Besides the furin cleavage site (FCS), SARS2 has another unique feature mentioned in DEFUSE not yet seen in any natural SARS-like viruses – an ablated N-linked glycan at position N370. This glycan was ablated via a T372A amino acid mutation that came about via a double nucleotide mutation of the original ACT codon into GCA (the latter, incidentally, is the same codon as the one coding for alanine – out of 4 possible alanine codons – in the PRRA insertion which has created an FCS in SARS2).
Importantly, the T327A mutation greatly increases SARS2 infectivity in human lung cells but, just like an FCS, this kind of a mutation seems to have selective pressure AGAINST it in ancestral bat viruses.
DEFUSE’s interest in N-linked glycans stems from a very curious observation about SARS1 whose bat progenitor seems to have temporarily lost two of its N-linked glycans in civet SARS1 progenitors before re-acquiring them, and this led virologists to hypothesize that those glycans could be relevant for host switching. This is described in DEFUSE in a somewhat convoluted way:
“N-linked glycosylation: Some glycosylation events regulate SARS-CoV particle binding DC-SIGN/L-SIGN, alternative receptors for SARS-CoV entry into macrophages or monocytes [76,77]. Mutations that introduced two new N-linked glycosylation sites may have been involved in the emergence of human SARS-CoV from civet and raccoon dogs [77]. While the sites are absent from civet and raccoon dog strains and clade 2 SARSr-CoV, they are present in WIV1, WIV16 and SHC014, supporting a potential role for these sites in host jumping. To evaluate this, we will sequentially introduce clade 2 disrupting residues of SARS-CoV and SHC014 and evaluate virus growth in Vero cells, nonpermissive cells ectopically expressing DC-SIGN, and in human monocytes and macrophages anticipating reduced virus growth efficiency. We will introduce the clade I mutations that result in N-linked glycosylation in rs4237 RBD deletion repaired strains, evaluating virus growth efficiency in HAE, Vero cells, or nonpermissive cells +/- ectopic DC-SIGN expression [77]. In vivo, we will evaluate pathogenesis in transgenic hACE2 mice.”
The [77] paper cited in DEFUSE is a 2007 work by Han et al. titled “Specific Asparagine-Linked Glycosylation Sites Are Critical for DC-SIGN- and L-SIGN-Mediated Severe Acute Respiratory Syndrome Coronavirus Entry”. It looked at the 5 civet progenitor strains of SARS1 and showed that initially those strains did not have glycans around positions N227 and N699 but then eventually acquired them in civet progenitors and kept in human SARS1.
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What the 2007 paper did not know at the time that the DEFUSE authors pointed out is that the bat progenitor strains like WIV1/Rs3367 or SHC014 also have glycans at those positions. This is what likely made the DEFUSE authors interested in the host jumping potential of these glycans and potentially genetically modifying them to further study their role:
3/ Circling back to the DEFUSE proposal, the N370 glycan in SARS2 is the same glycan as N357 in SARS1 which was implicated as being important for DC-SIGN binding in 2006:
Now, the loss of the N370 glycan by SARS2 has been shown to greatly increase its infectivity in human cells:
“Using a reverse genetics system to generate a SARS-CoV-2 mutant containing the putative ancestral SNP, we show that the A372T S mutant virus replicates over 60-fold less efficiently than WT SARS-CoV-2 in Calu-3 human lung epithelial cells (Figure 4d). Further, growth of the A372T S mutant was reduced greatly for multiple days, which may be indicative of an effect on viral shedding kinetics in humans. We also generated the D614G S mutant here—reported widely to increase SARS-CoV-2 infectivity (Korber et al., 2020)—which only increased viral titers by a maximum of 2.9-fold in Calu-3 cells compared with the WT, a finding that is consistent with previous results (Plante et al., 2021).”
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However, this mutation is unlikely to have arisen in bats as it is detrimental to oral-fecal transmission (which SARS-like CoVs rely on in bats; this is also likely why we don’t see an FCS in bat SARS-like CoVs):
“Why do all bat SC2r-CoVs retain T372, not A372, in their spike proteins, even though the A372 mutant showed substantially higher infectivity than T372? Since the fecal-oral route plays a vital role in bat CoV transmission among bats31,32, we hypothesized that fecal-oral transmission might favor S proteins in all "down" conformation during natural selection, and T372A change might cause some RBDs to assume “up” conformation, which might be detrimental for the survival of S proteins during their passage through the bat stomach. The pH of an insectivorous bat stomach is around 5.633. To test this hypothesis, WT and T372A mutant S pseudovirions were treated with TPCK trypsin at pH 5.5 at 37 °C, a condition roughly mimicking bat stomach digestion. With increase of trypsin concentration, both WT and T372A pseudovirions lost significant amount of infectivity (Fig. 4b, c). However, the speed and extent of infectivity loss varied significantly between WT and T372A mutants (Fig. 4b, c). While a brief 10 min treatment of trypsin at 2.5 μg/mL resulted in over 96.6% and 99.9% loss of infectivity for BANAL-20-52 T372A and BANAL-20-236 T368A mutants, respectively, WT BANAL-20-52 and BANAL-20-236 S pseudovirions retained more than 37% and 21% of infectivity (Fig. 4b, c). Moreover, even after 40 min digestion with trypsin at 2.5 μg/mL, WT BANAL-20-52 and BANAL-20-236 pseudoviruses still retain over 23% and 14% of infectivity, respectively, whereas T372A and T368A mutants almost completely lost infectivity (Fig. 4d, e).”
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All of this begs the question: how likely is it that these unique features of SARS2 — the FCS and the ablated N370 glycan — unseen in any natural SARS-like virus and unlikely to arise in bats due to selective pressure against them are the result of DEFUSE-inspired genetic engineering?
Alternatively, could they arise via serial passaging in civets or their cells? As DEFUSE stated, early SARS1 strains showed a loss of N-linked glycans, and WIV is known to have conducted infectivity experiments on live civets using SARS-like viruses:
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PS: As an aside, the fact that within months of the SARS1 outbreak, 5 different civet SARS1 progenitor strains were identified, but 3.5 years after the SARS2 outbreak we have nothing even remotely close to an intermediate host strain or even a potential bat progenitor — despite an additional two decades of progress in sequencing technology — only keeps adding to my skepticism about a natural origin of SARS2.
Here are the N-linked glycans in SARS2 vs. related bat or pangolin CoVs: all have the N370 glycan except SARS2.
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Latest SARS2 preprint provides additional evidence that not only was the novel FCS a key factor in its pandemic potential, but the way the FCS was acquired — via a 4 aa insertion — was also important. Re: "why would a genetic engineer use an insertion to create an FCS?"
I've hypothesized previously that the answers to "why use an insertion" and "why use a leading proline" lie in MERS, whose S1/S2 loop is 4 AAs longer than that of SARS-like CoVs, and also has the leading proline, making it more exposed to proteases:
So the desire to try a MERS-like FCS can explain both. Moreover, in the 3D structure of the spike protein, the S1/S2 loop in SARS2 with the insertion has its cleavage point ~3x closer to the MERS one than if the FCS in SARS2 arose w/o insertion:
Increasing neuroinvasiveness could indeed have been the research goal of a genetic engineer opting to use a “suboptimal” or “non-canonical” furin cleavage site like the one found in SARS2. And Baric did say that MHV and FIPV inspired the FCS interest of DEFUSE. Thread below:
Somewhat counterintuitively, in a human coronavirus, OC43, when it has the less efficient (“suboptimal”) RRSR furin cleavage site instead of the canonical RRSRR one, it is more lethal and more *neurovirulent* when tested in a mouse model:
But in a mouse hepatitis virus (MHV, one inspiration in DEFUSE) it is actually the JHM strain with the more efficient FCS, RRARR (reminiscent of RRAR in SARS2), that is *more* neuroinvasive and more lethal than the A59 strain with a suboptimal RRAHR FCS:
Excellent analysis of SARS2 cryptic lineages from persistent human infections (those nightmare scenarios happen when the virus colonizes your gut and remains there for months or years). Some implications for Covid origins there that I’d like to address in the thread below.
First an aside on extrapolating insertions found in SARS2 circulating in humans to the likelihood of insertions occurring (and getting fixed) in a SARS2 progenitor circulating in an intermediate host — SARS2 has now been circulating for years in hundreds of millions of humans, while in the wild it could not have had even a sliver of such a reservoir of non-bat hosts (and we all agree the FCS insertion could not have arisen in bats because it is detrimental to their preferred enteric tropism).
Of course, “a long time” for passaging in a lab could be just a few months, especially if we’re talking about in vitro passaging. Moreover, many novel mutations would arise quickly when the virus is put into cells from a novel host and organ system (respiratory vs. enteric).
🧵 Covid Origins: Lab leak critics claim the coincidence of a novel coronavirus emerging near the Wuhan Institute of Virology is dwarfed by the “counter-coincidence” of it first being noticed at a wildlife market. But the market was actually one of the most likely places for a lab-leaked virus to get noticed.
Here’s a clip from my podcast with @robertwrighter and a deep dive in the thread below:
Basically, if we imagine the map of Wuhan as a “probability landscape”, where each location has an inherent probability that a roaming lab-leaked virus would get noticed if it got there, the Huanan seafood market would dominate that landscape like Mount Everest.
Now, let’s walk through the thought experiment: How would a lab-leaked virus get noticed in Wuhan in late 2019?
Some critics of the Covid lab leak hypothesis say the SARS2 furin cleavage site is unlikely to have been engineered because in previous cases of creating novel FCSes in coronaviruses virologists have never inserted an FCS but rather created them in place via point mutations. But!
A close collaborator of Zhengli Shi and the Wuhan Institute of Virology, Shibo Jiang, in 2013 published a paper creating a furin cleavage site in a non-CoV synthetic vector via a 12-nt insertion (note the CGG codon for the leading arginine):
In 2016-17, WIV has created a novel reverse genetics system ("backbone") for the WIV1 virus, and Shibo Jiang subsequently coauthored a paper with Lei-Ping Zeng, the author of that backbone. (Lanying Du, another key collaborator of both WIV and Shibo Jiang, was also on the paper. She was also the editor for the 2013 paper inserting the RIRR FCS):
🚀 More amazing partial reprogramming news! This time from @davidasinclair — he mentioned some unpublished animal results for:
- *reversing* Alzheimer’s symptoms
- hearing loss
- ALS
- glaucoma (anticipating clinical trials in 2025!)
- rejuvenating skin, kidneys and liver
Great to hear that the FDA is ok with Tet-inducible (via rtTA3) partial reprogramming gene therapies, and that Life Bio is so close to the clinic. They presented encouraging results in monkeys in an eye stroke model (NIAON); I didn’t know they also tried a glaucoma monkey model.
PS: while Life Bio seems closest to human clinical trials among partial reprogramming companies, others aren’t far behind:
- Turn Bio with their skin therapy
- we at YouthBio with our Alzheimer’s therapy