lead to more furin cleaved S on the native virion. (the lentiviral work is misleading on this)
24.All this raises the question that the LL-engineer proponents never answer. If you were going to do what was suggested, why not just dump in the perfect FCS – RRSRR?
25.The above is fairly basic virological understanding. And it is why most of us find the idea that SC2 is some product of bioengineering fanciful in the extreme.
26.Can one completely rule out that someone isolated a natural SARS2 virus and contaminated themselves? But that is simply inconsistent with the known epidemiology and genetics making it, to use the new vernacular, improbable.
27.As for the case for the natural evolution of the FCS. Well FCS seemingly skip in and out of various other beta CoVs including the MERS-family quite regularly, and may even be associated with cross-species transmissions.
28.The S1/S2 boundary is very variable and tolerates insertions by copy-back recombination including with cellular sequences, and has been seen in SARS2 as highlighted by @PeacockFlu
29.And in European branch of the bat sarbecoviruses, there is evidence of viruses requiring a single AA change to make a potential FCS (and may even be already doing it).
30.And of course FCS is required for aerosol spread in SARS2, so skipping species to humans or an intermediate where respiratory transmission is a selective advantage, the acquisition of such a trait is not unlikely.
31.Not even going to bother with Sachs' and Ebright's ENAC stuff. All these arguments of intelligent design without any actual evidence to support them are simply creationism my another name.
32.Now off to teach my SARS2 lecture to undergraduates – cue the accusations of indoctrination!
33.Anyway, I trust the GOP committee will ask some of these questions of the “experts” on the panel today. I’m sure they will understand the details.
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