Binding of the Methyl Donor S-Adenosyl-l-Methionine to Middle East Respiratory Syndrome Coronavirus 2'-O-Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10

Aouadi W, et al., 91(5). pii: e02217-16, J Virol, 2017

Nonstructural protein 16 (nsp16) of Middle East respiratory syndrome coronavirus (MERS-CoV) is an S-adenosyl-L-methionine (SAM)-dependent 2'-O-methyltransferase (2'-O-MTase). Nsp 16 transforms virus cap-0 into cap-1 structure by methylating the ribose 2'-OH of the first transcribed nucleotide (N1) of viral RNA cap-0 structures, which blocks virus detection by cell innate immunity mechanisms. Reported herein are the biochemical characterization of nsp16 and the demonstration of nsp10 as a cofactor required for nsp16 2'-O-MTase enzymatic activity. Kinetic analysis of nsp10-nsp16 interaction was carried out using the Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED96 system equipped with Streptavidin-coated Biosensor probes was used in all BLI experiments. Biotinylated nsp10 was immobilized onto Streptavidin Biosensor tips. Association and dissociation of nsp16 was studied over various concentrations of nsp16. kon, koff and KD values were determined for the binding interactions. All binding curves were fitted using a 1:1 binding model. The steady-state curve of nsp10-nsp16 interaction was fitted using a site-specific binding equation. The effects of SAM and SAH on the off-rates of nsp16 were also evaluated. Overall results of this study might lead to the development of molecular inhibitors with an ability to target cap 2'-O-methylations and to restore the host antiviral response.

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