West Nile virus infection induces depletion of IFNAR1 protein levels

Abstract

Productive virus infection requires evasion, inhibition, or subversion of innate immune responses. West Nile virus (WNV), a human pathogen that can cause symptomatic infections associated with meningitis and encephalitis, inhibits the interferon (IFN) signal transduction pathway by preventing phosphorylation of Janus kinases and STAT transcription factors. Inhibition of the IFN signal cascade abrogates activation of IFN-induced genes, thus attenuating an antiviral response. We investigated the mechanism responsible for this inhibition and found that WNV infection prevents accumulation of the IFN-α receptor subunit 1 (IFNAR1). The WNV-induced depletion of IFNAR1 was conserved across multiple cell types. Our results indicated that expression of WNV nonstructural proteins resulted in activated lysosomal and proteasomal protein degradation pathways independent of the unfolded protein response (UPR). Furthermore, WNV infection did not induce serine phosphorylation, a modification on IFNAR1 that precedes its natural turnover. These data demonstrate that WNV infection results in a reduction of IFNAR1 protein through a non-canonical protein degradation pathway, and may participate in the inhibition of the IFN response.

FIG. 1.

WNV inhibits accumulation of IFNAR1. (A) HeLa cells were infected with WNV NY99 at an MOI of 0.5 or 2 for 24 h and lysates were analyzed by Western blotting with anti-IFNAR1, anti-NS3 (WNV), and anti-tubulin antibody. (B) Vero cells were infected with WNV NY99 at an MOI of 2 for 24 h and lysates were analyzed by Western blotting with anti-IFNAR1, anti-NS3 (WNV), and anti-tubulin antibody. (C) Graph representing densitometry of IFNAR1 bands in multiple experiments with HeLa and Vero cells. (D) HeLa and WNV replicon-bearing HeLa cells, KUNCD20, and cell lysates were analyzed by Western blotting with anti-IFNAR1, anti-STAT1, and anti-NS3 (WNV) antibody. Triplicate samples were run for comparison. (E) HeLa cells were transfected with plasmids expressing IFNAR1, IFNAR2, or IL-12Rβ1 and 48 h later were infected with WNV NY99 (MOI of 2) for 24 h. Cell lysates were analyzed by Western blotting with the indicated antibodies. Data from one representative experiment of three are shown.

FIG. 2.

WNV does not induce cell stress. (A) Schematic representation of the current understanding of ER stress. (B and C) HeLa cells were infected with WNV at an MOI of 2 for 24 and 48 h. Total RNA was analyzed for: (B) XBP1 splicing by reverse transcription PCR (arrow denotes spliced XBP1), and (C) expression of CHOP mRNA by quantitative PCR. (D) HeLa cells were infected with WNV at an MOI of 2 for 24 h or treated with tunicamycin (30 μM) for 3 h. Total protein was analyzed for: BiP induction by Western blot (TUN, tunicamycin). Data from one representative experiment of three are shown.

FIG. 3.

The WNV-induced decrease of IFNAR1 is mediated by a lysosomal and proteasomal degradation pathway. (A and B) HeLa cells were infected with WNV NY99 (MOI of 2) for 24 h. (A) Total RNA was analyzed for: IFNAR1 and IFNAR2 mRNA by quantitative PCR. (B) WNV-infected cells were treated with 1000 U/mL IFN for 30 min. Total cell lysates were analyzed for Tyk2 expression by Western blot with the appropriate antibodies. (C) HeLa cells transfected with wild-type or mutant IFNAR1S₅₃₅A were infected with WNV NY99 (MOI of 2) (lanes 2–4 and 6–8) for 8 h. WNV-infected cells were treated with NH₄Cl (30 mM) or MG132 (10 μM) for 16 h. Transfected cells that were mock-infected and untreated cells acted as controls (lanes 1 and 5). Cell lysates were analyzed by Western blotting using antibodies against IFNAR1, NS3, and tubulin. (D) Kunjin replicon-bearing cells were treated with NH₄Cl (30 mM) for 16 h. Cell lysates were analyzed by Western blotting using antibodies against IFNAR1, NS3, and tubulin. Data from one representative experiment of two are shown.

FIG. 4.

The WNV-induced decrease of IFNAR1 occurs through a non-canonical pathway. (A) Schematic representation of IFNAR1 protein. Potential tyrosine phosphorylation sites are noted. The enlarged sequence marks serines 535 and 539 important for IFNAR1 stability. (B) HeLa cells were infected with WNV NY99 at an MOI of 2 for 24 h. Mock-infected cells were left untreated (lane 1) or treated with IFN (1000 U/mL) for 30 min (lane 2). Cell lysates were immunoprecipitated with IFNAR1 antibodies. Immune complexes and lysates were analyzed by Western blotting with antibodies against IFNAR1, phospho-S535/S539-IFNAR1 (P-IFNAR1), phospho-STAT1 (P-STAT1), STAT1, tubulin, and WNV NS3.

FIG. 5.

WNV inhibits expression of cell surface IFNAR1 protein. (A) HeLa cells transfected with wild-type (Wt) IFNAR1, IFNAR1-S535A (Mt), or IL-12Rβ1 were infected with WNV NY99 (MOI of 2) for 24 h. Cells were surface biotin-labeled and precipitated with neutravidin beads. Lysates (WCE) and biotin-avidin complexes (Neut) were analyzed by Western blotting with the indicated antibodies. (B) The level of cell surface proteins expressed in transfected and infected cells shown in A were quantified and expressed as a percentage of the protein expressed in uninfected total lysate (WCE) lanes. Data from one representative experiment of two are shown.