title: Human parainfluenza virus type 2 V protein inhibits TRAF6-mediated ubiquitination of IRF7 to prevent TLR7- and TLR9-dependent interferon induction.
abstract: Paramyxovirus V proteins block Toll-like receptor 7 (TLR7)- and TLR9-dependent signaling leading to alpha interferon production. Our recent study has provided evidence that interaction of the V proteins with IRF7 is important for the blockade. However, the detailed mechanisms still remain unclear. Here we reexamined the interaction of the human parainfluenza virus type 2 (HPIV2) V protein with signaling molecules involved in TLR7/9-dependent signaling. Immunoprecipitation experiments in HEK293T cells transfected with V protein and one of the signaling molecules revealed that the V protein interacted with not only IRF7 but also TRAF6, IKKα, and MyD88. Whereas overexpression of TRAF6 markedly enhanced the level of V protein associating with IRF7, IKKα, and MyD88 in HEK293T cells, the level of V protein associating with TRAF6 was little affected by overexpression of IRF7, IKKα, and MyD88. Moreover, knockdown or knockout of endogenous TRAF6 in HEK293T or mouse embryonic fibroblast cells resulted in dissociation of the V protein from IRF7, IKKα, and MyD88. These results demonstrate that binding of the V protein to IRF7, IKKα, and MyD88 is largely indirect and mediated by endogenous TRAF6. It was found that the V protein inhibited TRAF6-mediated lysine 63 (K63)-linked polyubiquitination of IRF7, which is prerequisite for IRF7 activation. Disruption of the tryptophan-rich motif of the V protein significantly affected its TRAF6-binding efficiency, which correlated well with the magnitude of inhibition of K63-linked polyubiquitination and the resultant activation of IRF7. Taken together, these results suggest that the HPIV2 V protein prevents TLR7/9-dependent interferon induction by inhibiting TRAF6-mediated K63-linked polyubiquitination of IRF7.
authors.affiliation: Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kitagawa
authors.forename: Mayu
authors.initials: M
authors.lastname: Yamaguchi
authors.forename: Min
authors.initials: M
authors.lastname: Zhou
authors.forename: Machiko
authors.initials: M
authors.lastname: Nishio
authors.forename: Masae
authors.initials: M
authors.lastname: Itoh
authors.forename: Bin
authors.initials: B
authors.lastname: Gotoh
mesh_terms: D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D047468:Immunoprecipitation; D050839:Interferon Regulatory Factor-7 / Q000378:metabolism*; D016898:Interferon-alpha / Q000378:metabolism*; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D010223:Parainfluenza Virus 2, Human / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics / Q000378:metabolism*; D051199:Toll-Like Receptor 7 / Q000378:metabolism; D051217:Toll-Like Receptor 9 / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects; D014764:Viral Proteins / Q000378:metabolism* / Q000494:pharmacology
chemical_list: C494234:IRF7 protein, human; D050839:Interferon Regulatory Factor-7; D016898:Interferon-alpha; D053594:Myeloid Differentiation Factor 88; C495348:TLR7 protein, human; C495371:TLR9 protein, human; D048029:TNF Receptor-Associated Factor 6; D051199:Toll-Like Receptor 7; D051217:Toll-Like Receptor 9; C068854:V protein, human parainfluenza virus type 1; D014764:Viral Proteins; D051550:I-kappa B Kinase