Effects of natural RNA modifications on the activity of SARS‐CoV‐2 RNA‐dependent RNA polymerase

Abstract

RNA‐dependent RNA polymerase (RdRp) plays a key role in the replication of RNA viruses, including SARS‐CoV‐2. Processive RNA synthesis by RdRp is crucial for successful genome replication and expression, especially in the case of very long coronaviral genomes. Here, we analysed the activity of SARS‐CoV‐2 RdRp (the nsp12–nsp7–nsp8 complex) on synthetic primer–templates of various structures, including substrates with mismatched primers or template RNA modifications. It has been shown that RdRp cannot efficiently extend RNA primers containing mismatches and has no intrinsic RNA cleavage activity to remove the primer 3′‐end, thus necessitating the action of exoribonuclease for proofreading. Similar to DNA‐dependent RNA polymerases, RdRp can perform processive pyrophosphorolysis of the nascent RNA product but this reaction is also blocked in the presence of mismatches. Furthermore, we have demonstrated that several natural post‐transcriptional modifications in the RNA template, which do not prevent complementary interactions (N6‐methyladenosine, 5‐methylcytosine, inosine and pseudouridine), do not change RdRp processivity. At the same time, certain modifications of RNA bases and ribose residues strongly block RNA synthesis, either prior to nucleotide incorporation (3‐methyluridine and 1‐methylguanosine) or immediately after it (2'‐O‐methylation). The results demonstrate that the activity of SARS‐CoV‐2 RdRp can be strongly inhibited by common modifications of the RNA template suggesting a way to design novel antiviral compounds.

Trapping non-cognate nucleotide upon initial binding for replication fidelity control in SARS-CoV-2 RNA dependent RNA polymerase

“…In addition, experimental work on the nsp14 exonuclease enzyme of SARS-CoV-2 has shown that for excision of an incorrect nt, the nt needs to have a proper RNA sugar, 2’ and 3’ OH functional group [78], indicating that the enzyme may require some nucleotide selectivity to prevent dNTP’s from chemical incorporation. Furthermore, recent experimental studies have shown that an elongation complex soaked in solution with dNTPs had no catalytic activity [79]. Michaelis–Menten kinetics also showed the SARS-CoV-2 RdRp selectivity of ATP over dATP is ∼ 1000 ( = 0.02 in dATP and 23 in ATP) [80].…”

Trapping a non-cognate nucleotide upon initial binding for replication fidelity control in SARS-CoV-2 RNA dependent RNA polymerase

“…Furthermore, recent experimental studies have shown that an elongation complex soaked in solution with dNTPs had no catalytic activity. 89 Michaelis-Menten kinetics also showed the SARS-CoV-2 RdRp selectivity of ATP over dATP is B1000 ( V max K m ¼ 0:02 in dATP and 23 in ATP). 90 Similar trends were observed in PV in vitro biochemical studies with B117 discrimination factor…”

Safety and immunogenicity of VLPCOV-02, a SARS-CoV-2 self-amplifying RNA vaccine with a modified base, 5-methylcytosine

“…Most importantly, a nucleoside-modified base, 5-methylcytosine (5-MeC) was incorporated to reduce innate responses, resulting in reduced reactogenicity. 15 , 16 N1-Methylpseudouridine was utilized in existing mRNA vaccines to reduce the innate responses 17 , 18 ; however, N1-methylpseudouridine appears to inhibit the VEEV RNA-dependent RNA polymerase or transcriptase function. Therefore, innate responses were minimized in VLPCOV-02 by incorporation of 5-MeC, an approach supported by both preclinical in vitro and animal model observations.…”