Amino Acid Polymorphisms on the Brazilian Strain of Yellow Fever Virus Methyltransferase Are Related to the Host's Immune Evasion Mediated by Type I Interferon.

Since late 2016, a yellow fever virus (YFV) variant carrying a set of nine amino acid variations has circulated in South America. Three of them were mapped on the methyltransferase (MTase) domain of viral NS5 protein. To assess whether these changes affected viral infectivity, we synthesized YFV carrying the MTase of circulating lineage as well as its isoform with the residues of the previous strains (NS5 K101R, NS5 V138I, and NS5 G173S). We observed a slight difference in viral growth properties and plaque phenotype between the two synthetic YFVs. However, the MTase polymorphisms associated with the Brazilian strain of YFV (2016-2019) confer more susceptibility to the IFN-I. In addition, in vitro MTase assay revealed that the interaction between the YFV MTase and the methyl donor molecule (SAM) is altered in the Brazilian MTase variant. Altogether, the results reported here describe that the MTase carrying the molecular signature of the Brazilian YFV circulating since 2016 might display a slight decrease in its catalytic activity but virtually no effect on viral fitness in the parameters comprised in this study. The most marked influence of these residues stands in the immune escape against the antiviral response mediated by IFN-I.

Structure and Sequence Requirements for RNA Capping at the Venezuelan Equine Encephalitis Virus RNA 5' End.

Venezuelan equine encephalitis virus (VEEV) is a reemerging 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 nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The capping reaction starts with the methylation of GTP. The generated m7GTP is complexed to the enzyme to form an 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, m7GpppC, or m7GpppU. We show that capping is more efficient on small RNA molecules, whereas RNAs 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. IMPORTANCE The emergence, reemergence, and expansion of alphaviruses (genus of the family Togaviridae) are 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 or safe antivirals are available for human use. VEEV nonstructural protein 1 (nsP1) is the viral capping enzyme characteristic of the Alphavirus genus. nsP1 catalyzes 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.

Approved drugs screening against the nsP1 capping enzyme of Venezuelan equine encephalitis virus using an immuno-based assay.

Alphaviruses such as the Venezuelan equine encephalitis virus (VEEV) are important human emerging pathogens transmitted by mosquitoes. They possess a unique viral mRNA capping mechanism catalyzed by the viral non-structural protein nsP1, which is essential for virus replication. The alphaviruses capping starts by the methylation of a GTP molecule by the N7-guanine methyltransferase (MTase) activity; nsP1 then forms a covalent link with m7GMP releasing pyrophosphate (GT reaction) and the m7GMP is next transferred onto the 5'-diphosphate end of the viral mRNA to form a cap-0 structure. The cap-0 structure decreases the detection of foreign viral RNAs, prevents RNA degradation by cellular exonucleases, and promotes viral RNA translation into proteins. Additionally, reverse-genetic studies have demonstrated that viruses mutated in nsP1 catalytic residues are both impaired towards replication and attenuated. The nsP1 protein is thus considered an attractive antiviral target for drug discovery. We have previously demonstrated that the guanylylation of VEEV nsP1 can be monitored by Western blot analysis using an antibody recognizing the cap structure. In this study, we developed a high throughput ELISA screening assay to monitor the GT reaction through m7GMP-nsP1 adduct quantitation. This assay was validated using known nsP1 inhibitors before screening 1220 approved compounds. 18 compounds inhibiting the nsP1 guanylylation were identified, and their IC50 determined. Compounds from two series were further characterized and shown to inhibit the nsP1 MTase activity. Conversely, these compounds barely inhibited a cellular MTase demonstrating their specificity towards nsP1. Analogues search and SAR were also initiated to identify the active pharmacophore features. Altogether the results show that this HT enzyme-based assay is a convenient way to select potent and specific hit compounds targeting the viral mRNA capping of Alphaviruses.

mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research.

UNLABELLED: Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of an m(7)GMP-nsP1 adduct. Subsequent transfer of m(7)GMP onto the 5' end of the viral mRNA has not been demonstrated in vitro yet. Here we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalyzed by nsP1. The MTase and GT reaction activities were followed using a nonhydrolyzable GTP (GIDP) substrate and an original Western blot assay using anti-m3G/m(7)G-cap monoclonal antibody, respectively. The GT reaction is stimulated by S-adenosyl-l-homocysteine (Ado-Hcy), the product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m(7)GMP involves a phosphamide bond between the nucleotide and a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5'-diphosphate RNA oligonucleotide whose sequence corresponds to the 5' end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, and N375 revealed their respective roles in MT and GT reactions. Finally, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA), and ribavirin triphosphate on MTase and capping reactions were investigated, providing possible avenues for antiviral research. IMPORTANCE: Emergence or reemergence of alphaviruses represents a serious health concern, and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reaction sequence, to add to their mRNA a cap required for their protection against cellular nucleases and initiation of viral proteins translation. In this study, the capping of a 5' diphosphate synthetic RNA mimicking the 5' end of an alphavirus mRNA was observed in vitro for the first time. The different steps for this capping are performed by the nonstructural protein 1 (nsP1). Reference compounds known to target the viral capping inhibited nsP1 enzymatic functions, highlighting the value of this enzyme in antiviral development.