Slack / Private Message Drop — page 270

previously, computational prediction of O-linked glycosylation sites does not ensure that these sites are utilized by SARS-CoV-2 (Andersen et al., 2020). The sites may be used only in some cell types or under specific conditions or not utilized at all. This caveat also applies to the current analyses. However, the frequency that the O-glycosylation sites are predicted across spike proteins from various genuses suggests that their presence in not due to chance. Mucin-like domains which contain O-linked glycans are characterized by an abundance of serines, theonines and prolines. These amino acids form turns in protein structures. Turns potentially contribute to accessibility of furin cleavage sites, and therefore may be under positive selection. The pattern of proline and serine/threonine residues in or near insertions of the polybasic residues, as noted in SARS-CoV-2, MERS-CoV, HKU1 and Type I feline coronaviruses, has not to our knowledge been previously discussed. With notable exceptions, including a predicted mucin-like patch in the carboxy terminal domain of embecovirus spikes, predicted O-glycans are rare in coronavirus spikes other than near cleavage sites. While computational algorithms such as Net-O-Glyc are capable of determining sites that are likely to be O-glycosylated, this pattern would not have been obvious to anyone constructing SARS-CoV-2 in a laboratory, either for gain-of-function research or nefarious purposes. Previously one of us (WRG) presented additional strong evidence that the furin cleavage site insertion in SARS-CoV2 was generated via a natural process (Gallaher, 2020c). Although the 12 base insertion preserves the reading frame, the insertion is out-of-frame. It is highly implausible that any scientist attempting to insert a furin cleavage site would do so by making an out-of-frame insertion. Previous studies that introduced furin cleavage sites in SARS-CoV and MERS-CoV spike genes did not introduce insertions (Follis et al., 2006; Yang et al., 2015). Nor is it likely that any laboratorian would have engineered a change in the SARS-CoV-2 spike that purposefully resulted in prediction of O-linked glycan sites. [2020-11-21 07:38:13] [Robert Garry] Slightly different subject, but looking forward to an updated Nidovirus phylogenic tree with some of Eddie and company's new viruses. [2020-11-21 07:39:55] [Kristian Andersen] Bob, great post - get it on there! Now! [2020-11-21 07:40:46] [Kristian Andersen] Andrew, yes, thanks for sharing - great points in there. "Did the acquisition of the ACE2 RBD make the virus more species-promiscuous and thus facilitated emergence into farmed carnivores like raccoon-dogs/pangolins? High density transmission in carnivores may have evolved the FCS - i.e., like chickens & high-path AIV." I actually think that's due to the FCS - it's been shown before that FCS increases the hostrange of other coronaviruses, including MERS-like - one reference: https://pubmed.ncbi.nlm.nih.gov/31801868/. Key sentence here: "Together, these results indicate that proteolytic cleavage of the spike, not receptor binding, is the primary infection barrier for these two group 2c CoVs". [2020-11-21 07:40:56] [Robert Garry] some of the serpentoviruses and nonCoV nidos have interesting mucin-like domains [2020-11-21 07:44:05] [Kristian Andersen] The O-linked glycans very likely regulates the FCS site - I don't think it's a mucin-like domain [2020-11-21 07:45:37] [Robert Garry] agree - thou with some of the other Nido spikes there are more than a dozen o-linked sites so mucin-like [2020-11-21 07:48:16] [Kristian Andersen]