Human respiratory syncytial virus nonstructural protein NS2 antagonizes the activation of beta interferon transcription by interacting with RIG-I

Abstract

A wide variety of RNA viruses have been shown to produce proteins that inhibit interferon (IFN) production and signaling. For human respiratory syncytial virus (RSV), the nonstructural NS1 and NS2 proteins have been shown to block IFN signaling by causing the proteasomal degradation of STAT2. In addition, recombinant RSVs lacking either NS1 or NS2 induce more IFN production than wild-type (wt) RSV in infected cells. However, the mechanisms by which the NS proteins perform this function are unknown. In this study, we focused on defining the mechanism by which NS2 inhibits the induction of IFN transcription. We find that NS2 is required for the early inhibition of IFN transcription since the infection of cells with NS2-deletion RSV resulted in a higher level of IRF3 activation at early time points postinfection compared with that of wt or NS1-deletion RSV infection. In addition, NS2 expression inhibits IFN transcription induced by both the RIG-I and TLR3 pathways. Furthermore, we show that NS2 inhibits RIG-I-mediated IFN promoter activation by binding to the N-terminal CARD of RIG-I and inhibiting its interaction with the downstream component MAVS (IPS-1, VISA, Cardif). Thus, the RSV NS2 protein is a multifunctional IFN antagonist that targets specific components of both the IFN induction and IFN signaling pathways.

FIG. 1.

IRF3 activation in rRSV-infected cells. A549 cells were mock infected or infected by wt rRSV (rA2) or rRSV lacking NS1 (ΔNS1), NS2 (ΔNS2), or both (ΔNS1/2). At 9 (top), 12 (middle), and 16 (bottom) h p.i., cells were fixed, permeabilized, and stained with antibodies to IRF3 (αIRF3; red) or RSV F (αF; green) for immunofluorescence. Infected (RSV F-positive) cells were scored for the presence of IRF3 in the nucleus. Approximately 30 cells for each of 10 fields were scored for triplicate samples of each condition (∼900 cells/virus/time p.i.). The photomicrographs show examples of individual fields that were counted for each sample. Shown in the bar graphs on the right are percentages (± standard errors of the means) of infected cells displaying nuclear IRF3. Asterisks denote a P value of <0.01 versus rA2-infected cells by ANOVA.

FIG. 2.

Inhibition of RIG-I-induced IFN promoter activity by NS2. 293T cells were transfected by an expression construct encoding N-RIG and either pIFNβ-luc (a) or pISG56-luc (b). Increasing amounts of the NS2 expression plasmid were added to the transfection. phRL-TK was used as a transfection control and empty vector was used to standardize the amount of plasmid transfected. Luciferase activities were determined at 24 h posttransfection. Shown are the means (± standard errors of the means) of triplicate samples. One representative experiment (of three) is shown. Asterisks denote a P value of <0.01 versus that of the control by one-way ANOVA. (c) Expression levels of N-RIG, NS2, and β-actin (as a control) were determined by WB analysis. (d) The ISG56 reporter assay was performed as described above using N-RIG to induce promoter activity and increasing amounts of an expression plasmid encoding P. (e) WB analysis of protein expression from the experiment described for panel d.

FIG. 3.

NS2 inhibits TRIF-induced IFN promoter activity. 293T cells were transfected by an expression construct encoding TRIF and the pIFNβ-luc (a) or pISG56-luc (b) reporter constructs with increasing amounts of NS2 plasmids as in Fig. 2. Luciferase activities were determined at 24 h posttransfection. Shown are the means (± standard errors of the means) of triplicate samples. One representative experiment (of three) is shown. Asterisks denote a P value of <0.01 versus that of the control by one-way ANOVA. (c) The expression levels of TRIF, NS2, and β-actin were determined by WB analysis. (d) The ISG56 reporter assay was performed as described above using TRIF to induce promoter activity and increasing amounts of an expression plasmid encoding P. (e) WB analysis of protein expression from the experiment described for panel d.

FIG. 4.

NS2 inhibits IRF3 activation by RNA-activated RIG-I. 293T cells were transfected with pISG56-luc and expression constructs encoding full-length RIG-I and the indicated amounts of NS2. phRL-TK was used as a transfection control, and empty vector was used to standardize the amount of plasmid transfected. Twenty-four hours posttransfection, RIG-I was activated by the transfection of 5′-triphosphorylated RNA, and luciferase activities were determined 16 h later. Shown are the means (± standard errors of the means) of triplicate samples. One representative experiment (of two) is shown. Asterisks denote a P value of <0.01 versus that of the control by one-way ANOVA.

FIG. 5.

NS2 does not inhibit IRF3 activation by the IRF3 kinases or MAVS. The ISG56 promoter assay was performed as described in the legend to Fig. 2 using expression constructs encoding TBK-1 (a), IKKɛ (c), or MAVS (e) to induce luciferase activity and increasing amounts of NS2 expression plasmid. Shown are the means (± standard errors of the means) of triplicate samples for one representative experiment (of three) for each condition. Protein expression levels for the inducing protein, NS2, and β-actin, as determined by WB analysis, are shown in the bottom panels.

FIG. 6.

NS2 interacts with RIG-I. 293T cells were transfected by expression constructs encoding full-length RIG-I and either HA-NS1 or HA-NS2. Cytoplasmic extracts were subjected to immunoprecipitation with anti-Flag (αFlag) or anti-HA (αHA) antibodies. The presence of RIG-I or NS protein was detected by WB analysis of the precipitates using anti-Flag (a) or anti-HA (b) antibodies, respectively. (c) WB analysis of unprecipitated cytoplasmic extracts. (d and e) A549 cells were mock infected or infected by rA2 or rHA-NS2 at an MOI of 3. RIG-I expression was induced by treatment with IFN-α at 10 h p.i. for 6 h. Cytoplasmic extracts were then generated and subjected to immunoprecipitation using anti-HA antibody. Precipitates (top) and unprecipitated cytoplasmic extracts (Input, bottom) were analyzed by WB using anti-RIG-I antiserum (d) or anti-HA antibody (e).

FIG. 7.

NS2 interacts with the N-terminal CARD of RIG-I. 293T cells were transfected by expression constructs encoding NS2 and either full-length (FL) RIG-I or the N- or C-terminal fragments of RIG-I (N-RIG [N] or C-RIG [C], respectively). Immunoprecipitation with anti-Flag (αFlag; a) or anti-HA (αHA; b) antibodies and WB analysis for the transfected proteins were performed as described in the legend to Fig. 4. (c) WB analysis of unprecipitated cytoplasmic extracts. HC, immunoglobulin heavy chain.

FIG. 8.

NS2 disrupts N-RIG interaction with MAVS. 293T cells were transfected with the indicated combinations of expression vectors encoding Flag-N-RIG, HA-MAVS, and NS2. Cytoplasmic extracts were harvested 24 h posttransfection and subjected to immunoprecipitation using anti-Flag (αFlag; a) or anti-HA (αHA; b) antibodies. The presence of N-RIG, MAVS, and NS2 were detected by WB analysis using anti-Flag (top), anti-HA (middle), or anti-NS2 (bottom) antibodies, respectively. (c) WB analysis of unprecipitated cytoplasmic extracts.