Polypeptide encoded by the -1 PRF signal of SARS-CoV-2 controls multiple enzymatic activities of the viral NiRAN domain.

The Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain is located amino-terminally to the RNA-dependent RNA polymerase (RdRp) in nonstructural protein (nsp)12 of SARS-CoV-2. The GDP-polyribonucleotidyltransferase (GDP-PRNT) and UMPylation activities of NiRAN contribute to viral RNA biogenesis by forming an RNA capping precursor and an RNA synthesis initiation primer, respectively. Translation of nsp12 is mediated by -1 programmed ribosomal frameshifting (-1 PRF), via an RNA signal composed of a slippery sequence and a downstream stimulator structure. The -1 PRF signal encodes a polypeptide, yet its function remains unexplored. Here, we show that the -1 PRF signal-encoded polypeptide is required for NiRAN activity. Mutational analysis reveals that a phenylalanine encoded by part of the slippery sequence, as well as two specific amino acids encoded by the stimulator, are essential for the UMPylation and GDP-PRNT activities of NiRAN. Thus, in addition to affecting the expression level of nsp12 through RNA-mediated frameshifting efficiency, variation in the -1 PRF signal influences functional viral RNA production through polypeptide-mediated modulation of NiRAN activity. This dual role likely constrains the allowable sequence variation of the -1 PRF signal during viral propagation, suggesting that the entire -1 PRF signal is a better antiviral target than the stimulator alone.