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. 2006 Mar;80(6):2913-23.
doi: 10.1128/JVI.80.6.2913-2923.2006.

West Nile virus evades activation of interferon regulatory factor 3 through RIG-I-dependent and -independent pathways without antagonizing host defense signaling

Brenda L Fredericksen  1 , Michael Gale Jr
Affiliations

West Nile virus evades activation of interferon regulatory factor 3 through RIG-I-dependent and -independent pathways without antagonizing host defense signaling

Brenda L Fredericksen et al. J Virol. 2006 Mar.

Abstract

The ability of viruses to control and/or evade the host antiviral response is critical to the establishment of a productive infection. We have previously shown that West Nile virus NY (WNV-NY) delays activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical to the initiation of the antiviral response. Here we demonstrate that the delayed activation of IRF-3 is essential for WNV-NY to achieve maximum virus production. Furthermore, WNV-NY utilizes a unique mechanism to control activation of IRF-3. In contrast to many other viruses that impose a nonspecific block to the IRF-3 pathway, WNV-NY eludes detection by the host cell at early times postinfection. To better understand this process, we assessed the role of the pathogen recognition receptor (PRR) retinoic acid-inducible gene I (RIG-I) in sensing WNV-NY infection. RIG-I null mouse embryo fibroblasts (MEFs) retained the ability to respond to WNV-NY infection; however, the onset of the host response was delayed compared to wild-type (WT) MEFs. This suggests that RIG-I is involved in initially sensing WNV-NY infection, while other PRRs sustain and/or amplify the host response later in infection. The delayed initiation of the host response correlated with an increase in WNV-NY replication in RIG-I null MEFs compared to WT MEFs. Our data suggest that activation of the host response by RIG-I early in infection is important for controlling replication of WNV-NY. Furthermore, pathogenic strains of WNV may have evolved to circumvent stimulation of the host response until after replication is well under way.

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Figures

FIG. 1.
FIG. 1.
Effect of IRF-3 activation on WNV-NY replication. (A) 293 cells transfected with either IRF-3-5D, N-RIG, or EGFP were incubated for 24 h and infected with WNV-NY (MOI, 0.5). (B) pIC (100 μg/ml) was added to supernatants of PH5CH8 cells, and cultures were incubated for 8 h at 37°C prior to infection with WNV-NY (MOI, 0.05). (A and B) Culture supernatants were recovered at the time points indicated, and infectious particle production was assessed by plaque assay on Vero cells.
FIG. 2.
FIG. 2.
WNV-NY replication is required for activation of IRF-3. (A and B) Examination of IRF-3 activation in response to UV-inactivated WNV-NY. A549 cells were mock infected, infected with WNV-NY (MOI, 5), or exposed to UV-inactivated virus at a concentration equivalent to an MOI of 5. (A) Cell lysates were recovered at the indicated times and subjected to immunoblot analysis. Phosphorylation of IRF-3 was detected using an antibody specific for the phosphoserine 396 isoform of IRF-3 (IRF-3-P). Steady-state protein levels of total IRF-3, ISG56, WNV, and actin were also examined. (B) IRF-3 localization in mock-infected (a and d), WNV-NY-infected (b and e), or UV-inactivated WNV-NY- (c and f) treated A549 cells was detected by IFA. IRF-3 was detected using an IRF-3 polyclonal antiserum and an Alexa 488-conjugated secondary antibody (a, b, and c). WNV protein expression (d, e, and f) was detected using a mouse polyclonal anti-WNV antibody and rhodamine-conjugated secondary antibody. (C) Effect of cycloheximide on WNV-NY-induced expression of IRF-3 target genes. A549 cells were infected with WNV-NY (MOI, 1) in the presence or absence of cycloheximide (50 μg/ml). Induction of ISG15 and ISG56 was assessed by Northern blot analysis of total RNA harvested at the indicated times postinfection. Levels of GAPDH expression were also assessed to control for loading. (D) Activation of IRF-3 in cells harboring the WNV replicon. Cellular localization of IRF-3 (a and b) and WNV protein expression (c and d) were examined in parental Huh7 (a and c) and Huh7-WNV-2 replicon (b and d) cell lines by IFA.
FIG. 3.
FIG. 3.
Effects of WNV-NY infection on IRF-3 induction by SenV and VSV. (A) Parental Huh7 cells (a and b) and Huh-WNV-2 replicon cells (c and d) were mock infected (a and c) or infected with SenV (b and d). IRF-3 localization was assessed by IFA. (B) Phosphorylation state of IRF-3 in mock- (lanes 1 to 6), VSV-GFP- (lanes 7 to 11), WNV-NY- (lanes 12 to 17), or VSV-GFP- and WNV-NY- (lanes 18 to 22) infected A549 cells. WNV-NY-infected cultures were incubated for 6 h prior to superinfection with VSV-GFP. Whole-cell lysates were recovered at the indicated times postinfection with WNV-NY, and Western blot analysis was performed with an antibody specific for the phosphoserine 396 isoform of IRF-3 (IRF-3-P). Blots were stripped and reprobed with antisera against total IRF-3, WNV, VSV, or GAPDH.
FIG. 4.
FIG. 4.
Effect of WNV-NY infection on RIG-I-dependent induction of IRF-3. (A) Huh7 cells were infected in triplicate with WNV-NY (MOI, 5) for 3 h prior to transfection with pISG56-luc, pCMV-Renilla, and increasing concentrations of N-RIG (50, 250, and 500 ng). Cell extracts were recovered at 8 h posttransfection, and the level of luciferase expression was assessed using the Promega dual luciferase kit. A representative example from two independent experiments is shown. (B) Huh7 cells were infected with WNV-NY cells (MOI, 5) for 3 h and subsequently transfected with pISG56-luc, pCMV-Renilla, and pIC (1 μg). Cells were lysed at 4, 8, and 24 h posttransfection, and the level of luciferase expression was determined. A representative example from two independent experiments is shown.
FIG. 5.
FIG. 5.
TLR3-mediated activation of IRF-3. (A) 293T-pCDNA-TL3-YFP cells were treated with pIC or infected with WNV-NY at an MOI of 5. Whole-cell lysates were recovered at the indicated times, and steady-state levels of ISG56 expression were examined by Western blotting. Blots were stripped and reprobed for actin to control for loading. (B) pIC was added to the culture supernatants of WNV-NY-infected (MOI, 3.6) 293T-pCDNA-TL3-YFP cells at 6 h postinfection. (C) U-2 OS/NS3/4A cells were infected with WNV-NY (MOI, 3) for 4 h prior to the addition of pIC to the culture medium. (D) PH5CH8 cells infected with WNV-NY (MOI, 1) were treated with pIC at 3 h postinfection. In panels B, C, and D, whole-cell lysates collected at the indicated times postinfection were analyzed for steady-state levels of ISG56 by immunoblotting. Blots were stripped and reprobed for WNV protein to assess viral replication and GAPDH or actin to control for loading.
FIG. 6.
FIG. 6.
IRF-3 localization in WNV-NY-infected WT and RIG-I null MEFs. (A) The RIG-I null genotype was confirmed by immunoblot analysis of lysates prepared from WT and IRF-3 null MEFs incubated in the presence or absence of 200 U/ml mouse IFN-α. Steady-state levels of RIG-I were assessed using a rabbit polyclonal antiserum to RIG-I. (B) Cellular localization of IRF-3 in WT (a to c) and RIG-I null (e to g) MEFs was examined. Mock- (a and e), SenV- (b and f), and WNV-NY- (c and g) infected cells were probed for IRF-3 using an IRF-3 polyclonal antiserum and an Alexa 488-conjugated secondary antibody (a through c and e through g). WNV protein expression (d and h) was detected using a mouse polyclonal anti-WNV antibody and rhodamine-conjugated secondary antibody. (C) Percent IRF-3 nuclear localization in WNV-NY-infected WT and RIG-I null MEFs. The number of cells with nuclear IRF-3 was divided by the total number of cells present in nine individual fields of WT and RIG-I null MEFs.
FIG. 7.
FIG. 7.
Kinetics of activation of the host antiviral response in WNV-NY-infected WT and RIG-I null MEFs. (A) Comparison of the kinetics of expression of ISG56 mRNA levels in WNV-NY-infected WT and RIG-I null MEFs. Total RNA was recovered from WNV-NY-infected WT and RIG-I MEFs at the indicated times postinfection. Quantitative real-time PCR was used to determine the levels of ISG56 and GAPDH mRNA present at each time point. Bars show the level of ISG56 mRNA relative to GAPDH in each sample. (B) Western blot analysis of ISG54 expression. Whole-cell lysates were collected at the indicated times postinfection, and steady-state levels of ISG54, WNV, and GAPDH were examined. (C) Quantitation of ISG54 expression in WNV-NY-infected WT and RIG-I null MEFs.
FIG. 8.
FIG. 8.
Replication of WNV-NY in WT, RIG-I null, and TRIF null MEFs. (A) Virus-induced CPE in WT and RIG-I null MEFs. Mock-infected (a and c) and WNV-NY-infected (b and d) cultures of WT (a and b) and RIG-I null (c and d) MEFs were visualized at 56 h postinfection using a Zeiss light microscope, and images were captured with a digital camera. (B) Infectious particle production by WNV-NY-infected WT and RIG-I null MEFs. Culture medium was removed from infected MEFs and cleared of cell debris by low-spin centrifugation. The presence of infectious virus particles was determined as PFU per milliliter by titrating supernatants on Vero cells in duplicate. The average of three independent experiments is shown. Solid line, RIG-I null; broken line, WT MEFs. (C) Infectious particle production by WNV-NY-infected TRIF null MEFs. Titers for supernatants removed from WNV-NY-infected WT and TRIF null MEFS were determined on Vero cells in duplicate. The average of three independent experiments is shown. Solid line, TRIF null; broken line, WT MEFs.
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