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. 2007 Aug;81(15):8282-92.
doi: 10.1128/JVI.00235-07. Epub 2007 May 23.

Inhibition of interferon regulatory factor 7 (IRF7)-mediated interferon signal transduction by the Kaposi's sarcoma-associated herpesvirus viral IRF homolog vIRF3

Chul Hyun Joo  1 , Young C Shin, Michaela Gack, Liguo Wu, David Levy, Jae U Jung
Affiliations

Inhibition of interferon regulatory factor 7 (IRF7)-mediated interferon signal transduction by the Kaposi's sarcoma-associated herpesvirus viral IRF homolog vIRF3

Chul Hyun Joo et al. J Virol. 2007 Aug.

Abstract

Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway that is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate the host IFN-mediated immune response. Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homologs of the cellular IRFs, called vIRFs. Here, we report a novel immune evasion mechanism of KSHV vIRF3 to block cellular IRF7-mediated innate immunity in response to viral infection. KSHV vIRF3 specifically interacts with either the DNA binding domain or the central IRF association domain of IRF7, and this interaction leads to the inhibition of IRF7 DNA binding activity and, therefore, suppression of alpha interferon (IFN-alpha) production and IFN-mediated immunity. Remarkably, the central 40 amino acids of vIRF3, containing the double alpha helix motifs, are sufficient not only for binding to IRF7, but also for inhibiting IRF7 DNA binding activity. Consequently, the expression of the double alpha helix motif-containing peptide effectively suppresses IRF7-mediated IFN-alpha production. This demonstrates a remarkably efficient means of viral avoidance of host antiviral activity.

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Figures

FIG. 1.
FIG. 1.
Interaction of vIRF3 with IRF7. (A) Interaction between endogenous IRF7 and vIRF3 in KSHV-infected BCBL1 cells. TREx BCBL1/control and TREx BCBL1/vIRF3 cells were stimulated with doxycycline for 12 h and, subsequently, with IFN-β for 12 h to induce the expression of vIRF3 and IRF7, respectively. Whole cell lysates (WCL) were used for immunoprecipitation with an anti-IRF7 antibody, followed by immunoblotting with an anti-vIRF3 antibody. (B) vIRF3 binds to the two independent domains of IRF7. 293T cells were transfected with Myc-tagged vIRF3 and GST-fused IRF7 mammalian expression plasmids, as indicated to the right at the top; the numbers indicate the amino acid positions of the IRF7 domains. After 48 h, WCL were used for GST pull-down assay as described in Materials and Methods, followed by immunoblotting with an anti-Myc antibody (bottom). WCL were also used for immunoblotting with anti-Myc and anti-GST antibodies. (C, D, and E) Mapping of IRF7 binding regions in vIRF3. 293T cell were transfected with Flag-tagged IRF7 and GST-fused vIRF3 fragment expression plasmids as depicted at the top of each figure. The numbers on the right side indicate the amino acid positions in vIRF3. After 48 h, WCL were used for GST pull-down assay, followed by immunoblotting with anti-Flag antibody. WCL were used for immunoblotting with anti-Flag and GST antibodies. Molecular size markers are on the left of all panels. PLDN, GST pull-down assay; IP, immunoprecipitation; IB, immunoblotting.
FIG. 2.
FIG. 2.
The central 40 aa (aa 240-280) of vIRF3 are sufficient to bind IRF7. (A) Schematic diagram of the central double helix motifs of vIRF3 and its mutants. The vIRF3 Δ240-280 mutant carried the deletion of aa 240 to 280 that contain the putative helix-turn-helix structure. Aspartic acids (D) and glutamic acids (E) in the double helix motif of vIRF3 were replaced with asparagines (N) and glutamines (Q), respectively. The DH40m1, DH40m2, and DH40mB mutants carried the replacements at the first helix motif, the second helix motif, and both helix motifs, respectively. (B) Deletion mutation of the putative double helix motifs of vIRF3 abolishes IRF7 interaction. 293T cells were transfected with GST or the GST-IRF7 expression vector together with the V5-tagged WT vIRF3 or vIRF3 Δ240-280 mutant. At 48 h, whole cell lysates (WCL) were used for GST pull-down assay, followed by immunoblotting with anti-V5 antibody. WCL were also used for immunoblotting with anti-V5 and anti-GST antibodies to show the levels of expression of the proteins. The GST-K3 vector containing the KSHV K3 gene was used for a negative control. (C) The second helix motif of vIRF3 plays a more important role in IRF7 binding than the first helix motif. At 48 h after transfection of 293T cells with Flag-tagged IRF7 together with the GST, GST-DH40, GST-DH40m1, GST-DH40m2, or GST-DH40mB expression vector, WCL were used for GST pull-down assay, followed by immunoblotting with anti-Flag antibody. WCL were also used for immunoblotting with anti-Flag and anti-GST antibodies to show the levels of expression of the proteins. (D) The 40-aa fragment of vIRF3 binds independently to either the DBD or the IAD of IRF7. At 48 h after transfection of 293T cells with V5-DH40 (vIRF3 240-280) together with the GST, GST-WT IRF7, or GST-IRF7 truncation-mutant expression vector, WCL were used for GST pull-down assay, followed by immunoblotting with anti-V5 antibody. WCL were also used for immunoblotting with anti-V5 and anti-GST antibodies to show the levels of expression of the proteins. Molecular size markers are on the left of all gels. PLDN, GST pull-down assay; IB, immunoblotting.
FIG. 3.
FIG. 3.
vIRF3 inhibits the IRF7-mediated activation of IFN promoter activity. Amounts of 1 × 105 293T cells were seeded into 24-well plates 24 h prior to transfection and transfected with the pGL3-based IFN-α6 promoter construct (100 ng) and pRL-SV40 (10 ng). The total amounts of transfected DNA were adjusted to 200 ng by adjusting the empty pcDNA5 vector. The transfection and the luciferase assay were performed as described in Materials and Methods. (A) 293T cells were transfected with increasing amounts of IRF7 with or without 10 ng of vIRF3. At 24 h posttransfection, the cells were stimulated with 25 hemagglutination (HA) units of Sendai virus for 12 h. (B and C) 293T cells were transfected with increasing amounts of WT vIRF3 (B) or vIRF3 Δ240-280 (C) with or without IRF7 (1 ng). At 24 h posttransfection, the cells were stimulated with 25 HA units of Sendai virus for 12 h. (D and E) 293T cells were transfected with increasing amounts of TBK1 (D) or IKKɛ (E) in combination with vIRF3 (10 ng) and IRF7 (1 ng). (F) Amounts of 1 × 105 293T cells were seeded into 6-well plates and subsequently transfected with IRF7 (4 ng) with or without vIRF3, followed by stimulation with 100 HA units of Sendai virus for 12 h. Real-time qRT-PCR was performed as described in Materials and Methods.
FIG. 4.
FIG. 4.
vIRF3 interaction shows no effect on phosphorylation, protein level, or dimerization of IRF7. (A) vIRF3 does not affect IRF7 phosphorylation. At 48 h posttransfection with V5-IRF7, Flag-IKKɛ and/or Flag-vIRF3 expression vectors in various combinations as indicated at the top and 293T whole cell lysates (WCL) were separated through SDS-PAGE, followed by immunoblotting with anti-IRF7 or anti-Flag antibody. pIRF7 indicates the phosphorylated form of IRF7. (B) vIRF3 does not affect IRF7 protein level. TREx BCBL1/control and TREx BCBL1/vIRF3 cells were stimulated with doxycycline for the indicated times. WCL were used for immunoblotting with anti-IRF7, anti-vIRF3, and antiactin antibodies. (C and D) WT vIRF3, but not the vIRF3 Δ240-280 mutant, inhibits the transcriptional activity of constitutively active IRF7 S477D/S479D (IRF7 SSDD). Amounts of 1 × 105 293T cells were seeded into 24-well plates 24 h prior to transfection and transfected with the pGL3-IFN-α6 promoter construct (100 ng) and IRF7 S477D/S479D (1 ng) with increasing amounts of WT vIRF3 or the vIRF3 Δ240-280 mutant. The levels of luciferase were measured at 36 h posttransfection. (E) vIRF3 does not affect IRF7 dimerization. At 48 h posttransfection with GST-IRF7 and Flag-IRF7, together with increasing amounts of WT vIRF3 or the vIRF3 Δ240-280 mutant, WCL were used for GST pull-down assay, followed by immunoblotting with anti-Flag antibody or anti-V5 antibody to detect IRF7 homodimerization and IRF7-vIRF3 interaction, respectively. WCL were also used for immunoblotting with anti-GST, Flag, and V5 antibodies to detect the levels of expression of each protein. Molecular size markers are on the left of panels A and E. PLDN, GST pull-down assay; IB, immunoblotting.
FIG. 5.
FIG. 5.
vIRF3 interaction inhibits the DNA binding activity of IRF7. 293T cells were transfected with IRF7 (no tag), V5-vIRF3, V5-vIRF3 Δ240-280, Flag-IKKɛ (A) or Flag-TBK1 (B) in various combinations, as indicated. At 48 h posttransfection, cell lysates were subjected to EMSA as described in Materials and Methods (bottom panels). The same samples were also used for immunoblotting (IB) with anti-V5, anti-Flag, and anti-IRF7 antibodies to show the levels of expression of the proteins (top). *, position of the shifted probe containing IRF7 protein; **, position of the supershifted (SS) probe containing anti-IRF7 antibody. The specificity of IRF7 DNA binding activity was tested by competition with the cold probe (CC). Molecular size markers are on the left of each panel.
FIG. 6.
FIG. 6.
The central double helix motifs of vIRF3 are sufficient to bind to and inhibit IRF7. (A) The central double helix motifs of vIRF3 are sufficient to inhibit the DNA binding activity of IRF7. 293T cells were transfected with IRF7 (no tag), GST-DH40, GST-DH40mB, and/or Flag-IKKɛ in various combinations, as indicated. At 48 h posttransfection, whole cell lysates were subjected to EMSA as described in Materials and Methods. The same samples were also used for immunoblotting (IB) with anti-GST, anti-Flag, and anti-IRF7 antibodies to show the levels of expression of the proteins (top). Molecular size markers are on the left. *, position of the shifted probe containing IRF7 protein; **, position of the supershifted (SS) probe containing anti-IRF7 antibody. The specificity of IRF7 DNA binding activity was tested by competition with the cold probe (CC). (B) The central double helix motifs of vIRF3 are sufficient to inhibit IRF7 transcription factor activity. 293T cells were transfected with the pGL3-IFN-α6 promoter construct (100 ng) and IRF7 (1 ng) together with increasing amounts of GST, GST-DH40, or GST-DH40mB. At 24 h posttransfection, the cells were stimulated with 25 hemagglutination (HA) units of Sendai virus for 12 h. Luciferase assay was performed as described in Materials and Methods, and the percent inhibition was calculated by comparison with cells transfected with the GST expression vector. (C) The central double helix motifs of vIRF3 are sufficient to suppress IFN-α1, IFN-α4, and IFN-α6 mRNA induction upon Sendai virus infection. Amounts of 1 × 105 293T cells were seeded into 6-well plates and subsequently transfected with IRF7 (4 ng) along with GST, GST-DH40, or GST-DH40mB, followed by stimulation with 100 HA units of Sendai virus for 12 h. Real-time qRT-PCR was performed as described in Materials and Methods.
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