2'-O methylation of the viral mRNA cap evades host restriction by IFIT family members

Abstract

Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2'-O positions of the 5' guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2'-O methylation has remained uncertain since its discovery 35 years ago. Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2'-O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2'-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2'-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2'-O methylation of the 5' cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2'-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells.

Figure 1. WNV-E218A is attenuated in wild-type mice and cells but is virulent in Ifnar1^−/− mice and cells.

a, Survival curves of wild-type and Ifnar1^−/− C57BL/6 mice after subcutaneous infection with WNV-WT or WNV-E218A. b, Virus replication in wild-type mice in blood (day 4), spleen (day 4) or brain (day 8) after subcutaneous infection with WNV-WT or WNV-E218A. c, Survival curves of wild-type mice after intracranial infection with WNV-WT (10¹) or WNV-E218A (10⁵ plaque-forming units (PFU)). d, Viral burden in the serum, spleen, kidney, spinal cord and brain from Ifnar1^−/− mice at day 3 after infection. e, f, Replication of WNV-WT and WNV-E218A in wild-type or Ifnar1^−/− MEFs (e) or Mφ (f). Results are the average of three experiments performed in triplicate. Error bars, s.d.; dashed line, limit of sensitivity of the assay. PowerPoint slide

Figure 2. 2′-O methylation of viral RNA alters the sensitivity of WNV to the antiviral effects of IFN.

a, IFN-β gene induction in Ifnar1^−/− MEF after WNV-WT or WNV-E218A infection. Results are representative of three independent experiments performed in duplicate. b, Viral replication in IPS-1^−/− MEF after IFN-β pretreatment. The data are the average of two independent experiments performed in triplicate, and the asterisks indicate differences that are statistically significant (***P < 0.0001; **P < 0.005; *P < 0.05). Error bars, s.d. IU, international units. PowerPoint slide

Figure 3. WNV-E218A is more sensitive to the antiviral actions of Ifit genes.

a–c, Viral replication of WNV-WT or WNV-E218A in 3T3 MEFs transgenically expressing GFP (a–c), ISG20 (a), IFIT-2 (b) or IFIT-1 (c). The data are the average of three experiments performed in duplicate. d, siRNA knockdown of IFIT-2 enhances replication of WNV-E218A. 3T3 cells were transfected with a non-target (NT) or IFIT-2 siRNA and then infected with WNV-E218A. One day post-infection cells were collected and (top) viral RNA was assayed by quantitative reverse transcriptase PCR. The data are the average of three experiments performed in duplicate. Bottom, knockdown of IFIT-2 protein was confirmed by western blot. e, f, Murine IFIT-2 expression prevents accumulation of negative- and positive-strand viral RNA in WNV-E218A-infected cells. g, Replication of WNV-E218A is attenuated in wild-type and Ifit2^−/− Mφ but restored in Ifit1^−/− cells. h, Survival curves of wild-type or Ifit1^−/− mice after intracranial challenge with 10⁵ plaque-forming units of WNV-WT or WNV-E218A. Error bars, s.d.; dashed line, limit of sensitivity of the assay. PowerPoint slide

Figure 4. Poxvirus and coronavirus mutants lacking 2′-O methylation are more sensitive to the antiviral effects of murine IFIT-2.

a–d, Studies with VACV. a, Viral replication of VACV-WT or VACV-J3-K175R in wild-type or Ifnar1^−/− Mφ (a) or 3T3 MEF expressing GFP (b–d), ISG20 (b), Ifit2 (c) or Ifit1 (d). e, Viral replication of MHV-WT or MHV-D130A in 3T3 cells expressing GFP or IFIT-2. f, Viral replication of EMCV in 3T3 cells expressing GFP or IFIT-2. Error bars, s.d.; dashed line, limit of sensitivity of the assay. PowerPoint slide