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

Abstract

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.

Regional astrocyte IFN signaling restricts pathogenesis during neurotropic viral infection

“…VCAM-1-VLA-4 interactions facilitate immune trafficking during CNS infection (38,53). Although CNS immune infiltration is required for viral clearance following WNV infection (9,54), high levels of inflammatory cell infiltration are associated with pathology and bystander injury (11,45,55 (4). Immunohistochemical characterization of primary human and murine astrocytes using anti-S100-β (Abcam; ab41548) and anti-ALDH1A1 (Abcam; ab52492) antibodies in conjunction with nuclear stains (TO-PRO-3) revealed a greater than 99% purity.…”

A Role for Ifit2 in Restricting West Nile Virus Infection in the Brain

“…As IFIT genes are implicated in the recognition and control of flaviviruses, coronaviruses, and poxviruses lacking 2=-O methylation of the 5= viral RNA cap structures (17,18,36), we also tested whether an absence of Ifit2 affected pathogenicity of WNV-NS5-E218A. Previous studies established that this WNV mutant virus lacked 2=-O methyltransferase activity and was attenuated in wildtype mice (26) yet more virulent in Ifit1 Ϫ/Ϫ mice (17,18). Unexpectedly, WNV-NS5-E218A remained attenuated in Ifit2 Ϫ/Ϫ adult mice, as no illness or mortality was observed after subcutaneous or intracranial challenge (data not shown).…”

“…Initial experiments indicated that Ifit2 could restrict infection of several viruses by binding to and inhibiting subunits of eIF3, a key protein involved in initiation of host translation (14). More recent structural and functional studies suggest that IFIT family members also inhibit infection of cytoplasmic RNA and DNA viruses by directly binding nonself RNA, including moieties displaying 5=-ppp RNA (15,16) and possibly 5= cap 0 structures (7mGpppN) lacking 2=-O methylation (17,18). Ifit2, in particular, has been demonstrated to have antiviral activity in cell culture against WNV (19).…”

Recent advances in human flavivirus vaccines

“…T cells were also shown to play an important role in clearing WNV and limiting disease severity [26][27][28]. However, the protective role of T cells during infection has been questioned in several studies [28][29][30].…”