Effects of Epitranscriptomic RNA Modifications on the Catalytic Activity of the SARS‐CoV‐2 Replication Complex**

Abstract

SARS-CoV-2 causes individualized symptoms. Many reasons have been given. We propose that an individual's epitranscriptomic system could be responsible as well. The viral RNA genome can be subject to epitranscriptomic modifications, the modifications can be different for different individuals, and thus epitranscriptomics can affect many events including RNA replication differently. In this context, we studied the effects of modifications including pseudouridine (Ψ), 5methylcytosine (m 5 C), N 6 -methyladenosine (m 6 A), N 1 -methyladenosine (m 1 A) and N 3methylcytosine (m 3 C) on the activity of SARS-CoV-2 replication complex (SC2RC). We found that Ψ, m 5 C, m 6 A and m 3 C had little effects, while m 1 A inhibited the enzyme. Both m 1 A and m 3 C disrupt canonical base-pairing, but they had different effects. The fact that m 1 A inhibits SC2RC implies that the modification can be difficult to detect. The fact also implies that individuals with upregulated m 1 A including cancer, obesity and diabetes patients may have milder symptoms. However, this contradicts clinical observations. Relevant discussions are provided.

The Naturally Occurring m¹A RNA Modification Can Be Efficiently Incorporated into RNA by SARS-CoV-2 RdRp

“…From the above experiments and the data we published earlier regarding inhibition of SARS-CoV-2 RdRp by m 1 A in RNA template [8], it is clear that m 1 ATP based prodrugs may be investigated for the treatment of COVID-19. The reason is that once m 1 ATP is formed from a prodrug in SARS-CoV-2 infected cells, the viral RdRp could incorporate m 1 A into its RNA genome.…”

“…Besides m 1 A, there are over 300 epitranscriptomic RNA modifications [12][13][14]. Among them, we have found that m 3 C in RNA template does not inhibit SARS-CoV-2 RdRp [8], which was surprising considering that m 1 A inhibits the RdRp and both m 1 A and m 3 C disrupt canonical base pairing. However, even though m 3 C does not inhibit RdRp, it is unlikely that the nucleotide incorporated across it is precisely G due to G-C base pair disruption by the modification.…”

“…The difference in the modifications may affect viral life cycles by inhibiting or activating the RdRp, and as a result may contribute to the difference of clinical outcomes of COVID-19. Among other observations during the studies, we found that the m 1 A modification in an RNA template is capable to stop the RNA polymerization reaction catalyzed by SARS-CoV-2 RdRp [8]. With this finding, we were curious to know if m 1 A could be incorporated into RNA by the same RdRp via its triphosphate (m 1 ATP).…”

“…This observation has led to the invention of vaccines [4][5][6]. During the pandemic, driven by the curiosity to find additional causes for individualized symptoms of infectious diseases beyond those described in the literature such as induced immunity [7], we studied the effects of epitranscriptomic RNA modifications on the catalytic activity of SARS-CoV-2 RNA dependent RNA polymerase (RdRp) [8,9]. We reasoned that the epitranscriptomic systems of different individuals are not identical, and therefore they can modify the viral RNA genome differently.…”

The regulation of antiviral innate immunity through non-m6A RNA modifications

“…Viral infection has been shown to impact the expression levels of m 1 A writers and erasers on host mRNA, and some of them may have proviral effects ( 203 ). Intriguingly, upregulated m 1 A on host RNA inhibits the activity of the replication complex of SARS-COV-2 and thus achieves an antiviral effect, an effect that does not appear to be related to the steric hindrance caused by m 1 A ( 233 ). As for the impact on immune cells, research on m 1 A has mainly focused on cancer, such as its influence on immune cell infiltration ( 111 ).…”