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. 2008 Jan;82(2):609-16.
doi: 10.1128/JVI.01305-07. Epub 2007 Oct 31.

Establishment and maintenance of the innate antiviral response to West Nile Virus involves both RIG-I and MDA5 signaling through IPS-1

Brenda L Fredericksen  1 , Brian C Keller, Jamie Fornek, Michael G Katze, Michael Gale Jr
Affiliations

Establishment and maintenance of the innate antiviral response to West Nile Virus involves both RIG-I and MDA5 signaling through IPS-1

Brenda L Fredericksen et al. J Virol. 2008 Jan.

Abstract

RIG-I and MDA5, two related pathogen recognition receptors (PRRs), are known to be required for sensing various RNA viruses. Here we investigated the roles that RIG-I and MDA5 play in eliciting the antiviral response to West Nile virus (WNV). Functional genomics analysis of WNV-infected fibroblasts from wild-type mice and RIG-I null mice revealed that the normal antiviral response to this virus occurs in two distinct waves. The initial response to WNV resulted in the expression of interferon (IFN) regulatory factor 3 target genes and IFN-stimulated genes, including several subtypes of alpha IFN. Subsequently, a second phase of IFN-dependent antiviral gene expression occurred very late in infection. In cells lacking RIG-I, both the initial and the secondary responses to WNV were delayed, indicating that RIG-I plays a critical role in initiating innate immunity against WNV. However, another PRR(s) was able to trigger a response to WNV in the absence of RIG-I. Disruption of both MDA5 and RIG-I pathways abrogated activation of the antiviral response to WNV, suggesting that MDA5 is involved in the host's defense against WNV infection. In addition, ablation of the function of IPS-1, an essential RIG-I and MDA5 adaptor molecule, completely disabled the innate antiviral response to WNV. Our data indicate that RIG-I and MDA5 are responsible for triggering downstream gene expression in response to WNV infection by signaling through IPS-1. We propose a model in which RIG-I and MDA5 operate cooperatively to establish an antiviral state and mediate an IFN amplification loop that supports immune effector gene expression during WNV infection.

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Figures

FIG. 1.
FIG. 1.
Microarray analysis of WT and RIG-I−/− MEFs. Graphic representation of the total number of host cell genes whose expression was differentially modulated in WNV-infected cells compared to mock-infected cultures. Genes were selected based on two criteria: a greater-than-99% probability of being differentially expressed (P < 0.01) and an overall change in expression of twofold or greater. The total number and frequency of genes that fit these criteria are indicated below each bar.
FIG. 2.
FIG. 2.
Expression profiles of WNV-responsive genes. (A) WNV-responsive genes with similar kinetics of induction in WT and RIG I−/− MEFs. (B) Genes exhibiting a delayed induction of expression in RIG-I−/− MEFs compared to WT MEFs. (C) Genes that were not induced in RIG-I−/− MEFs compared to mock-infected cells at either time point.
FIG. 3.
FIG. 3.
Analysis of the induction kinetics of WNV-responsive genes at late times postinfection. (A) Comparison of the kinetics of expression of ISG54 and ISG15 in WT and RIG-I−/− MEFs infected with WNV. WT and RIG-I−/− MEFs were infected with WNV and whole-cell lysates were collected at the indicated times postinfection (p.i.). Steady-state levels of ISG54, ISG15, WNV, and GAPDH were examined by Western blotting. (B) WT and RIG-I−/− MEFs were infected with WNV in the presence of control α-IgG or α-IFN-α/β antibodies (400 U/ml). Whole-cell lysates were assessed for steady-state levels of ISG15, STAT1, and GAPDH. α-, anti.
FIG. 4.
FIG. 4.
WNV infection of MDA5−/− and WT MEFs. Whole-cell lysates collected at the indicated times postinfection were analyzed for steady-state levels of ISG54, WNV protein, and GAPDH by Western blot analysis.
FIG. 5.
FIG. 5.
Cellular localization of IRF-3 in RIG-I−/− MEF coinfected with WNV and SenV. Mock (a, e, and i), SenV (b, f, and j), and WNV (c, g, and k) cultures, along with cultures coinfected with SenV and WNV (d, h, and l), were fixed at 72 h postinfection and probed for WNV protein expression (a to d), IRF-3 (e to h), and SenV protein expression (i to l). α-, anti.
FIG. 6.
FIG. 6.
IPS-1 plays a central role in modulating the innate antiviral response to WNV. (A) Induction of IRF-3 nuclear localization by WNV infection. Cellular localizations of IRF-3 (panels a and b) and HCV NS3/4A (panels c and d) protein expression were examined by IFA in U-2 OS/NS3/4A cells propagated in the presence (panels a and c) or absence (panels b and d) of tetracycline. (B) Effect of HCV NS3/4A on WNV-induced expression of IRF-3 target genes. U-2 OS/NS3/4A cells propagated in the presence or absence of tetracycline were infected with WNV (MOI = 3). Induction of ISG56 was assessed by Western analysis of whole-cell lysates harvested at the indicated times postinfection. Blots were stripped and reprobed for HCV NS3/4A to confirm the induction of expression in the absence of tetracycline. The levels of GAPDH expression were also assessed to control for loading. (C) Activation of the innate antiviral response in IPS-1−/− MEFs. WT and IPS-1−/− MEFs were infected at an MOI of 5 (based on titer from Vero cells), and whole-cell lysates were collected at 48 h postinfection. Expression of ISG54, ISG56, WNV, and GAPDH was assessed by Western blotting.
FIG. 7.
FIG. 7.
Model of the innate antiviral response to WNV. Details are described in the text.
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