Structural Basis of RNA Cap Modification by SARS-CoV-2 Coronavirus

Abstract

24The novel severe acute respiratory syndrome coronoavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused over 2 million infections worldwide in four months. In SARS coronaviruses, the non-structural protein 16 (nsp16) methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. 28We report here the high-resolution structure of a ternary complex of full-length nsp16 and nsp10 of SARS-CoV-2 in the presence of cognate RNA substrate and a methyl donor, S-adenosyl methionine. The nsp16/nsp10 heterodimer was captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We reveal large conformational 32 changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This structure provides new mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discovered a distantly located ligand-binding site unique to SARS-CoV-2 that may serve as an alternative target site for antiviral development.of the mechanisms that permit the virus to invade cells and evade host immune restriction. SARS-CoV-2 is an enveloped, positive-sense single-stranded β-coronavirus with a large, complex RNA genome 2 . To hijack the host translation machinery for propagation, enzymes encoded by the genome of coronaviruses (CoVs) modify the 5'-end of virally encoded mRNAs by creating a cap 3 . 44RNA capping in CoVs involves activities of several nonstructural proteins (nsps): nsp13, a bifunctional RNA/NTP triphosphatase (TPase) and helicase; nsp14, a bifunctional 3'à5' mismatch exonuclease and mRNA cap guanine-N7 methyltransferase; nsp16, a ribose 2'-O methyltransferase; and an elusive guanylyl transferase 4-7 . 48Nsp16 forms an obligatory complex with nsp10 to efficiently convert client mRNA species from the Cap-0 ( me7 GopppA1) to the Cap-1 form ( me7 GopppA1m), by methylating the ribose 2'-O of the first nucleotide (usually adenosine in CoVs) of the nascent mRNA using SAM (S-adenosyl 52 methionine) as the methyl donor 4, 8 . This Cap-1 form serves to avoid induction of the innate immune response 9-11 . Hence, ablation of nsp16 activity should trigger an immune response to CoV infection and limit pathogenesis 9, 10 . It has been shown that live vaccination with nsp16defective SARS-CoV-1 or an immunogenic disruption of the nsp16-nsp10 interface protects mice 56 from an otherwise lethal challenge 12, 13 , making nsp16/nsp10 an attractive drug target.Crystal structures of SARS-CoV-1 nsp16/nsp10 in complex with SAM/SAH or sinefungin, but without an RNA substrate, have been reported 4,8,14 , so that key information about the catalytic 60 mechanism of mRNA capping in CoVs, and SARS-CoV-2 in particular, is still missing. To understand the determinants of RNA cap modification and help guide the development of SARS-CoV-2 antiviral therapies, we have now succeeded in solving the high-resolution structure (to 1.8

A direct RNA-protein interaction atlas of the SARS-CoV-2 RNA in infected human cells

“…As shown in Figure 1b, we found five known RMRP components among the 10 most significantly enriched proteins in RMRP purifications (Supplementary Table 1). This is consistent with our previous results 13 As expected, the SARS-CoV-2 N protein, which is thought to occupy the majority of the viral RNA, was one of the two most strongly enriched viral proteins, followed by several known viral RNA-binders, such as the endoribonuclease NSP15 22 , the RNA-dependent RNA polymerase (RdRP) NSP12 23 , the methyltransferase NSP16 24 , the single-stranded RNAbinding protein NSP9 25 , the capping factor NSP10 24 , the primase NSP8 23 , and the multifunctional protein NSP3 26 . In addition to NSPs, we also found ORF3A, which binds the 5'-end of the SARS-CoV-1 genomic RNA 27 , as well as the Spike protein (S), and the Membrane protein (M) among strongly enriched proteins.…”

The folate antagonist methotrexate diminishes replication of the coronavirus SARS-CoV-2 and enhances the antiviral efficacy of remdesivir in cell culture models

“…In addition to purine synthesis, MTX also limits the transfer of C1 bodies (methyl groups) through MTHFR to regenerate S-adenosylmethionine (SAM), a major substrate for methyltransferases in the cell (Cronstein and Aune, 2020). Since SAM is required for the capping of the viral (as well as cellular) RNA (Viswanathan et al, 2020), this might further limit the production of infectious RNA. Indeed, the characterization of cellular metabolomics in response to SARS-CoV-2 infection, and the observed reduction in spermidine, at least suggested that SAM levels were 451 reduced in infected cells (Gassen et al, 2020).…”

How Signaling Games Explain Mimicry at Many Levels: From Viral Epidemiology to Human Sociology

“…i) Replication organelles, inside which the virus replicates [44], are a form of camouflage. ii) The addition of a cap-like structure onto the 5'end of viral mRNA by SARS-CoV-2 nsp16 [45], produces virus deceiver mRNAs, as discussed in Section 3.1. These are Batesian mimics of normal cellular mRNAs, which constitute a Müllerian mimicry ring, and is invaded by the viral deceiver mRNAs.…”