Slack / Private Message Drop — page 269

The _Merbecovirus_ subgenus of the _Betacoronaviridae_ includes bat coronaviruses HKU4 and HKU5 as well as Middle Eastern Respiratory Syndrome corononavirus (MERS-CoV), which infects camels and humans. Phylogenic analyses place HKU4 at a basal position leading separately to the HKU5 and MERS-CoV lineages (Lau et al., 2013). Relative to the HKU4 spike, the HKU5 spike has an insertion of 3 amino acids (RFR) at the S1/S2 junction (Fig. 1B). This insertion generates an optimal furin cleavage site (RFRR). The S1/S2 junction of the HKU5 spike is predicted to contain two O-linked glycans. Relative to HKU4, the MERS-CoV spike displays an insertion of 6 amino acids (LTPRSV). The insertion in the MERS-CoV spike produces a minimal furin cleavage site (RSVR), albeit without predicted O-linked glycans. The _Embecovirus_ subgenus of the _Betacoronaviridae_ includes the seasonal coronaviruses OC43 and HKU1. Of additional importance is Betacoronavirus 1, an embecovirus notable for its spread to a large number of diverse animal species (Corman et al., 2018). Betacoronavirus 1 is represented here by a bovine coronavirus. The rat embecovirus HKU24 is ancestral to each of these viruses (Lau et al., 2015). HKU24, OC43 and Betacoronavirus 1 spikes have optimal furin cleavage sites (Fig. 1A). The junctional sequences in Betacoronavirus 1 and OC43 spikes are predicted to contain 4 and 2 O-linked glycans, respectively. Relative to HKU24 spike some variants of HKU1 spike have an insert of 6 amino acids (PSSSS) near the S1/S2 junction. Other variants have inserts of 2 amino acids (PS). It is unclear whether the HKU1 spike inserts have occurred independently or sequentially. HKU1 S1/S2 junctions are predicted to contain 2 or 3 O-linked glycans. Mouse hepatitis virus (MHV), a well-studied embecovirus, further illustrates the natural variability in the S1/S2 junction of betacornavirus spikes, with examples of monobasic, minimal furin and optimal furin cleavage sites existing in spike of various isolates (Fig. 1B). As with certain other betacoronaviruses, various serine and threonine residues at the MHV spike S1/S2 junctions are predicted O-linked glycosylation sites. The MHV spike, like other coronavirus spikes undergoes additional proteolytic cleavages, including cleavage at a site referred to as S2' (Belouzard et al., 2009; Millet and Whittaker, 2014). Cleavage at S2' exposes a fusion peptide that interacts with a host cell membrane permitting fusion with the viral envelope. Compared to the spike of ancestral embecovirus HKU24 the S2' site of MHV spike contains insertions of variable lengths, although deletions may also contribute to variability at the S2' site in spike of different MHV variants. Furin cleavage sites are present in spikes of other coronaviruses. The S1/S2 junctions of feline alphacoronavirus spikes bear similarities to the corresponding region in sarbeco- and merbecovirus spikes. Feline alphacoronaviruses are divided into two different types (Jaimes et al., 2020). Type I feline coronavirus spikes have an optimal furin cleavage site, whereas Type II feline coronavirus spikes lack either a monobasic or polybasic cleavage site at the S1/S2 junction (Fig. 1C). Relative to Type II feline coronavirus spikes, the optimal furin cleavage sites in Type I spikes are included in 17 or 18 amino acid insertion at the S1/S2 junction. The modification adds 2 or 3 predicted O-linked glycosylation sites. Type I feline coronavirus spikes have a monobasic cleavage site at S2', whereas feline coronaviruses have three amino acid indel at S2'. In some cases (for example strain KUK-HL) the S2' junction contains a minimal furin cleavage site. Gammacoronavirus spikes, including infectious bursal disease virus contain optimal furin cleavage sites at the S1/S2 junction (Fig. 1D). Some avian gammacoronavirus spikes have predicted O-linked glycans at the S1/S2 cleavage site. Avian gammacoronavirus spikes display sequence variability at the S2' cleavage site; most are monobasic, but infectious bursal disease strain Beaudette spike has acquired an optimal furin cleavage site. The S2' site of Infectious bursal disease virus variant SES_15SK spike provides an example of a predicted O-linked glycosylation site that is associated with a monobasic cleavage site. Because the evolutionary history of gammacoronaviruses is not well-described, it cannot be inferred whether or not evolution of their S1/S2 or S2' spike cleavage sites has involved insertions or deletions. *Discussion* Furin cleavage sites have been generated naturally in the spike proteins of members of at least 3 of 4 orthocoronavirus genuses, including three betacoronavirus subgenuses, via insertions and/or deletions. Both the S1/S2 and S2' junction s of coronavirus spike genes are hot spots for RNA recombination. Furthermore, deletions, but not insertions, in the S1/S2 junction appear to arise commonly on serial passage of SARS-CoV-2 in cell culture, and have also been detected as quasi-species in infected humans (Lau et al., 2020; Liu et al., 2020). These observation stand in direct contrast to proponents of theories that SARS-CoV-2 has a nonnatural origin. A recent analysis by one of us (WRG) suggests that this variability is facilitated by short oligonucleotide "breakpoint sequences" that direct recombination to certain positions in the genome (Gallaher, 2020b). Further evidence for the natural origin of the furin cleavage site in the SARS-CoV-2 spike is the observation that the indels in other coronavirus spikes often encode sequences with a propensity for O-linked glycosylation. As noted