A conserved motif within the NSP2 of SARS-CoV-2 is required for processing of the distal NSP1/NSP2 junction by NSP3

In 2019, the severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) started to spread globally and caused the COVID-19 pandemic. SARS-CoV-2, like other members of the Coronaviridae, has a single-stranded, positive sense RNA genome about 30 kb in length, which is translated to generate 16 non-structural proteins (NSPs); a set of sub-genomic mRNAs encode the structural and accessory proteins. The ORF1a precursor includes NSP1-11 and is processed by virus-encoded proteases to produce the mature proteins. We recently identified a short, highly conserved motif (YCPRP) within the structural protein precursor of foot-and-mouth disease virus (FMDV), a member of the Picornaviridae. This motif is conserved among picornaviruses and is found as (W/F/Y)-x-P-R-(P/A). The motif has a major influence on the processing of the FMDV capsid precursor (P1-2A) by the viral protease 3C^pro. We have now identified a similar motif (WVPRA) within the NSP2 of SARS-CoV-2. Interestingly, this motif is required for the efficient processing of the NSP1-NSP2 junction by the SARS-CoV-2 protease PL^pro (NSP3) and a single amino acid substitution within the motif can abrogate cleavage of this junction. We hypothesise that this motif acts, within NSP1-NSP2, to enable this precursor to fold correctly and allow efficient processing of the NSP1/NSP2 junction.