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Dengue virus (DENV) is the most prevalent mosquito-borne virus pathogen in humans. There is currently no antiviral therapeutic or widely available vaccine against dengue infection. The DENV RNA genome is methylated on its 5′ cap by its NS5 protein. DENV bearing a single E216A point mutation in NS5 loses 2′-O-methylation of its genome. While this mutant DENV is highly attenuated and immunogenic, the mechanism of this attenuation has not been elucidated. In this study, we find that replication of this mutant DENV is attenuated very early during infection. This early attenuation is not dependent on a functional type I interferon response and coincides with early activation of the innate immune response. Taken together, our data suggest that 2′-O-methylation of DENV genomic RNA is important for evasion of the host immune response during the very early stages of infection as the virus seeks to establish infection. [ABSTRACT FROM AUTHOR]

Abstract: Methyltransferases (MTases) from the genus Flavivirus encode both N-7 and 2′-O activities needed for type 1 (m7GpppNm) cap structure formation. We performed kinetic studies to understand the mechanisms of its progressive N-7 and 2′-O methylations. Sequential N-7 to 2′-O methylation occurred via a random bi bi and processive mechanism that does not involve enzyme–RNA dissociation. Analyses of steady state kinetic parameters showed that N-7 precedes 2′-O methylation as it turnovers RNA faster (k cat) resulting in 2.4-fold higher catalytic efficiency. Michaelis constants for S-adenosyl-methionine (AdoMet) in both reactions were about 10-fold lower than for their respective RNA substrates, suggesting that the rate-limiting steps in methylase reactions were associated with RNA templates. In the context of long viral RNA sequences, and compared to S-adenosyl-homocysteine, sinefungin was about 60- and 12-folds more potent against dengue N-7 and 2′-O MTase activity, exhibiting IC50 values of 30 and 41nM, respectively. [Copyright &y& Elsevier]