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Mutations in the Coronavirus Spike Protein

The last post talked about antibodies to the spike protein of the coronavirus, and one of the main things that everyone has to keep an eye on are the mutations in that area. That has implications for monoclonal antibody therapy, for vaccine production, and for the behavior of the coronavirus itself. Antibodies against the Spike protein could range from neutralizing ones that will stop the virus in its tracks all the way to others that would cause antibody-dependent enhancement and make the viral infection even worse (see below), and we don’t know how the mutational landscape might alter the activity of any given monoclonal candidate. A new preprint on spike muations (from researchers at Los Alamos, Duke, and Sheffield) has gotten a great deal of attention in the last couple of days, and I think that a detailed look at it would be useful to help explain these issues.

Most of this manuscript focuses on a particular mutant called D614G. For those outside the field, that’s a standard notation that translates as “at amino acid residue 614, take the existing amino acid (aspartic acid, single-letter code D) and change it to a glycine (single-letter code G)” That change happens through the triplet code in the RNA sequence that codes for the amino acids – a single random-mutation A-to-G switch in the right spot is enough to flip the resulting amino acid from an aspartate to a glycine (GAU and GAC both code for aspartic acid, and GGU and GGC both code for glycine). And those are pretty different beasts: aspartic acid has a polar, charged side chain, while glycine is the no-side-chain-at-all generic amino acid. This is not expected to be a silent mutation, in other words.

It’s important to realize that this mutant did not just suddenly come into view in the last few days. In fact, readers of this blog have encountered it before, in this post on coronavirus mutations back on April 21. Which of course seems like about six months ago, but you all know how that goes, right? (That one might be good background for this post, if you haven’t read it). I reproduced a graphic from the Nextstrain.org folks that showed mutations in the virus’ sequence in terms of entropy – that is, how different the amino acid residues were from the canonical sequence. Here it is again:

As that earlier post explains, moving from left to right you have the whopper ORF1, which codes for the equally whopping polypeptide that is the self-unzipping archive of a lot of the key viral proteins (it really is alarming how these things assemble themselves; there’s no easy analogous example out here in the macroscopic world that gets the weirdness of it across). Then you have the green region marked “S”, and that’s our friend the Spike protein. Note right smack in the middle of it is that big tall line: that is the G to A switch that gives you the D614G mutation itself, and it’s so tall because that entropy measurement is the degree to which the amino acid has changed. So people who are into coronavirus mutations (I mean, who isn’t) have had their eye on this one for some time.

Part of the reason for that is that this particular mutant has seemingly been on the rise. It was one of the founding viral types in Europe, and gained a good foothold there before being spread to other regions – and in those, the glycine mutant seems to have increased versus the original aspartate one. The tricky part is to distinguish between founder effects (what virus type got there first and started spreading) versus selective advantage (one that could show up later and outcompete what came before). In general, we don’t have quite detailed enough mutation-through-time data sets to be sure about distinguishing those two. The preprint applies several statistical tools to that purpose, and brings in another interesting piece of evidence from the Sheffield part of the collaboration: when you look at the “cycle threshold” for an RT-PCR test to pick up on the viral RNA, it looks like the glycine mutant needs a bit fewer cycles – which would mean that those patients the samples came from likely had a higher viral load to start with. Virologist Trevor Bedford at the University of Washington, whose name (and whose expertise) coronavirus aficionados will recognize immediately, has said on Twitter that the UW samples seem to show this effect as well. The preprint advances the idea that this mutant is more transmissible, and Bedford says that while there is some evidence for that, it’s not conclusive yet.

If, though, there is a difference, what could be behind it? The preprint notes that there are two broad possibilities. One is based on protein structure, and there are two main possibilities in that bin. The first is that that there’s an effect on the Spike’s receptor binding domain interaction with the human ACE2 protein. That’s harder to get a handle on, because this mutation is nowhere near the RBD. It would have to be some allosteric long range effect, and while that’s certainly possible, it’s difficult-to-impossible to try to model such things. You’re looking at a number of very small energetic changes lining up in the same direction to have an effect on a completely different region of the protein, and we just don’t have the ability to pick up on those computationally at the level of detail needed. The second structural possibility is that the interaction between the two main parts of the Spike protein (S1 and S2) has been affected by the loss of the polar Asp residue. It could well be forming a hydrogen bond with a nearby Threonine residue, which would disappear in the Glycine mutant form, and perhaps this “loosening up” facilities the viral entry process as the Spike goes about attacking the human cell membrane.

But the other broad possibility is immunological: the mutation is in a protein region that turned out to be important in antibody recognition during the original SARS epidemic. It showed high immunogenicity (many antibodies isolated from patients reacted to it) and it was also implicated in antibody-dependent enhancement. That effect has been mentioned here several times; it’s a major concern. Recall that this happens when antibodies bind to the virus but don’t neutralize it – in some of these cases, that antibody binding actually enhances the ability of the virus to get inside the human cell, which just makes everything worse. Not only do some SARS antibodies do that in the patients that developed them, but the same problem was seen in antibodies raised after potential SARS vaccine treatment. The good news is that everyone working on vaccines and monoclonals is very much on alert for this, and that so far more than one vaccine candidate has been reported to show no signs of it in animal models). So immunologically, the D614G mutant might be enabling antibody-dependent enhancement, possibly by favoring the generation of non-neutralizing antibodies to this region of the protein.

There’s even a mechanism that might bridge these two proposals: there’s more than one mechanism for ADE, and one of these (see the first link in the above paragraph) involves altering the Spike/ACE2 interaction. So you could have a direct effect on protein structure and target binding that also ends up with an immunological effect once the antibody response gets going.

It’s important to realize, though, that none of these mechanisms are proven. In fact, the very hypothesis that this mutant is more transmissible is as yet unproven – it’s not unlikely, but it could be wrong. Here’s an important question: what about the patients who are infected with with the D614G form? Turns out that the Sheffield group was able to match up hospitalization data with the sequencing of 453 individual patients, and they found no correlation of  hospitalization status with the mutant form. Trevor Bedford and the UW group independently checked for this and had the same result: no apparent effect on severity of disease. At most, this mutation may be more transmissible (bad enough, to be sure), but it does not appear deadlier once a patient has been infected.

What about an effect on vaccine and monoclonal antibody development? We don’t have all the details, but the great majority of work I’ve been seeing on the monoclonals and on the antigen proteins targeted as vaccine candidates has focused on the RBD region of the Spike protein. This D614G mutation isn’t in that part of the protein, which is good. If that allosteric hypothesis has something to it, though, there could conceivably be an effect on the overall shape of the RBD, which could in turn affect antibody binding and selectivity. But this is two levels of un-proven-ness, at the very least. It’s also worth keeping in mind that that some of the monoclonal antibody candidates are from B cells of recovered patients, who themselves may well have been infected with the D614G mutant to start with. And the fact that there seems to be no difference in severity of disease between the two forms argues that the antibody response in general is unimpaired. So I’m not sounding any alarms on this based on the data we have.

 

37 comments on “Mutations in the Coronavirus Spike Protein”

  1. Barry says:

    Pretty sure that
    “that is the G to A switch that gives you the Dg14G mutation”
    should read “that is the G to A switch that gives you the Dg614G mutation”

    1. loupgarous says:

      I think Derek meant D614G when he typed “Dg14G”. Google search for “Dg14G” comes up dry. while “D614G” in the article he quoted from refers only to “D614G”.

  2. Poinsy says:

    In the 7th paragraph, a small frameshift typo?
    “There is reason to believe that (The good news . . . models).”

    1. Athaic says:

      In an article about mutations, frameshift typos are to be expected.

  3. SP123 says:

    If a mutation is associated with higher viral load but no difference in hospitalization (as a proxy for disease severity), doesn’t that suggest the 614G virus is less virulent? All else being equal you’d expect more virus to be more harmful.

    1. eub says:

      Yeah, this.

      Or else they’re picking up bits of noise and fitting them into a picture.

  4. Buzz says:

    Derek, I am a bit disappointed in your usual excellent analysis.First, you accept the argument that this change can plausibly lead to ADE- yet clinical outcomes are unchanged???! It makes no sense to infer that the mutation triggers ADE , but outcomes are the same as the original virus. The PCR data in the paper is even worse. There is no data as to when in the infection course nasal swabs were obtained from patients Which will majorly impact viral load. This is to argue that PCR turning positive 1-2 cycles faster (24 vs. 25) is Of any clinical relevance an implication for a “worse, more dangerous strain of the virus” after the clinical data as far as hospital admission showed suggests an alarmist group looking for press and going overboard with conclusions without any functional data to back them up. The PCR cycle graph looks like P hacking IMO

  5. Kent Matlack says:

    Just a note on the statement “it really is alarming how these things assemble themselves”. There is a very large and growing literature that all viruses receive a great deal of assistance from host factors to achieve their assembly. I see an analogy to protein folding: individual pure proteins can (often) fold by themselves into functional form, but in vivo they receive a tremendous amount of assistance. Similarly, purified viral capsid proteins can be made to assemble in vitro, but that assembly is inefficient; in vivo – i.e. in infected cells in people – they receive a tremendous amount of help. But, yes, however it’s done, and whoever participates, the structural assembly of viruses is very impressive.

    1. See: https://www.pnas.org/content/early/2020/03/04/1920695117 MicroRNAs organize intrinsic variation into stem cell states 3/5/20

      “…naturally arising cell-to-cell variation, sometimes described as stochastic fluctuation, is in fact coherently organized biology.”

      The problem with Derek Lowe’s misrepresentations of top-down causation starts when he ignores the fact that light-activated microRNA biogenesis links the Creation of ATP to the ATP-dependent creation of RNA, and RNA-dependent RNA polymerase biophysically constrains viral latency in all eukaryotes. Remdesivir treatment of coronavirus pathology exemplifies that fact.

      1. Tom A says:

        James: I had a moment of confusion there but now I understand:
        https://microrna.pro/microrna-biogenesis-is-light-activated-biblical-genesis-3/

      2. Some Dude says:

        The corona crisis sure has brought the crazies to this blog.

        1. johnnyboy says:

          Indeed – you thought Lane and his peroxynitrites were bad, but there’s a whole deeper dimension of crazy out there

          1. Alguido says:

            @johnnyboy, at this stage I would almost welcome something from Lane

            ………..wait………what’s that……….Zinc ionophores linked to peroxynitrite generation……………Arrrrrggghhhhh…….

  6. Ted says:

    Hi:

    Yet another great post.

    For some reason, probably childhood imprinting, I always imagine Sylvester McMonkey McBean when I think of viral unpacking:

    https://youtu.be/qPhOZzsi_6Q?t=270

    On a more serious note, I wonder/worry if D614G has different blood brain barrier permeability kinetics? Based on the clinical outcomes, it appears not, but I have no experience in virology nor CNS.

    -t

    1. Some idiot says:

      I didn’t need to look at the video… The Sneetches, by Dr Seuss…! Most of them are solidly imprinted on my brain. And now I will never think of viral unpacking in the same way again…! (-:

  7. NotARealName says:

    So Los Alamos Labs has quietly been dialed up to defuse this thing.
    And the antivirals, they’re not much, that lot is some Slim Pickens’.
    Sounds like Doomsday.

    – Hat, coat, I’ll show myself out 😉

  8. The Science Mechanic says:

    On the topic of April 21 seeming like six months ago, I heard a joke today: “This is the weirdest leap year ever. February had 29 days, March had 10 weeks and April had 5 years!”

    Also, as someone who came into life sciences from a different field in mid-career, I found “residue” to be among the weirdest pieces of jargon. When aiming for a lay audience I suggest “position”.

    1. eub says:

      The terminology I was taught was that the amino acid within a polypeptide is a residue, the ribonucleotide is the residue in RNA — the residue is the ‘monomer’. Is that not the use in practice?

      (From that it still makes sense to say “changed at residue 614” like you would say “at car 17 in the train” — you’re counting along the cars or along the amino acids.)

      For the term “residue” I have no source and possibly I just assumed it, but: that these substances are what make up the residual mixture after you break apart the macromolecule by acid hydrolysis etc.

      1. Athaic says:

        the residue is the ‘monomer’

        While inserted into the polymeric chain, yes.

        I believe the idea is to distinguish in our speech the free-floating monomer molecule from the form it takes once inserted. Usually the difference is just that the equivalent of a water molecule is missing from the residue form. It’s trivial but could be important.
        Well, it’s mostly important whenever you want to talk about their mass (proteomics mass spec specialist here). I should not use the same value for preparing a glycine-containing buffer or for calculating the mass added by a glycine inside a peptide chain.
        (let’s not go into average mass versus monoisotopic mass, that’s another layer of complexity)

        Using the word ‘residue’ may also be a convenient shorthand for “two chemical parts of the monomer molecule being busy forming a polymer by connecting to other monomers, let’s consider now the biological/chemical effects of the monomer-specific residual part of the monomer”.

        1. eub says:

          That does make sense, to distinguish between the amino acid and the residue that’s down one water of polymerization, but boy, people refer to the “glycine at position 614” all the time even if the residue is a few atoms short.

  9. Marko says:

    Countries in east Asia , plus Australia and NZ , along with states on the west coast of the US , seem to have managed the Covid-19 outbreak much better than Europe or east coast US states. This maps quite well with the global distribution of the two variants :

    https://pbs.twimg.com/media/EUY9IIuUMAAb95E.jpg

    Map is from this Bedford tweet :

    https://twitter.com/trvrb/status/1244750395630444544

    This isn’t a slam-dunk , but it’s pretty suggestive that something might be going on.

  10. Athaic says:

    @ eub

    Oh, I forget that in my previous comment:

    “these substances are what make up the residual mixture after you break apart the macromolecule ”

    Actually, no. When you break apart a macromolecule, you either get back the monomers (especially if using enzymes) or some broken/modified molecules.

  11. Simon Auclair the Great and Terrible says:

    Doesn’t anyone have an opinion on remdesivir getting FDA emergency approval?
    On the back of dr Fauci’s NIAID study showing30% reduction in hospitalization time and25% reduction in mortality?
    After lobbying by Gilead?

    1. JasonP says:

      Perhaps they are waiting for the preprint or data to be released? Me thinks these guys (Derek, et al) like to base opinions on an analysis of data, which is forthcoming.

      I too am anxious to see what that data reveals in the eyes of trained experts.

  12. ezra abrams says:

    Given the severity and cost of c19 (>2.5 trillion us dollars in direct spending by US fed gov’t alone) I would hope that there is money to fund multiple labs looking at each possible epitope on the virus surface

    I think it took us decades to find buried epitopes on HIV and influenza that are very promising for nAbs

    based on this we need really large scale mAb screening (regeneron ? )
    I mean, tens of thousands of 96 well plates

  13. luysii says:

    Glycine has the least bulky side chain (hydrogen) of any of the amino acids. This should give that area more conformational flexibility, and make allosteric effects more likely. It would work the same way that ” loss of the polar Asp residue. It could well be forming a hydrogen bond with a nearby Threonine residue, which would disappear in the Glycine mutant form, and perhaps this “loosening up” does. A mutation of the aspartic acid to anything other than Asparagine, Glutamine or glutamic acid would destroy the putative hydrogen bond with the nearby threonine.

    1. ghost of q.mensch says:

      Very cool. I very much like the way you analyse the effects of such a single point change on the spike protein conformation at the chemical environment level.

      Then there is this on the the furin cleavage site PRRA insertion (not mutation) segment in ncov-19 ACE2 binding spike protein coding RNA:
      https://www.youtube.com/watch?time_continue=928&v=uZUJhKUbd0k&feature=emb_title

      And this, from which some of the first link was sourced:

      https://medium.com/@yurideigin/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748

      1. SV says:

        The fact that a Harvard head of chemistry dpt was marched off menacled in jan or feb for misdeamanors linked to a nanotechnology lab in …wuhan, could also be folded in this story.
        Nanotech and coronavirus ….
        Coincidences???

        1. matt says:

          It is not even a co-incident, it is a moderately-far-away-incident with no correlation that you are only managing to bring together by the diamond anvil of towering ignorance. Or, perhaps that was just sarcasm, stretching a bit. It’s hard to tell, sometimes, especially when the moon is full.

          1. SV says:

            Around 2014 we briefly looked at combining coronavirus and nanopart. Ignorant yes, but not that much!1

      2. Safe says:

        It is easy for virologists to convert a non-virulent virus into a highly pathogenic virus a.o. by insertion of a polybasic cleavage site(e.g. avian influenza: https://doi.org/10.1099/vir.0.031591-0). Therefore it is not only conspiracy thinkers who want to know the origin of SARS-Cov2, but also scientists who are concerned about the fact that scientists are human beings making mistakes and that their findings can also be misused by the ‘dark site of the force’. Chemists created important drugs, plastic, TNT and nerve gas, physicist created silicon chips and atomic bombs; anyway, the good, the bad and the ugly sooner or later ‘escape’ from the lab……..so perhaps better for life on earth to stop creating new highly pathogenic viruses in the lab, but of course that is naive thought and the labs are safe, sure they are and super computers show this is not an engineered virus, look at that…

  14. steve says:

    The idea that viruses with the D614G mutation are more virulent is pure speculation that is not supported by any data in the paper. A more recent study from Glasgow debunks the notion that there are different strains of virus; rather, there are a number of random mutations in a single virus type (search MacLean in the journal Virus Evolution if the link doesn’t convey). https://academic.oup.com/ve/advance-article/doi/10.1093/ve/veaa034/5827470?searchresult=1

  15. Science Junkie says:

    What therapeutic trials on old drugs are showing results? The antivirals seem to steal the show, but is there anything else out there?

  16. John Monahan says:

    I am familiar with the concept of conservative vs nonconservative missense mutations, and D614G is a whooper of a change. Either the loss of an aspartic acid residue or the gain of a glycine would deserve a critical look for changes in the 3D structure and interactions of the spike protein. I would bet against it being a neutral substitution,
    I’m not familiar with the Nextstrain.org folks entropy axis. I have spent some time at their site but could not find an explanation or reference. Could someone help pointing me to an explanation of their entropy scale? Thanks.

    1. matt says:

      So biological warfare specialists, evil Nazi scientists brought from the ruins of Hitler’s war machine, working for decades on terrible Cold War weapons, unleashed a disease that kills 5-10 people a year, and mostly just makes a few tens of thousands feel tired and achy?

      Should I not be seeing the mighty Stonehenge being lowered from above, with little pointy-shoed people dancing around?

  17. Justin says:

    In the ref sequence NC_045512.2 the base at position 23403 is an ‘a’ and not a ‘g’ as cited in the original paper. Any thoughts??

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