Nonstructural protein 16 – 2’-O-ribose methyltransferase

The nonstructural protein 16 (nsp16) is involved in capping of viral mRNA to protect it from host degradation, and it has been demonstrated that it has to be associated with nsp10 to be active.   Mutation that increases hydrophobic interaction between nsp10 and the hydrophobic pocket of nsp16, increases methyltransferase activity of nsp16. Conversely, a mutation in the RNA binding site of nsp16, completely abolished its methyltransferase activity. All these key amino acids are conserved in SARS-CoV-2 nsp16, but non-conservative substitutions in their vicinity may have a steric effect.

Narrative

The nonstructural protein nsp16 is involved in capping of viral mRNA to protect it from host degradation, and it has been demonstrated that it has to be associated with nsp10 to be active. One potential method of ablating nsp16 activity is to disrupt the binding interface between nsp16 and nsp10. In SARS-CoV-1, the following interfacial mutations in nsp16 were shown to ablate nsp16 2’-O-methyltransferase activity: Ile⁴⁰Ala, Met⁴¹Ala, Val⁴⁴Ala, Val⁷⁸Ala, Arg⁸⁶Ala, Val¹⁰⁴Gly, Leu²⁴⁴Ala and Met²⁴⁷Ala.(Decroly et al. 2011) In a separate study, a double mutant (His⁸³Ala/Pro⁸⁴Ala) and a triple mutant (Tyr⁷⁶Ala/Cys⁷⁷Ala/Arg⁷⁸Ala) of nsp10 abolished SAM and m7GppA-RNA binding to nsp16 (Chen et al. 2011), further supporting the essential role of nsp10 in activating nsp16. In terms of the physicochemical interfacial properties that are essential for activating nsp16, it appears that certain hydrophobic interactions play a critical role and that enhancement of these may increase nsp16. This is exemplified by the Tyr⁹⁶Phe mutation in nsp10 that increases methyltransferase activity by increasing hydrophobic interaction between nsp10 and the hydrophobic pocket of nsp16, which includes the sidechain of Val⁸⁴ and the mainchain portions of Gln⁸⁷ and Arg⁸⁶ (Chen et al. 2011). In SARS-Cov-1, residue Tyr³⁰ in the RNA binding site of nsp16, when mutated to either alanine or phenylalanine, abolished methlyltransferase activity, suggesting the critical role of this residue in RNA binding and, consequently, the enzymatic function (Decroly et al. 2011). All these key amino acids are conserved in SARS-CoV-2 nsp16, but non-conservative substitutions in the vicinity of Tyr³⁰, namely Asn³³Ser and Val³⁵Thr may affect RNA binding, as well as conservative substitutions (Glu³²Adp and Ile³⁶Leu) that may have a steric effect.