Thread by @ydeigin

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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:

ncbi.nlm.nih.gov/pmc/articles/P…

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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.