The NY-1 hantavirus Gn cytoplasmic tail coprecipitates TRAF3 and inhibits cellular interferon responses by disrupting TBK1-TRAF3 complex formation

Abstract

Pathogenic hantaviruses replicate within human endothelial cells and cause two diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. In order to replicate in endothelial cells pathogenic hantaviruses inhibit the early induction of beta interferon (IFN-beta). Expression of the cytoplasmic tail of the pathogenic NY-1 hantavirus Gn protein is sufficient to inhibit RIG-I- and TBK1-directed IFN responses. The formation of TBK1-TRAF3 complexes directs IRF-3 phosphorylation, and both IRF-3 and NF-kappaB activation are required for transcription from the IFN-beta promoter. Here we report that the NY-1 virus (NY-1V) Gn tail inhibits both TBK1-directed NF-kappaB activation and TBK1-directed transcription from promoters containing IFN-stimulated response elements. The NY-1V Gn tail coprecipitated TRAF3 from cellular lysates, and analysis of TRAF3 deletion mutants demonstrated that the TRAF3 N terminus is sufficient for interacting with the NY-1V Gn tail. In contrast, the Gn tail of the nonpathogenic hantavirus Prospect Hill virus (PHV) failed to coprecipitate TRAF3 or inhibit NF-kappaB or IFN-beta transcriptional responses. Further, expression of the NY-1V Gn tail blocked TBK1 coprecipitation of TRAF3 and infection by NY-1V, but not PHV, blocked the formation of TBK1-TRAF3 complexes. These findings indicate that the NY-1V Gn cytoplasmic tail forms a complex with TRAF3 which disrupts the formation of TBK1-TRAF3 complexes and downstream signaling responses required for IFN-beta transcription.

FIG. 1.

NY-1V Gn cytoplasmic tail inhibits TBK1- and TRAF2-directed NF-κB activation. HEK 293 cells were transfected with 250 ng ISRE promoter (A) or NF-κB luciferase reporters (B and C) with or without TBK1 (500 ng) or TRAF2 (500 ng) expression vector. Where indicated, cells were transfected with increasing amounts of NY-1V or PHV Gn cytoplasmic tail expression vectors (1 to 2 μg), NY-1V nucleocapsid protein expression vector (2 μg), or control vector in order to transfect cells with a constant amount of DNA. Luciferase reporter activity was assayed 48 h posttransfection, was normalized to Renilla luciferase levels, and is reported as the increase over controls lacking TBK1 or TRAF2 activation.

FIG. 2.

The NY-1V Gn cytoplasmic tail binds TRAF3. (A) HEK 293 cells were transfected with TBK1, NY-1V Gn tail, or PHV Gn tail expression plasmids as indicated. Cells were treated with MG132 (50 μM) for 6 h prior to cell lysis. Cells were lysed 2 days posttransfection. GAL4- or myc-tagged proteins were immunoprecipitated (IP) with anti-GAL4 or anti-myc antibodies. Immunocomplexes were isolated on protein A/G agarose beads, and immunoprecipitated proteins were detected by anti-GAL4 (Gn tail) or anti-myc (TBK1) Western blotting (WB). (B) HEK 293 cells were transfected with TRAF3, NY-1V Gn tail, PHV Gn tail, or GAL4 expression plasmids as indicated. Transfected cells were treated with MG132 (50 μM) for 6 h prior to cell lysis. FLAG-tagged TRAF3 protein expression was analyzed by anti-FLAG Western blotting and is shown in the bottom panel. GAL4-tagged Gn tail proteins were immunoprecipitated, and immunocomplexes were isolated as described for panel A. Immunoprecipitated Gn tail proteins were analyzed by anti-GAL4 Western blotting (center panel), and coimmunoprecipitated FLAG-TRAF3 protein was analyzed by anti-FLAG Western blotting (top panel). (C) HEK 293 cells were transfected with NY-1V Gn tail expression vector alone (lane 1) or TRAF3 expression vector alone (lane 2) or cotransfected with both NY-1V Gn tail and TRAF3 expression vectors (lanes 3 and 4). The coprecipitation of TRAF3 by the NY-1V Gn tail was performed in the presence (lane 3) or absence (lane 4) of MG132. HC, IgG heavy chain; LC, IgG light chain.

FIG. 3.

The NY-1V Gn cytoplasmic tail binds the N-terminal domain of TRAF3. HEK 293 cells were cotransfected with GAL4, NY-1V Gn, or PHV Gn expression plasmids along with FLAG-TRAF3 (N415). Transfected cells were treated with MG132 (50 μM) for 6 h prior to cell lysis and analysis. Two days posttransfection and following anti-GAL4 antibody immunoprecipitation (IP), NY-1V and PHV Gn tail expression was analyzed by anti-GAL4 Western blotting (center panel). Immunocomplexes were isolated on protein A/G agarose beads, and coimmunoprecipitated TRAF3 (N415) protein was analyzed by anti-FLAG Western blotting (WB) (top panel). TRAF3 (N415) expression was determined by anti-FLAG Western blotting (bottom panel). IgG heavy chain (HC) and light chain (LC) are indicated.

FIG. 4.

Expression of the NY-1V Gn cytoplasmic tail disrupts TBK1-TRAF3 interactions. HEK 293 cells were cotransfected with TBK1 and TRAF3 expression vectors and either NY-1V Gn tail or GAL4 expression vector. Transfected cells were treated with MG132 (50 μM) for 6 h prior to cell lysis. Two days posttransfection, cells were lysed and TBK1 was immunoprecipitated (IP) using a monoclonal anti-myc antibody. Coprecipitated FLAG-TRAF3 was detected by anti-FLAG Western blotting (WB) (top panel). TBK1 and TRAF3 expression was determined by anti-myc or anti-FLAG Western blotting (middle and bottom panels). HC, IgG heavy chain.

FIG. 5.

NY-1V but not PHV infection disrupts TBK1-TRAF3 interactions. Vero E6 cells were transfected with TBK1 and TRAF3 expression plasmids, and 24 h posttransfection cells were infected with NY-1V or PHV or mock infected. One day postinfection cells were lysed and TBK1 was immunoprecipitated (IP) with anti-myc antibody. Coprecipitated TRAF3 was detected by anti-FLAG Western blotting (WB). Immunoprecipitated TBK1 and input TRAF3 were determined by Western blotting (center panels). NY-1V- and PHV-infected cells were determined by Western blotting for nucleocapsid protein (N-protein) 24 h postinfection by using an anti-N-protein rabbit polyclonal antiserum (bottom panel). HC, IgG heavy chain.