The Middle East respiratory syndrome coronavirus (MERS-CoV) nonstructural protein 16 (nsp16) is an S-adenosyl-L-methionine (SAM)-dependent 2=-Omethyltransferase (2=-O-MTase) that is thought to methylate the ribose 2=-OH of the first transcribed nucleotide (N 1 ) of viral RNA cap structures. This 2=-O-MTase activity is regulated by nsp10. The 2=-O methylation prevents virus detection by cell innate immunity mechanisms and viral translation inhibition by the interferon-stimulated IFIT-1 protein. To unravel the regulation of nsp10/nsp16 2=-O-MTase activity, we used purified MERS-CoV nsp16 and nsp10. First, we showed that nsp16 recruited N7-methylated capped RNA and SAM. The SAM binding promotes the assembly of the enzymatically active nsp10/nsp16 complex that converted 7m GpppG (cap-0) into 7m GpppG 2=Om (cap-1) RNA by 2=-OH methylation of N 1 in a SAM-dependent manner. The subsequent release of SAH speeds up nsp10/nsp16 dissociation that stimulates the reaction turnover. Alanine mutagenesis and RNA binding assays allowed the identification of the nsp16 residues involved in RNA recognition forming the RNA binding groove (K46, K170, E203, D133, R38, Y47, and Y181) and the cap-0 binding site (Y30, Y132, and H174). Finally, we found that nsp10/nsp16 2=-O-MTase activity is sensitive to known MTase inhibitors, such as sinefungin and cap analogues. This characterization of the MERS-CoV 2=-O-MTase is a preliminary step toward the development of molecules to inhibit cap 2=-O methylation and to restore the host antiviral response.IMPORTANCE MERS-CoV codes for a cap 2=-O-methyltransferase that converts cap-0 into cap-1 structure in order to prevent virus detection by cell innate immunity mechanisms. We report the biochemical properties of MERS-CoV 2=O-methyltransferase, which is stimulated by nsp10 acting as an allosteric activator of the nsp16 2=-Omethyltransferase possibly through enhanced RNA binding affinity. In addition, we show that SAM promotes the formation of the active nsp10/nsp16 complex. Conversely, after cap methylation, the reaction turnover is speeded up by cap-1 RNA release and nsp10/ nsp16 complex dissociation, at the low intracellular SAH concentration. These results suggest that SAM/SAH balance is a regulator of the 2=-O-methyltransferase activity and raises the possibility that SAH hydrolase inhibitors might interfere with CoV replication cycle. The enzymatic and RNA binding assays developed in this work were also used to identify nsp16 residues involved in cap-0 RNA recognition and to understand the action mode of known methyltransferase inhibitors.KEYWORDS RNA processing, RNA virus, biochemistry

The Flavivirus Zika virus (ZIKV) is the causal agent of neurological disorders like microcephaly in newborns or Guillain-Barre syndrome. Its NS5 protein embeds a methyltransferase (MTase) domain involved in the formation of the viral mRNA cap. We investigated the structural and functional properties of the ZIKV MTase. We show that the ZIKV MTase can methylate RNA cap structures at the N-7 position of the cap, and at the 2=-O position on the ribose of the first nucleotide, yielding a cap-1 structure. In addition, the ZIKV MTase methylates the ribose 2=-O position of internal adenosines of RNA substrates. The crystal structure of the ZIKV MTase determined at a 2.01-Å resolution reveals a crystallographic homodimer. One chain is bound to the methyl donor (S-adenosyl-L-methionine [SAM]) and shows a high structural similarity to the dengue virus (DENV) MTase. The second chain lacks SAM and displays conformational changes in the ␣X ␣-helix contributing to the SAM and RNA binding. These conformational modifications reveal a possible molecular mechanism of the enzymatic turnover involving a conserved Ser/Arg motif. In the second chain, the SAM binding site accommodates a sulfate close to a glycerol that could serve as a basis for structure-based drug design. In addition, compounds known to inhibit the DENV MTase show similar inhibition potency on the ZIKV MTase. Altogether these results contribute to a better understanding of the ZIKV MTase, a central player in viral replication and host innate immune response, and lay the basis for the development of potential antiviral drugs. IMPORTANCE The Zika virus (ZIKV) is associated with microcephaly in newborns,and other neurological disorders such as Guillain-Barre syndrome. It is urgent to develop antiviral strategies inhibiting the viral replication. The ZIKV NS5 embeds a methyltransferase involved in the viral mRNA capping process, which is essential for viral replication and control of virus detection by innate immune mechanisms. We demonstrate that the ZIKV methyltransferase methylates the mRNA cap and adenosines located in RNA sequences. The structure of ZIKV methyltransferase shows high structural similarities to the dengue virus methyltransferase, but conformational specificities highlight the role of a conserved Ser/Arg motif, which participates in RNA and SAM recognition during the reaction turnover. In addition, the SAM binding site accommodates a sulfate and a glycerol, offering structural information to initiate structure-based drug design. Altogether, these results contribute to a better understanding of the Flavivirus methyltransferases, which are central players in the virus replication.KEYWORDS methyltransferase, RNA processing, Zika virus, flavivirus, protein structure-function