2′-O Methylation of the Viral mRNA Cap by West Nile Virus Evades Ifit1-Dependent and -Independent Mechanisms of Host Restriction In Vivo

Prior studies have shown that 2′-O methyltransferase activity of flaviviruses, coronaviruses, and poxviruses promotes viral evasion of Ifit1, an interferon-stimulated innate immune effector protein. Viruses lacking 2′-O methyltransferase activity exhibited attenuation in primary macrophages that was rescued in cells lacking Ifit1 gene expression. Here, we examined the role of Ifit1 in restricting pathogenesis in vivo of wild type WNV (WNV-WT) and a mutant in the NS5 gene (WNV-E218A) lacking 2′-O methylation of the 5′ viral RNA cap. While deletion of Ifit1 had marginal effects on WNV-WT pathogenesis, WNV-E218A showed increased replication in peripheral tissues of Ifit1 −/− mice after subcutaneous infection, yet this failed to correlate with enhanced infection in the brain or lethality. In comparison, WNV-E218A was virulent after intracranial infection as judged by increased infection in different regions of the central nervous system (CNS) and a greater than 16,000-fold decrease in LD50 values in Ifit1 −/− compared to wild type mice. Ex vivo infection experiments revealed cell-type specific differences in the ability of an Ifit1 deficiency to complement the replication defect of WNV-E218A. In particular, WNV-E218A infection was impaired in both wild type and Ifit1 −/− brain microvascular endothelial cells, which are believed to participate in blood-brain barrier (BBB) regulation of virus entry into the CNS. A deficiency of Ifit1 also was associated with increased neuronal death in vivo, which was both cell-intrinsic and mediated by immunopathogenic CD8+ T cells. Our results suggest that virulent strains of WNV have largely evaded the antiviral effects of Ifit1, and viral mutants lacking 2′-O methylation are controlled in vivo by Ifit1-dependent and -independent mechanisms in different cell types.
Author Summary We recently showed that a West Nile virus (WNV) mutant in NS5 (WNV-E218A) lacking 2′-O methyltransferase activity was attenuated in primary macrophages but replicated well in cells lacking type I interferon (IFN) signaling or expression of Ifit1, an IFN-stimulated gene. Here, we follow-up these studies by examining the pathogenesis in Ifit1−/− mice of WNV-E218A, the mutant virus lacking 2′-O methyltransferase activity. Because a deficiency of Ifit1 did not alter pathogenesis of wild type WNV, we conclude that the viral 2′-O methyltransferase encoded by NS5 largely overcomes Ifit1-mediated control of infection. In comparison, WNV-E218A showed increased infection in peripheral tissues of Ifit1 −/− mice after subcutaneous infection, yet this did not result in enhanced replication in the brain. However, WNV-E218A caused lethal infection when it was directly introduced into the brain. We observed cell-type specific differences in the ability of an Ifit1 deficiency to rescue replication of WNV-E218A; for example, WNV-E218A showed equivalently impaired infection in wild type and Ifit1 −/− brain endothelial cells, which potentially allow virus access into the brain. Our results suggest that virulent strains of WNV have evaded the antiviral effects of Ifit1, and mutants lacking 2′-O methylation are restricted by cell-type specific Ifit1-dependent and -independent mechanisms.