Inhibition of interferon signaling by the New York 99 strain and Kunjin subtype of West Nile virus involves blockage of STAT1 and STAT2 activation by nonstructural proteins

Abstract

The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-alpha/beta) and gamma IFN (IFN-gamma) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-alpha. IFN-alpha signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins responsible for this inhibition.

FIG. 1.

IFN-α and IFN-γ did not inhibit KUN replicon RNA replication and expression. HEp2 cells stably expressing the KUN replicon RNA repPAC/β-gal (23) in a 96-well plate were treated with different concentrations of IFN-α2A (Roferon-A; Roche, Basel, Switzerland) and IFN-γ (I-1520; Sigma) as indicated. Forty-eight hours after IFN treatment, cells were lysed and analyzed for β-galactosidase expression to compare the replication and expression efficiencies of KUN replicon RNAs. The values of β-galactosidase expression are averages from triplicate samples, and the error bars show standard deviations.

FIG. 2.

IFN signaling is inhibited in Vero cells stably (A-C) or transiently (D) transfected with KUN (KUNrep) or NY99 (WNrep) replicon RNAs. (A-C) Normal Vero cells and Vero cells stably transfected with KUN replicon RNA (C20SDrepVero) (24) in 24-well plates (four wells from each transfection mixture) were cotransfected with the control plasmid pCMV-β (Clontech, Palo Alto, Calif.) and either the IFN-α/β-responsive (ISRE) luciferase reporter plasmid p(9-27)4thΔ(−39)Lucter (18) or the IFN-γ-responsive (GAS) luciferase reporter plasmid p(IRF-1*GAS)6tkΔ(−39)Lucter (18). Forty hours after transfection, cells were treated with 1,000 U of IFN-αA (I-4276; Sigma) per ml (A), 1,000 IU of IFN-β (I-4151; Sigma) per ml (B), or 50 ng of IFN-γ (I-1520; Sigma) per ml (C) for 14 h and then assayed for luciferase and β-galactosidase expression. (D) Vero cells were infected with VLPs containing KUN replicon RNA (C20DXHDVrep, derivative of C20UbHDVrep) (31) or NY99 replicon RNA (29) at an MOI of 10. Replicon VLPs were produced by transfection of replicon RNAs into the tetKUNCprME packaging cell line as described previously (11). Four hours after infection, cells were cotransfected with plasmid p(9-27)4thΔ(−39)Lucter and the control plasmid pCMV-β. Forty hours after transfection, cells were treated with 1,000 U of IFN-αA (I-4276; Sigma) per ml for 14 h and assayed for luciferase and β-galactosidase expression. The luciferase activity in panels A to D is expressed in relative light units normalized to the expression of β-galactosidase from the cotransfected pCMV-β plasmid and represent average values from duplicate samples. The error bars represent standard deviations.

FIG. 3.

The nuclear translocation of STAT1 and STAT2 in response to IFN treatment is blocked in HEp2 cells stably transfected with KUN (KUNrep) and WN (WNrep) replicon RNAs. (A) Normal HEp2 cells and HEp2 cells stably transfected with KUN (23) or NY99 (29) replicon RNAs were treated with 1,000 U of IFN-αA (I-4276; Sigma) per ml for 30 min at 37°C. Cells were fixed in 4% formaldehyde-phosphate-buffered saline for 10 min at room temperature, permeabilized with ice-cold acetone-methanol (1:1) for 30 min at −20°C, and stained sequentially with KUN NS1 antibodies (9) and with STAT1 (SC-345; Santa Cruz Biotechnology, Santa Cruz, Calif.) or STAT2 (SC-476; Santa Cruz Biotechnology) antibodies at concentrations of 1 μg/ml essentially as described by the manufacturer. (B) Block in nuclear translocation of STAT1 in KUN or WN replicon cells in response to treatment with 50 ng of IFN-γ/ml.

FIG. 4.

The nuclear translocation of STAT1 and STAT2 in response to IFN treatment is blocked in HEp2 cells infected with KUN and NY99. HEp2 cells on coverslips in 24-well plates were infected with KUN and NY99 at an MOI of 0.01. KUN (MRM61C strain) (32) and NY99 (NY99-4132 strain, provided by Roy Hall, University of Queensland) were grown and titrated on Vero cells. Seventy-two hours after infection, cells were treated for 30 min with 1,000 IU of IFN-αA (I-4276; Sigma) per ml (A) or 50 ng of IFN-γ (I-1520; Sigma) per ml (B), fixed, permeabilized with acetone-methanol, and stained sequentially with KUN NS1, STAT1, and STAT2 antibodies as described for Fig. 3. Arrows show cells positively infected with either KUN or NY99.

FIG. 5.

Western blot analysis of STAT1 and STAT2 expression and phosphorylation in HEp2 cells stably transfected with KUN and NY99 replicon RNAs. H-KUNrep and H-WNrep cells were treated with IFN-αA (I-4276; Sigma) (A) and IFN-γ (I-1520; Sigma) (B) as described for Fig. 4, and cell lysates were used for the detection of STAT1 and STAT2 expression and phosphorylation by Western blot analysis with antibodies recognizing nonphosphorylated and phosphorylated (P) forms of STAT1 and STAT2, respectively (anti-P-STAT1 antibody 550428; Pharmingen, San Diego, Calif.; anti-P-STAT2 antibody SC-21689 and the anti-STAT1 and anti-STAT2 antibodies described in the legend to Fig. 3; Santa Cruz Biotechnology). Controls included the detection of replicon RNA expression by cross-reacting KUN anti-NS3 antibodies and of host cell protein expression by anti-α-actin antibodies. The detection of reacted proteins on the blotted membrane was performed with an ECL Plus chemiluminescence kit (Amersham). In panels A and B, the membrane was exposed to X-ray film for 1 to 2 min, and in panel C, the membrane was exposed for 10 min.

FIG. 6.

Inhibition of IFN-α signaling by KUN nonstructural proteins. (A) Inhibition of ISRE-driven transcription by KUN nonstructural proteins in response to IFN-α treatment. Vero cells were cotransfected with pcDNA plasmids expressing the indicated KUN nonstructural proteins, the ISRE reporter plasmid p(9-27)4thΔ(−39)Lucter (19), and the pCMV-β plasmid. The controls included pcDNA3 vector plasmid (for positive IFN stimulation) and a plasmid expressing a known inhibitor of IFN signaling, V protein of SV5 (4). Thirty-two hours after transfection, cells were treated or not treated with 1,000 U of IFN-αA (I-4276; Sigma) per ml for 14 h and assayed for luciferase and β-galactosidase expression. Luciferase activity, expressed in relative light units, was normalized to β-galactosidase activity. The error bars represent standard deviations. (B) The nuclear translocation of STAT2 in response to IFN-α treatment is blocked by KUN nonstructural (NS) proteins. A549 cells on coverslips in 24-well plates were transfected with pcDNA plasmids expressing the indicated KUN nonstructural proteins. Forty-eight hours after transfection, cells were treated with 800 IU of IFN-α2A (Roferon-A; Roche) per ml for 30 min at 37°C. Cells were fixed and stained sequentially with STAT2 antibody and with either KUN anti-NS1 and anti-NS5 antibodies or anti-C-myc antibodies (15).