Structure and sequence requirements for RNA capping at the Venezuelan Equine Encephalitis Virus RNA 5'-end.

Venezuelan equine encephalitis virus (VEEV) is a re-emerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5'-end. The capping process is performed by the non-structural protein nsP1, which bears methyl and guanylyltransferases activities. The capping reaction starts by the methylation of GTP. The generated m7GTP is complexed to the enzyme to form a m7GMP-nsP1 covalent intermediate. The m7GMP is then transferred onto the 5'-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m7GMP to yield m7GpppA,C, or U. We show that capping is more efficient on small RNA molecules whereas RNA longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short conserved stem loop, downstream to the cap, is an essential regulatory element for the capping process.IMPORTANCEThe emergence, the re-emergence and the expansion of alphaviruses (genus of the family Togaviridae) is a serious public health and epizootic threat. Venezuelan equine encephalitis virus (VEEV) causes encephalitis in human and domesticated animals, with a mortality rate reaching 80% in horses. To date no efficient vaccine nor safe antivirals are available for human use. VEEV non structural protein 1 (nsP1) is the viral capping enzyme characteristic of the alphavirus genus. NsP1 catalyses methyltransferase and guanylyltransferase reactions, representing a good therapeutic target. In the present report, we provide insights into the molecular features and specificities of the cap acceptor substrate for the guanylylation reaction.