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

SARS-CoV-2 spike (S) glycoprotein furin cleavage site is a key functional determinant of SARS-CoV-2 virulence and COVID-19 pathogencity. Located at the S1/S2 junction, it is unique among sarbecoviruses but is also found among other betacoronaviruses. Recent evidence suggests that this proteolytic site extends to a ten amino acids long polyfunctional domain with two additional functional motifs: a pat7 nuclear localization signal (NLS) and two flanking O-glycosite Thr/Ser residues. However, a systematic analysis of coronavirus S protein sequences bearing this polyfunctional domain has been missing. Here we report that among spike protein sequences of genus Betacoronavirus and outside of the SARS-CoV-2 clade an analogous polyfunctional domain was found in only one other virus: an artificial MERS infectious clone, described already in 2017, which constitutes an adapted and rationally selected genotype from serial passage in genetically humanized mice. In contrast to this artificial MERS, which has no natural primary host and which during passage acquired the full pat7 motif, S protein sequences from all other betacoronaviruses did not present a polyfunctional S1/S2 junction domain analogous to SARS-CoV-2. As the evolutionarily closest betacoronaviruses outside of the SARS-CoV-2 clade lack all its three functional features, this critical S1/S2 polyfunctional domain becomes an unlikely product of natural evolution alone. This evidence implicates a possible artificial origin of SARS-CoV-2 and extends the current view on its pathogenesis.