Dissecting nucleotide selectivity in viral RNA polymerases

Abstract

Designing antiviral therapeutics is of great concern per current pandemics caused by novel coronavirus or SARS-CoV-2. The core polymerase enzyme in the viral replication/transcription machinery is generally conserved and serves well for drug target. In this work we briefly review structural biology and computational clues on representative single-subunit viral polymerases that are more or less connected with SARS-CoV-2 RNA dependent RNA polymerase (RdRp), in particular, to elucidate how nucleotide substrates and potential drug analogs are selected in the viral genome synthesis. To do that, we first survey two well studied RdRps from Polio virus and hepatitis C virus in regard to structural motifs and key residues that have been identified for the nucleotide selectivity. Then we focus on related structural and biochemical characteristics discovered for the SARS-CoV-2 RdRp. To further compare, we summarize what we have learned computationally from phage T7 RNA polymerase (RNAP) on its stepwise nucleotide selectivity, and extend discussion to a structurally similar human mitochondria RNAP, which deserves special attention as it cannot be adversely affected by antiviral treatments. We also include viral phi29 DNA polymerase for comparison, which has both helicase and proofreading activities on top of nucleotide selectivity for replication fidelity control. The helicase and proofreading functions are achieved by protein components in addition to RdRp in the coronavirus replication-transcription machine, with the proofreading strategy important for the fidelity control in synthesizing a comparatively large viral genome.

Keywords: Fidelity control; Kinetic modeling; Molecular dynamics (MD) simulation; Nucleotide selection; RNA dependent RNA polymerase (RdRp); RNA/DNA polymerase (RNAP/DNAP).

Fig. 1

The representative single-subunit viral polymerase structures and conserved motifs. (A) The RdRp structure (or 3D^pol) from Polio virus (PV; left; PDB:3OLB) , with fingers, palm, thumb subdomains shown in pink, blue, and green, respectively; and with motifs A-F shown (right), colored red for A, green for B, light green for C, blue for D, magenta for E, and purple for F (motif G not shown); (B) The RdRp structure (or NS5B) from hepatitis C virus (HCV; left; PDB:4WTA) , and motifs A-F (right); (C) The core structure of SARS-CoV2 RdRp (left; PDB:7BTF; shown for residues S367-F920, no N-terminal part) , and motifs A to F (right); (D) The structure of T7 RNAP (left; PDB:1S0V) , and the motifs A-D (right). (E) The structure of human mitochondrial RNAP (or POLRMT) (left; PDB:4BOC; similar to T7 RNAP) , and the motifs A-D (right); (F) The structure of phi29 DNAP (left; PDB:2PYL) , and the motifs A-C (right). (G) The molecular schematics of the triphosphate-form nucleotide analogs from remdesivir (RDV) and sofosbuvir (SBV) along with corresponding natural nucleotide substrates ATP and UTP, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

The key residues involved in NTP entry, NTP binding or selection, and catalysis in the individual viral polymerases. For clarity, the catalysis/NTP entry related residues are shown (left) separately from those involved in the NTP binding/selection (right). (A) The key residues shown for Polio virus (PV) RdRp (or 3D^pol; PDB:3OLB; the NTP binding/selection residues identified are shaded and detailed in text). (B) The key residues shown for HCV RdRp (NS5B; PDB:4WTA; the NTP entry residues identified are shaded and addressed in text). (C) The key residues shown for SARS-CoV-2 RdRp (PDB:7BTF; the conserved catalytic residues and the residues potentially involved in the delayed chain termination are shaded and addressed in text, along with the key residues to be involved in the NTP-entry, binding/selection). (D) The key residues shown for T7 RNAP, with the O-helix and Y-helix on the fingers subdomain highlighted (PDB:1S0V; the NTP binding/selection residues are addressed in the related work [10]). (E) The key residues shown for human mitochondrial (hmt) RNAP (or POLRMT), with the O-helix and Y-helix highlighted (PDB:4BOC; the residues are shown similarly as in T7 RNAP). (F) The key residues shown for phi29 DNAP, with a comparable helix from the fingers subdomain highlighted [residue 375–390 overlap with the motif B; PDB:2PYL; the proofreading/cleavage involved residues are shaded (with Asp to Ala mutations); other residues are addressed in text].