ORF45 of Kaposi's sarcoma-associated herpesvirus inhibits phosphorylation of interferon regulatory factor 7 by IKKε and TBK1 as an alternative substrate

Abstract

Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus (KSHV) is an immediate-early and tegument protein that plays critical roles in antagonizing host antiviral responses. We have previously shown (Zhu et al, Proc. Natl. Acad. Sci. U. S. A., 99:5573-5578, 2002) that ORF45 suppresses activation of interferon regulatory factor 7 (IRF7), a crucial regulator of type I interferon gene expression, by blocking its virus-induced phosphorylation and nuclear accumulation. We report here further characterization of the mechanisms by which ORF45 inhibits IRF7 phosphorylation. In most cell types, IRF7 is phosphorylated and activated by IKKε and TBK1 after viral infection. We found that phosphorylation of IRF7 on Ser477 and Ser479 by IKKε or TBK1 is inhibited by ORF45. The inhibition is specific to IRF7 because phosphorylation of its close relative IRF3 is not affected by ORF45, implying that ORF45 does not inactivate the kinases directly. In fact, we found that ORF45 is phosphorylated efficiently on Ser41 and Ser162 by IKKε and TBK1. We demonstrated that ORF45 competes with the associated IRF7 and inhibits its phosphorylation by IKKε or TBK1 by acting as an alternative substrate.

Fig 1

KSHV ORF45 specifically inhibits phosphorylation of IRF7 on Ser477 and Ser479 by IKKε or TBK1. (A) Phosphorylation of human IRF7 on Ser477 and Ser479 by IKKε and TBK1. HEK293T cells were transfected with 1 μg of Flag-tagged IRF7 and 100 ng of HA-IKKε/TBK1 or their kinase-dead mutant (S38A) plasmids. Cell lysates were prepared at 48 h after transfection and immunoblotted with anti-pIRF7 (Ser477/479) antibody. (B) Specific recognition of IRF7 phosphorylation on Ser477 and Ser479 by the anti-pIRF7 (Ser477/479) antibody. HEK293T cells were transfected with 100 ng HA-IKKε plus 1 μg of Flag-tagged expression plasmids encoding wild-type IRF7 or the S477A, S479A, or S477/479A mutant. At 48 h after transfection, cell lysates were prepared and immunoblotted with antibodies as indicated. (C and D) ORF45 inhibits phosphorylation of IRF7 by IKKε and TBK1. HEK293T cells were transfected with 1 μg of Flag-IRF7 together with 100 ng of HA-IKKε (C) or HA-TBK1 (D) expression plasmid in the presence or absence of 2 μg of pCR3.1-ORF45. At 48 h after transfection, cell lysates were prepared and analyzed by Western blotting with antibodies as indicated. (E and F) ORF45 does not inhibit phosphorylation of IRF3 by IKKε or TBK1. HEK293T cells were transfected with 1 μg Flag-IRF3 together with 100 ng of HA-IKKε (E) or HA-TBK1 (F) expression plasmids in the presence or absence of 2 μg of pCR3.1-ORF45. Cell lysates were prepared and analyzed by Western blotting with the specified antibodies.

Fig 2

KSHV ORF45 does not interfere with interaction between the IKKε/TBK1 kinases and their substrates. (A and B) ORF45 does not interfere with the association between IRF7 and IKKε. Plasmids expressing Flag-IRF7 (5 μg), Flag-IRF3 (5 μg), HA-IKKε (1 μg), and ORF45 [5 μg (A) or 2, 5, and 10 μg (B)] were cotransfected into HEK293T cells in different combinations as indicated. At 48 h after transfection, cell lysates were prepared and subjected to immunoprecipitation with anti-Flag M2 affinity beads. The input whole-cell lysates (WCL) and immunoprecipitation (IP) complexes were analyzed by immunoblotting with anti-Flag, anti-HA, and anti-ORF45 antibodies. (C) ORF45, IRF7, and IKKε associate with each other in the same complexes in cells. HEK293T cells were transfected with Flag-IRF7-, HA-IKKε-, and ORF45-expressing plasmids in different combinations as indicated. Cell lysates were immunoprecipitated with anti-Flag M2 affinity beads and eluted with 3×Flag peptides. The eluates were then immunoprecipitated with anti-HA affinity beads and eluted with 3×HA peptides. Cell lysates and eluates were analyzed by immunoblotting with the specified antibodies. (D) ORF45 cofractionates with IRF7 and IKKε but not with IRF3. Lysates of HEK293T cells expressing Flag-IRF7 and HA-IKKε in the presence or absence of ORF45 were fractionated on a Superdex 200 HR 10/30 column. Fractions were analyzed by immunoblotting with the indicated antibodies.

Fig 3

KSHV ORF45 is phosphorylated by IKKε or TBK1 on Ser41 and Ser162. (A and B) KSHV ORF45 is phosphorylated by IKKε and TBK1. Four ORF45 fragments, GST-ORF45 (aa 1 to 115), GST-ORF45 (aa 116 to 237), GST-ORF45 (aa 238 to 332), and GST-ORF45 (aa 333 to 407), as well as GST-IRF7 (aa 452 to 503) and control GST-S6 and GST proteins were purified from Escherichia coli and used for in vitro kinase assays with IKKε (A) or TBK1 (B). (C and D) Identification of the primary sites of ORF45 phosphorylated by IKKε or TBK1. A series of serine-to-alanine mutant proteins of GST-ORF45 (aa 1 to 115) and GST-ORF45 (aa 116 to 237) were expressed and purified from E. coli. Equal amounts of these proteins were subjected to in vitro kinase assays with IKKε (C) or TBK1 (D).

Fig 4

KSHV ORF45 is phosphorylated by IKKε or TBK1 in cells. (A) Phosphorylation of ORF45 is increased by IKKε. HEK293T cells were transfected with 5 μg of Flag-ORF45 in the absence or presence of 500 ng of HA-IKKε expression vectors. At 48 h after transfection, cell lysates were prepared and immunoprecipitated with anti-Flag M2 affinity beads. The IP complexes were immunoblotted with anti-phospho-serine/threonine antibody for detection of gross phosphorylation of ORF45. The input WCL and IP complexes were also analyzed with the indicated antibodies as controls. (B) Phosphorylation of ORF45 by IKKε is reduced by mutations of Ser41 and Ser162. HEK293T cells were transfected with 5 μg of wild-type ORF45 or ORF45-S41A, ORF45-S162A, or ORF45-S41/162A in the absence or presence of 500 ng of HA-IKKε expression plasmids. At 48 h after transfection, cell lysates were prepared and immunoprecipitated with anti-ORF45 antibody. The input WCL and IP complexes were analyzed by immunoblotting with anti-phospho-serine/threonine and other antibodies as indicated. (C) Phosphorylation of ORF45 induced by TLR3 ligand poly(I·C) is reduced by mutation of Ser41 and Ser162. HEK293-TLR3 cells were transfected with 5 μg of wild-type ORF45 or ORF45-S41/162A mutant expression vectors. At 48 h after transfection, the cells were treated with poly(I·C) for 30 min. Cell lysates were prepared, immunoprecipitated, and analyzed as described above. (D) Phosphorylation of ORF45 is increased by Sendai virus infection. HEK293T cells were transfected with 5 μg of Flag-ORF45 expression vector. At 24 h after transfection, the cells were treated with Sendai virus for 24 h. Cell lysates were prepared, immunoprecipitated, and analyzed as described above. (E) Phosphorylation of ORF45 is reduced by knockdown of TBK1 and IKKε by siRNAs. HEK293T cells were transfected with commercially validated siRNAs against TBK1 and IKKε or the accompanying control. The cells were further transfected with Flag-ORF45 expression plasmid 24 h later. Phosphorylation of ORF45 was analyzed as described above.

Fig 5

KSHV ORF45 competitively inhibits IRF7 phosphorylation by IKKε or TBK1. (A) ORF45 is phosphorylated more efficiently than IRF7 by IKKε and TBK1. Short GST-fused peptides containing the phosphorylation sites of IRF7 (Ser477/479), ORF45 Ser41, Ser162, and chimeric Ser41/Ser162, and the corresponding S41A and S162A mutants were expressed and purified from E. coli. Equal amounts of the proteins were subjected to in vitro kinase assays with HA-IKKε as described above (left). The sequences of the peptides were aligned to the consensus substrate phosphorylation motif of IKKε identified by Hutti et al. (24) (right). (B) ORF45 competitively inhibits phosphorylation of IRF7 by IKKε and TBK1 in vitro. GST-IRF7 (aa 283 to 503) proteins were mixed with increasing amounts of full-length (FL) ORF45 proteins in reactions for in vitro kinase assays. The assays were performed as described for Fig. 4 except that IRF7 substrate phosphorylation was detected by Western blotting with anti-pIRF7(Ser477/479) antibody. (C) ORF45 competitively inhibits phosphorylation of IRF7 by IKKε/TBK1 in cells. Plasmids expressing Flag-IRF7 (1 μg), IKKε (100 ng), and increasing amounts of FL-ORF45 (100 ng, 500 ng, 2 μg, and 4 μg) were transfected into HEK293T cells. At 48 h after transfection, cell lysates were prepared, immunoprecipitated, and analyzed by Western blotting with antibodies as indicated. (D) ORF45 does not inhibit phosphorylation of IRF3 by IKKε/TBK1 in cells. Plasmids expressing Flag-IRF7 (1 μg), IKKε (100 ng), and increasing amounts of FL-ORF45 (100 ng, 500 ng, 2 μg, and 4 μg) were transfected into HEK293T cells. At 48 h after transfection, cell lysates were prepared, immunoprecipitated, and analyzed by Western blotting with antibodies as indicated. (E) A short GST-fused peptide containing ORF45 phosphorylation sites (GST-ORF45-S41/162) competitively inhibits phosphorylation of IRF7 by IKKε/TBK1 in vitro. GST-IRF7 (aa 283 to 503) proteins were mixed with increasing amounts of GST-ORF45-S41/162 peptide (shown in panel A) in reactions for in vitro kinase assays. The assays were performed as described for panel B. (F) GST-ORF45-S41/162 peptide does not inhibit phosphorylation of IRF7 by IKKε/TBK1 in cells. Plasmids expressing Flag-IRF7 (1 μg), IKKε (100 ng), and increasing amounts of mammalian GST expression vector pEBG-ORF45-S41/162 (100 ng, 500 ng, 2 μg, and 4 μg) were transfected into HEK293T cells. At 48 h after transfection, cell lysates were prepared and analyzed by Western blotting with antibodies as indicated. Identical results were obtained when IKKε was replaced with TBK1 in these experiments and thus were not shown.

Fig 6

Ser41 and Ser162 of KSHV ORF45 contribute to inhibition of IRF7 phosphorylation and transactivation activity. (A) Ser41 and Ser162 of KSHV ORF45 are required for inhibition of IKKε- or TBK1-induced IRF7 phosphorylation. HEK293T cells were transfected with 1 μg of Flag-IRF7, 100 ng of HA-IKKε, and 2 μg of wild-type ORF45 or ORF45-S41/162A mutant expression plasmids. At 48 h after transfection, cell lysates were prepared and analyzed by Western blotting with the indicated antibodies. Similar results were obtained with TBK1 (data not shown). (B) Mutation of Ser41 and Ser162 of ORF45 does not affect IRF7/ORF45 or IRF7/IKKε interaction. HEK293T cells were transfected with 5 μg of Flag-IRF7, 500 ng of HA-IKKε, and 10 μg of wild-type ORF45 or ORF45-S41/162A mutant expression vectors. At 48 h after transfection, cell lysates were prepared and immunoprecipitated with anti-Flag M2 affinity beads. The input WCL and IP complexes were analyzed by immunoblotting with antibodies as indicated. (C) Mutation of both Ser41 and Ser162 of ORF45 does not affect interactions between ORF45 and IKKε or TBK1. HEK293T cells were transfected with IKKε (500 ng) or TBK1 (500 ng) with wild-type ORF45 or ORF45-S41/162A mutant expression vectors. At 48 h after transfection, cell lysates were prepared and immunoprecipitated with anti-Flag M2 affinity beads. The input WCL and IP complexes were analyzed by immunoblotting with antibodies as indicated. (D) Mutation of both Ser41 and Ser162 of ORF45 impairs the inhibition of virus-induced IRF7 transactivation activity. HEK293T cells were transfected with IFN-α1 promoter reporter, pRL-TK, IRF7, and increasing amounts of ORF45 expression plasmids as indicated. Eight hours after transfection, each well was infected with 80 hemagglutinin units of Sendai viruses (SV). At 24 h after transfection, cell lysates were prepared and used for dual-luciferase assays. The relative luciferase activity was expressed in arbitrary units by normalization of firefly luciferase activity to Renilla luciferase activity. Values are averages ± standard deviations from three experiments. (E) Mutation of both Ser41 and Ser162 of ORF45 impairs the inhibition of IKKε/TBK1-induced IRF7 transactivation activity. HEK293T cells were transfected with the IFN-α1 promoter reporter, IRF7, and 60 ng ORF45 with or without 2 ng IKKε expression plasmids. At 24 h after transfection, cell lysates were prepared and used for dual-luciferase assays. (F) ORF45 contributes to inhibition of IRF7 during KSHV lytic replication. The iSLK/BAC36-wt and iSLK/BAC-stop45 cells seeded in 24-well plates were transfected with IFN-α1 promoter reporter, pRL-TK, and IRF7 plasmids using the Fugene 6 reagent. Six hours after transfection, cells were treated with 2 μg/ml doxycycline (Dox) and 1 mM sodium butyrate. At 24 h after transfection, cell lysates were prepared and used for dual-luciferase assays and for Western blot analysis. (G) Ser41 and Ser162 are required for inhibition of IRF7 by ORF45 during KSHV lytic replication. The iSLK/BAC36-wt and iSLK/BAC-stop45 cells were transfected with IFN-α1 promoter reporter, pRL-TK, IRF7, and increasing amounts of the wild-type ORF45 or the S41/162A mutant plasmids as indicated. Six hours after transfection, the cells were treated and assayed as described above.

Fig 7

Schematic diagram of ORF45-mediated specific inhibition of IRF7 phosphorylation by IKKε/TBK1. ORF45 interacts with IRF7 but not with IRF3. The IRF7-bound ORF45 competes with IRF7 for phosphorylation by IKKε/TBK1. Because IRF3 does not interact with ORF45, its phosphorylation by IKKε/TBK1 is not affected.