The measles virus V protein binds to p65 (RelA) to suppress NF-kappaB activity - PubMed (original) (raw)

The measles virus V protein binds to p65 (RelA) to suppress NF-kappaB activity

Kerstin M Schuhmann et al. J Virol. 2011 Apr.

Abstract

Nuclear factor κB (NF-κB) transcription factors are involved in controlling numerous cellular processes, including inflammation, innate and adaptive immunity, and cell survival. Here we show that the immunosuppressive measles virus (MV; Morbillivirus genus, Paramyxoviridae) has evolved multiple functions to interfere with canonical NF-κB signaling in epithelial cells. The MV P, V, and C proteins, also involved in preventing host cell interferon responses, were found to individually suppress NF-κB-dependent reporter gene expression in response to activation of the tumor necrosis factor (TNF) receptor, RIG-I-like receptors, or Toll-like receptors. NF-κB activity was most efficiently suppressed in the presence of V, while expression of P or C resulted in moderate inhibition. As indicated by reporter gene assays involving overexpression of the IκB kinase (IKK) complex, which phosphorylates the inhibitor of κB to liberate NF-κB, V protein targets a downstream step in the signaling cascade. Coimmunoprecipitation experiments revealed that V specifically binds to the Rel homology domain of the NF-κB subunit p65 but not of p50. Notably, the short C-terminal domain of the V protein, which is also involved in binding STAT2, IRF7, and MDA5, was sufficient for the interaction and for preventing reporter gene activity. As observed by confocal microscopy, the presence of V abolished nuclear translocation of p65 upon TNF-α stimulation. Thus, MV V appears to prevent NF-κB-dependent gene expression by retaining p65 in the cytoplasm. These findings reveal NF-κB as a key target of MV and stress the importance of the V protein as the major viral immune-modulatory factor.

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Figures

FIG. 1.

FIG. 1.

Suppression of TNF-α-mediated NF-κB activation by measles virus P, V, and C proteins. (A) The P gene of MV encodes the P protein and the nonstructural proteins V and C. The V mRNA is generated by insertion of an additional guanosine between nucleotides 751 and 752 of the mRNA by RNA editing. Therefore, the MV P and V proteins share an amino-terminal domain (PVNTD) stretching from aa 1 to 231 but have distinct carboxy-terminal domains (PCTD; VCTD). The C protein is produced by translation of an alternative open reading frame (ORF) initiated 19 nucleotides downstream of the P/V start codon. (-)ssRNA, negative-sense single-stranded RNA. (B) Increasing amounts (200 ng, 400 ng, 600 ng) of expression plasmids encoding measles virus (MV) proteins (P, V, C), rabies virus (RV) P protein, or an empty vector (EV) were cotransfected into HEK-293T cells with the NF-κB-dependent reporter plasmid p55A2-luc and pRL-CMV for normalization. After 18 h, cells were stimulated with 10 ng/ml recombinant human TNF-α and incubated for an additional 6 h, followed by cell lysis. NF-κB-driven luciferase activity was determined by a dual-luciferase assay. Values given are averages and standard deviations of results from two independent experiments. Depicted are the results of a representative experiment of four repeats. (Lower panel) Cell lysates were subjected to SDS-PAGE, and separated proteins were probed with anti-MV P/V, anti-MV C, or anti-RV P antibodies by Western blotting to determine the expression levels.

FIG. 2.

FIG. 2.

NF-κB activation by pattern recognition receptors is suppressed by MV P gene products. Increasing amounts (200 ng, 400 ng, 600 ng) of vectors encoding the indicated measles virus (MV) proteins, rabies virus (RV) P protein, or an empty vector (EV) were cotransfected into HEK-293T cells with either 200 ng of an expression plasmid for the ΔRIG-I mutant (A), MyD88 (B), IPS-1 (C), or TRIF (D) and the NF-κB-dependent reporter system (100 ng p55A2, 10 ng pRL-CMV). After 24 h, cells were lysed and NF-κB activity was determined by a dual-luciferase assay. Values given are averages and standard deviations of results from two independent experiments. Depicted are the results of representative experiments (of three repeats). (Lower panels) Expression of the viral proteins was assessed by Western blotting.

FIG. 3.

FIG. 3.

MV V inhibits NF-κB signaling downstream of the IKK complex. (A) HEK-293T cells were cotransfected with expression plasmids encoding IKKα, IKKβ, and IKKγ (100 ng each) together with increasing amounts (200 ng, 400 ng, 600 ng) of vectors for the indicated proteins or an empty vector (EV) and the NF-κB-dependent reporter system (100 ng p55A2, 10 ng pRL-CMV). After 12 h, the cells were lysed and the luciferase activity was measured by a dual-luciferase assay. Values given are averages and standard deviations of results from two independent experiments. The results of a representative experiment (out of three repeats) are shown. (Lower panel) Expression levels were determined by Western blotting. (B) Plasmids encoding p65 and p50 (150 ng each) and increasing amounts (300 ng, 600 ng) of vectors for the indicated proteins or an empty vector (EV) were cotransfected into HEK-293T cells together with a dual-luciferase reporter system. Cells were lysed 12 h after transfection, followed by determination of normalized NF-κB-dependent luciferase activity. The given values are averages and standard deviations of results from two independent experiments. Shown are the results of a representative experiment (of three repeats). (Lower panel) Expression levels of viral proteins were assessed by Western blotting.

FIG. 4.

FIG. 4.

MV V binds the NF-κB subunit p65. (A) HEK-293T cells were used to express p65 in combination with the indicated Ig-tagged proteins or the Ig tag (Ig) itself (3 μg each). After 24 h, cells were lysed under native conditions and Ig-tagged proteins were pulled down using protein A-conjugated Sepharose beads. Binding of p65 to measles proteins was visualized by Western blotting. Depicted are the results of a representative experiment (of four repeats). PO, horseradish peroxidase. (B) p50 was coexpressed in HEK-293T cells with the indicated flag proteins or an empty vector (EV) (3 μg each). Cells were lysed 24 h posttransfection, and flag-tagged measles proteins were immunoprecipitated using anti-flag M2 affinity gel. The interaction of p50 and flag-tagged proteins was analyzed by Western blotting (WB). The results of a representative experiment (of three) is shown. (C) HEK-293T cells were cotransfected with vectors encoding the indicated flag-tagged constructs or an empty vector (EV) and the RHD of p65 (aa 1 to 309). A CoIP assay was performed as described, and RHD-p65 was stained using anti-p65 (Cell Signaling; catalog no. 3035). The results of a representative experiment (of three) are shown. (D) MV V was coexpressed with the indicated flag-tagged proteins or an empty vector (EV) (3 μg each) in HEK-293T cells. CoIP experiments were performed as described above, and MV V was stained using anti-MV VCTD. Depicted are the results of a representative experiment (of three repeats).

FIG. 5.

FIG. 5.

V prevents nuclear translocation of p65. HEp2 cells were transfected with a vector for flag-MV V or an empty vector. Twenty-four hours posttransfection, cells were either treated with 10 ng/ml TNF-α for 30 min (+) or left untreated (−). Subsequently, cells were fixed and stained with the indicated antibodies. Green, p65 (stained with specific antibody); red, flag-MV V (stained with anti-FLAG M2); blue, ToPro3 nuclear staining; open arrow, not a flag-MV V-expressing cell; filled arrow, flag-MV V-expressing cell. Depicted are the results of a representative experiment (of three repeats).

FIG. 6.

FIG. 6.

The CTD of MV V is required and sufficient for p65 binding and suppression of p65/p50-mediated NF-κB activity. (A) HEK-293T cells were cotransfected with vectors encoding the indicated Ig-tagged constructs or the Ig tag itself (Ig) and p65. A CoIP assay was performed as described in the text. The results of a representative experiment of four are shown. (B) Increasing amounts (300 ng, 600 ng) of the indicated Ig-tagged constructs were coexpressed in HEK-293T cells together with p65, p50 (150 ng each), and the NF-κB reporter system (100 ng p55A2, 10 ng pRL-CMV). Twelve hours posttransfection, cells were lysed and NF-κB activity was determined by a dual-luciferase assay. Values given are averages and standard deviations of results from two independent experiments. Depicted are the results of a representative experiment (of three repeats). (Lower panel) Expression levels were determined by Western blotting.

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