Nonstructural proteins 7, 8 and 12 – replication complex

The complex formed by nsp7, 8, 9, 10, and 12 is responsible for the replication and transcription of the viral genome. Nsp12 encodes the RNA-dependent RNA polymerase (RdRp) domain. Nsp7 forms a hexadecameric complex with nsp8 that may act as a processivity clamp for the RNA polymerase. When compared to SARS-CoV-1, key functional residues are fully conserved in SARS-CoV-2, while all non-conservative substitutions are located in the complex surface. Several of these substitutions involve residues that  are potential sites for post-translational modifications, indicating a variation in post-translational patterns relative to the SARS-CoV-1 RNA polymerase complex.

Narrative

After infection the virus assembles a multi-subunit RNA-synthesis complex consisting of several nonstructural proteins (nsps), namely nsp7, 8, 9, 10, and 12 (Smith and Denison 2013). This complex is responsible for the replication and transcription of the viral genome. The complex of nsp12, 7, and 8 (total 160 kDa) constitutes the minimum set of nsps required for nucleotide polymerization (Kirchdoerfer and Ward 2019). Nsp12 encodes the RNA-dependent RNA polymerase (RdRp) domain. Nsp7 and nsp8 are nsp12 cofactors. Nsp7 forms a hexadecameric complex with nsp8 and together these may act as a processivity clamp for the RNA polymerase (Zhai et al. 2005). In the replication complex there are two well defined regions of nsp8: nsp8(I), forming the heterodimer with nsp7, and nsp8(II), binding to nsp12 (Kirchdoerfer and Ward 2019).

Structural analysis and comparison with SARS-CoV-1 nsp7-nsp8-nsp12 complex– A study conducted by Kirchdoerfer and Ward(2019) showed that the polymerase domain of SARS-CoV-1 nsp12 (a.a 398-919) consists of the fingers domain (a.a 398–581, 628–687), palm domain (a.a. 582–627, 688–815) and a thumb domain (a.a. 816–919). The Nidovirus-unique N-terminal extension (aka NiRAN) is situated between a.a. 1–397. There are two metal (Zn) binding sites in nsp12: The first is in the NiRAN extension and is coordinated by residues His²⁹⁵, Cys³⁰¹, Cys³⁰⁶, and Cys³¹⁰. The second site is in the fingers domain and is coordinated by Cys⁴⁸⁷, His⁶⁴²,Cys⁶⁴⁵, and Cys⁶⁴⁶. The binding site for the nsp7-nsp8 heterodimer overlaps with the conserved regions of polymerase functional domains (fingers (a.a 398–581, 628–687) and thumb domains (a.a. 816–919). The binding site between nsp8(II) and nsp12 is on the N-terminal region (77–126) of nsp8(II).

Several studies have pointed to functional residues in nsp7, 8, and 12 sequences. The study conducted by Lehmann et al. (2015), shows conserved sequence motifs in the NiRAN domain named A_N, B_N, and C_N. These motifs are conserved throughout all members of the order Nidovirales, of which CoVs are members (Lehmann et al. 2015). Another example is covalent modification of the Lys⁷³ residue in nsp12 (using GTP or UTP) that reduced viral growth and recovery in the equine arterivirus (Lehmann et al. 2015). In vitro polymerase activity assays further enabled the identification of other key functional sites, Lys⁷, His³⁶, and Asn³⁷, which, being replaced by alanine, are associated with decreased polymerase activity (Subissi et al. 2014). Four other mutations in nsp8 were detrimental to polymerase activity namely, Pro¹⁸³Ala, Asp⁹⁹Ala, Pro¹¹⁶Ala, and Arg¹⁹⁰Ala. These mutations are associated with a defective fold of nsp8 and disruption of nsp8(II)-nsp12 binding (Subissi et al. 2014).

The nucleoside analog GS-5734 is capable of impairing CoV RNA synthesis by targeting the viral RNA synthesis machinery. The side chain in a motif of the fingers domain in nsp12 is involved in GS-5734 interaction through Val⁵⁵⁷ residue (Agostini et al. 2018; C. J. Gordon et al. 2020).

All the mentioned residues are fully conserved in SARS-CoV-2. All non-conservative substitutions are located in the complex surface, mostly in nsp12. Several of these substitutions involve residue that are potential sites for post-translational modifications (e.g., cysteines, serines, threonines, asparagines and tyrosines), indicating a variation in post-translational patterns relative to the SARS-CoV-1 RNA polymerase complex.