Meta-Biol

• Pathways

After synthesizing the complementary minus RNA of the plus strand viral genomic RNA, virally encoded RNA-dependent RNA polymerase (nsp12, also known as RdRP), as part of the replication-transcription complex (RC), uses the minus strand as a template to generate viral genomic RNA that can be packaged into virions. SARS-CoV-2-derived nsp12, in complex with nsp7 and nsp8, was shown to have RNA polymerization activity on a poly-U template (Yin et al. 2020). Structures of actively replicating SARS-CoV-2 RdRP show no drastic rearrangements upon RNA binding, and that the active site is formed by highly conserved residues in nsp12 (reviewed by Hillen, 2021). Magnetic tweezer experiments lead to a description of the replication cycle as a series of bursts of nucleotide addition interrupted by pauses of various durations (Bera et al, 2021). There is also evidence for backtracking, the reversible sliding of polymerase along RNA or DNA, facilitated by the nsp13 helicase (Malone et al, 2021). Other details of SARS-CoV-2 replication are inferred from SARS-CoV-1. Purified SARS-CoV-1 nsp12 shows both primer dependent and primer-independent RNA synthesis activity in vitro. nsp12 is able to initiate RNA synthesis with as little as 37 nucleotides of RNA from the 3’ end of the minus strand viral RNA (complementary to the 5’-UTR of the plus strand genomic RNA - c5’-UTR). Similar to the 3'-UTR of the plus strand, the 3' end of the minus strand (c5’-UTR) is predicted to form a stable stem-loop structure and seems to be the minimal cis-acting RNA element required for nsp12 to initiate RNA synthesis using the minus strand as a template (Ahn et al. 2012). It is unclear if replication of the minus strand is primer-dependent. The complex of nsp7 and nsp8 confers processivity to nsp12 (Subissi et al. 2014).