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. 2012 May;86(10):5708-18.
doi: 10.1128/JVI.00207-12. Epub 2012 Feb 29.

West Nile virus noncoding subgenomic RNA contributes to viral evasion of the type I interferon-mediated antiviral response

Andrea Schuessler  1 , Anneke Funk, Helen M Lazear, Daphne A Cooper, Shessy Torres, Stephane Daffis, Babal Kant Jha, Yutaro Kumagai, Osamu Takeuchi, Paul Hertzog, Robert Silverman, Shizuo Akira, David J Barton, Michael S Diamond, Alexander A Khromykh
Affiliations

West Nile virus noncoding subgenomic RNA contributes to viral evasion of the type I interferon-mediated antiviral response

Andrea Schuessler et al. J Virol. 2012 May.

Abstract

We previously showed that a noncoding subgenomic flavivirus RNA (sfRNA) is required for viral pathogenicity, as a mutant West Nile virus (WNV) deficient in sfRNA production replicated poorly in wild-type mice. To investigate the possible immunomodulatory or immune evasive functions of sfRNA, we utilized mice and cells deficient in elements of the type I interferon (IFN) response. Replication of the sfRNA mutant WNV was rescued in mice and cells lacking interferon regulatory factor 3 (IRF-3) and IRF-7 and in mice lacking the type I alpha/beta interferon receptor (IFNAR), suggesting a contribution for sfRNA in overcoming the antiviral response mediated by type I IFN. This was confirmed by demonstrating rescue of mutant virus replication in the presence of IFNAR neutralizing antibodies, greater sensitivity of mutant virus replication to IFN-α pretreatment, partial rescue of its infectivity in cells deficient in RNase L, and direct effects of transfected sfRNA on rescuing replication of unrelated Semliki Forest virus in cells pretreated with IFN-α. The results define a novel function of sfRNA in flavivirus pathogenesis via its contribution to viral evasion of the type I interferon response.

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Figures

Fig 1
Fig 1
Replication of sfRNA-deficient WNVKUN is rescued in IRF-3−/− × IRF-7−/− MEFs. (A) Northern blot of viral RNA using a 3′ UTR-specific probe in wt and IRF-3−/− × IRF-7−/− MEFs infected at an MOI of 1 with wt (FLSDX) and sfRNA-deficient (FL-IRAΔCS3) viruses. Ethidium bromide staining of rRNA is included as a loading control. (B) Corresponding titers of infectious virus in the supernatant of the infected cells. The results are the average of two (wt MEFs) or four (IRF-3−/− × IRF-7−/− MEFs) independent experiments. Error bars indicate standard deviations. (C) Virus titers in the supernatants of IRF-3−/− × IRF-7−/− MEFs infected at an MOI of 0.1 and 0.01 with wt (FLSDX) or sfRNA-deficient (FL-IRAΔCS3) virus.
Fig 2
Fig 2
sfRNA-deficient virus is more sensitive to IFN-α, and its replication is rescued by neutralizing antibodies to IFNAR. (A) Northern blot of viral RNA in IRF-3−/− × IRF-7−/− MEFs infected with wt (FLSDX) or sfRNA-deficient (FL-IRAΔCS3) virus after pretreatment with IFN-α (0 to 1,000 IU/ml for 8 h prior to infection at an MOI of 1). Supernatants and cells were harvested at 48 hpi for plaque assay and Northern blotting with a 3′ UTR-specific probe. Ethidium bromide staining of rRNA is included as a loading control. (B) Corresponding titers of infectious virus in the supernatant of the infected cells. Results are representative of two independent experiments. (C) Northern blot of viral RNA in wt MEFs infected with wt (FLSDX) and sfRNA-deficient (FL-IRAΔCS3) viruses in the presence of indicated concentrations of IFNAR-neutralizing antibody (ab) MAR1-5A3 or isotype control antibody. wt MEFs were infected with the wt and mutant viruses for 2 h at an MOI of 1, after which MAR1-5A3 or an isotype control antibody was added. Cells were harvested 4 days later, and Northern blotting was performed to detect viral RNA using a 3′ UTR-specific probe. Ethidium bromide staining of rRNA is included as a loading control.
Fig 3
Fig 3
Rescue of pathogenesis of sfRNA-deficient virus in mice deficient in IFN induction or signaling. (A) Weanling (1- to 20-day-old) wt C57BL/6 mice were inoculated with 103 PFU of wt (FLSDX) or sfRNA-deficient (FL-IRAΔCS3) virus by footpad injection and followed for mortality for 21 days. Survival data were combined from three independent experiments with a total of 16 to 18 mice per group (P < 0.002). (B) Adult (8- to 10-week-old) IRF-3−/− × IRF-7−/− mice were inoculated with 103 PFU of wt (FLSDX) or sfRNA-deficient (FL-IRAΔCS3) virus by footpad injection and followed for mortality for 21 days. Survival data were combined from two independent experiments with a total of 6 to 10 mice per group (P < 0.05). (C to E) Adult (8- to 10-week-old) IFNAR−/− mice were inoculated with 103 (C), 102 (D), or 101 (E) PFU of wt (FLSDX) or sfRNA-deficient (FL-IRAΔCS3) virus by footpad injection and followed for mortality. Survival data were combined from three independent experiments with a total of 5 to 9 mice per group (103 PFU, P < 0.02; 102 and 101 PFU, P < 0.001). (F to I) Viral RNA levels were determined from serum samples harvested on the indicated days after infection of weanling mice with 103 PFU or from adult IFNAR−/− mice infected with 103, 102, or 101 PFU of wt (FLSDX) or sfRNA-deficient (FL-IRAΔCS3) virus using qRT-PCR. Data are shown as log10 viral RNA equivalents per ml from 11 to 18 (wt) or 4 to 9 (IFNAR−/−) mice per time point. The error bar indicates standard error of the mean, and the dotted line represents the limit of sensitivity of the assay. Asterisks and corresponding P values shown represent differences that are statistically significant by two-way ANOVA.
Fig 4
Fig 4
sfRNA reduces the inhibitory effect of IFN treatment on SFV replication. Wild-type MEFs were electroporated with in vitro transcribed sfRNA or sfRNA containing IRAΔCS3 mutations (IΔRNA) and treated with 1,000 IU/ml mouse IFN-α for 8 h. Cells were infected subsequently with SFV at an MOI of 1 for 1 h. Samples were harvested at 10 h postelectroporation (before SFV infection) and 12 h after SFV infection. (A) Northern blot of RNA isolated before SFV infection showing different sfRNA species using a 3′ UTR-specific probe. RNA from cells infected with FLSDX (F) and FL-IRAΔCS3 (IΔ) viruses were used as controls for detecting sfRNA1 and sfRNA3, respectively. Ethidium bromide staining of rRNA is included as a loading control. (B) Northern blot detecting SFV gRNA with a probe for the SFV nsp4 gene. Ethidium bromide staining of rRNA is included as a loading control. A representative blot from three independent experiments is shown. (C) Fold reduction of SFV titers after IFN treatment in the presence of sfRNA or IΔRNA. Values are the mean of three independent experiments. Error bars indicate standard errors of the means. Statistical significance as analyzed by unpaired t test is indicated by an asterisk (*, P < 0.05).
Fig 5
Fig 5
Replication of sfRNA mutant WNVKUN is partially rescued in RNase L−/− but not in PKR−/− MEFs. (A) Northern blot of viral RNA in wt and PKR−/− MEFs infected with wt (FLSDX) and sfRNA-deficient (FL-IRAΔCS3) viruses. (B) Corresponding titers of infectious virus in the supernatant of the infected cells. The data are the average of two independent experiments. Error bars indicate standard deviations. (C) Northern blot of viral RNA in wt MEFs or RNase L−/− MEFs infected with wt (FLSDX) and sfRNA-deficient (FL-IRAΔCS3) viruses. (D) Corresponding titers of infectious virus in the supernatant of the infected cells. The data are the average of two independent experiments performed in duplicate. Error bars indicate standard deviations. Viral RNA was detected in all Northern blots with a 3′ UTR-specific probe. Ethidium bromide staining of rRNA is included as a loading control for each blot.
Fig 6
Fig 6
WNV RNA is sensitive to RNase L degradation in vitro, and sfRNA fails to protect WNV RNA from RNase L degradation. (A) Radiolabeled RNase L-resistant PV RNA, RNase L-sensitive PV G5761A RNA, and WNV RNA at 150 nM were incubated for the indicated periods of time in reaction mixtures containing 20 nM RNase L and 20 nM 2-5A or 20 nM RNase L without 2-5A. Reactions were terminated in SDS buffer, and products were phenol-chloroform extracted, ethanol precipitated with tRNA carrier, and fractionated by electrophoresis in 1.2% agarose-morpholinepropanesulfonic acid (MOPS)-formaldehyde. RNA was detected by ethidium bromide staining and UV light (left panels) and by phosphorimaging (right panels). (B) Radiolabeled WNV RNA (50 nM) was incubated for 60 min in reaction mixtures containing 20 nM RNase L and 20 nM 2-5A (lanes 2 to 11) or 20 nM RNase L without 2-5A (lane 1). sfRNA (lanes 3 to 5), PV2122 ciRNA (lanes 6 to 8), and PV2121 RNA (lanes 9 to 11) at 500, 1,000, and 2,000 nM were included in the reaction mixtures. Reactions were terminated in SDS buffer, and products were phenol-chloroform extracted, ethanol precipitated with tRNA carrier, and fractionated by electrophoresis in 1.2% agarose-MOPS-formaldehyde. RNA was detected by ethidium bromide staining and UV light (left panel) and by phosphorimaging (right panel). The mobilities of sfRNA and poliovirus RNAs are indicated with asterisks in the left panel.
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