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, 486 (3), 700-705

KSHV-encoded Viral Interferon Regulatory Factor 4 (vIRF4) Interacts With IRF7 and Inhibits Interferon Alpha Production

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KSHV-encoded Viral Interferon Regulatory Factor 4 (vIRF4) Interacts With IRF7 and Inhibits Interferon Alpha Production

Sung-Woo Hwang et al. Biochem Biophys Res Commun.

Abstract

Before an infection can be completely established, the host immediately turns on the innate immune system through activating the interferon (IFN)-mediated antiviral pathway. Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes a unique antagonistic mechanism of type I IFN-mediated host antiviral immunity by incorporating four viral interferon regulatory factors (vIRF1-4). Herein, we characterized novel immune evasion strategies of vIRF4 to inhibit the IRF7-mediated IFN-α production. KSHV vIRF4 specifically interacts with IRF7, resulting in inhibition of IRF7 dimerization and ultimately suppresses IRF7-mediated activation of type I IFN. These results suggest that each of the KSHV vIRFs, including vIRF4, subvert IFN-mediated anti-viral response via different mechanisms. Therefore, it is indicated that KSHV vIRFs are indeed a crucial immunomodulatory component of their life cycles.

Keywords: IFN-alpha; IRF7; Innate immunity; KSHV; vIRF4.

Figures

Fig. 1
Fig. 1. Expression of KSHV vIRF4 robustly facilitates VSV replication
(a) Effect of KSHV vIRF4 on VSV-GFP propagation. TREx293 pcDNA and TREx293 vIRF4/AU cells upon Doxy (1 μg/ml) treatment were infected with VSV carrying GFP at an MOI of 0.01. At 24 h and 48 h post-infection, the level of VSV replication was monitored by microscopic observation of VSV-induced cytopathic effect. (b) Plaque assay of VSV-GFP upon KSHV vIRF4 expression. Cells were infected with VSV carrying GFP at an MOI of 0.01. The cell culture media was collected at 48 h post-infection. The viral titer was determined by plaque assays. The data are represented as means of triplicate infection ± s.d. An asterisk (*) represents P < 0.001.
Fig. 2
Fig. 2. KSHV vIRF4 suppresses IFN-α production
(a–c) TREx293 pcDNA and TREx293 vIRF4/AU cells together with Doxy (1 μg/ml) treatment were infected with SeV. At 24 h post-infection, total RNA was extracted from the cells infected with 20 hemagglutination (HA) units of SeV. The expression of IFN-α1, IFN-α4, and IFN-α6 were examined by real time PCR. The data are represented as means of triplicate PCR analysis ± s.d. (d) The vIRF4 expression level was monitored by immunoblot (IB) analyses.
Fig. 3
Fig. 3. Expression of vIRF4 Inhibits IRF7-mediated activation of IFN promoter activity
293T cells were transfected with 100 ng of pGL3-based IFN-α4 (IFN-α6 or ISRE) promoter construct and 10 ng of pRL-SV40 renilla. Luciferase activity was measured as described in material and method. (a) 293T cells were transfected with increasing amounts of vIRF4 with or without IRF7. At 24 h post-transfection, the cells were stimulated with 20 HA units of SeV infection. (b) 293T cells were transfected with increasing amounts of vIRF4, followed by stimulation of SeV infection for 12 h after 24 h post-transfection. (c and d) 293T cells were transfected with IFN-α4 or IFN-α6 promoter-directed luciferase (luc) reporter, renilla luciferase reporter, IRF7, and IKKε plasmid along with increasing amount of vIRF4.
Fig. 4
Fig. 4. Interaction of vIRF4 with IRF7 Inhibits dimerization of IRF7
Interaction between vIRF4 and IRF7. (a) 293T cells were transfected with either GST-fused IRF7 or GST-fused IRF3 together with V5-tagged vIRF4 as indicated. Cell lysates were used for GST-pull down assay, followed by IB with anti-V5 antibody. (b) TRExBCBL1-vIRF4 cells were stimulated with Doxy (1 μg/ml) for 12 h, followed by treatment with IFN-β for 12 h to induce expression of vIRF4 and IRF7. Cell lysates were used for immunoprecipitation with an anti-IRF7 antibody and subsequently, for IB analysis with anti-V5 antibody. (c) Cells were transfected with GST-IRF7 and Flag-IRF7 together with increasing amount of vIRF4 (wt). At 48 h post-transfection, cell lysate were used for GST pull-down assay, followed by IB with anti-Flag antibody or anti-V5 antibody to detect IRF7 homodimerization. (d) Cells were transfected with GST-IRF7 and Flag-IRF3 together with increasing amounts of vIRF4 (wt) and subsequently analyzed in GST pull-down assay, followed by IB with anti-Flag antibody or anti-V5 antibody to detect IRF7-IRF3 heterodimerization.

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