Pre-pandemic artificial MERS analog of pathogenic polyfunctional SARS-CoV-2 S1/S2 furin cleavage site domain is unique among betacoronaviruses

Abstract SARS-CoV-2 spike (S) glycoprotein furin cleavage site is a key determinant of SARS-CoV-2 virulence and COVID-19 pathogencity. Located at the S1/S2 junction, it is unique among sarbecoviruses but frequently found among betacoronaviruses. Recent evidence suggests that this site includes two additional functional motifs: a pat7 nuclear localization signal and two flanking O-glycosites. However, a systematic overview of spikes bearing this polyfunctional sequence domain has been missing. Here we report that among spike sequences of genus Betacoronavirus and outside of the SARS-CoV-2 clade an analogous domain was found in only one other virus: an artificial MERS infectious clone, described already in 2017, which was rationally selected from serial passage in genetically humanized mice. As the evolutionarily closest betacoronaviruses outside of the SARS-CoV-2 clade lack all its three functional motifs, this critical domain becomes an unlikely product of natural evolution alone, which extends the current view on SARS-CoV-2 origins, evolution and pathogenesis.