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Contribution of Ser386 and Ser396 to activation of interferon regulatory factor 3.

ABSTRACT: IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors, functions in innate immune defense against viral infection. Upon infection, host cell IRF-3 is activated by phosphorylation at its seven C-terminal Ser/Thr residues: (385)SSLENTVDLHISNSHPLSLTS(405). This phosphoactivation triggers IRF-3 to react with the coactivators, CREB-binding protein (CBP)/p300, to form a complex that activates target genes in the nucleus. However, the role of each phosphorylation site for IRF-3 phosphoactivation remains unresolved. To address this issue, all seven Ser/Thr potential phosphorylation sites were screened by mutational studies, size-exclusion chromatography, and isothermal titration calorimetry. Using purified proteins, we show that CBP (amino acid residues 2067-2112) interacts directly with IRF-3 (173-427) and six of its single-site mutants to form heterodimers, but when CBP interacts with IRF-3 S396D, oligomerization is evident. CBP also interacts in vitro with IRF-3 double-site mutants to form different levels of oligomerization. Among all the single-site mutants, IRF-3 S396D showed the strongest binding to CBP. Although IRF-3 S386D alone did not interact as strongly with CBP as did other mutants, it strengthened the interaction and oligomerization of IRF-3 S396D with CBP. In contrast, IRF-3 S385D weakened the interaction and oligomerization of IRF-3 S396D and S386/396D with CBP. Thus, it appears that Ser385 and Ser386 serve antagonistic functions in regulating IRF-3 phosphoactivation. These results indicate that Ser386 and Ser396 are critical for IRF-3 activation, and support a phosphorylation-oligomerization model for IRF-3 activation.

SUBMITTER: Chen W

PROVIDER: S-EPMC2410038 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Contribution of Ser386 and Ser396 to activation of interferon regulatory factor 3.

Chen Weijun W Srinath Hema H Lam Suvana S SS Schiffer Celia A CA Royer William E WE Lin Kai K

Journal of molecular biology 20080403 2

IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors, functions in innate immune defense against viral infection. Upon infection, host cell IRF-3 is activated by phosphorylation at its seven C-terminal Ser/Thr residues: (385)SSLENTVDLHISNSHPLSLTS(405). This phosphoactivation triggers IRF-3 to react with the coactivators, CREB-binding protein (CBP)/p300, to form a complex that activates target genes in the nucleus. However, the role of each phosphorylation sit ...[more]

PMID: 18440553

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Project description:Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRFs 1 to 4), all of which are expressed during lytic replication and inhibit a variety of antiviral signaling pathways. Viral IRFs 1, 2, and 3 are also expressed during latency in primary effusion lymphoma (PEL) cells, and vIRF-1 and vIRF-3 have been reported to promote PEL cell viability. Viral IRFs 1, 3, and 4 are known to interact with ubiquitin-specific protease 7 (USP7); interactions of vIRF-1 and vIRF-3 with USP7 promote PEL cell viability and regulate productive replication. Here, we report that vIRF-2 also targets USP7, utilizing a PSTS motif matching the USP7 N-terminal domain-binding A/PxxS consensus, but uniquely requires catalytic domain residues for intracellular interaction. In functional and mechanistic analyses, tumor necrosis factor receptor-associated factor (TRAF)-mediated signaling and associated polyubiquitination of TRAFs 3 and 6, specifically, were regulated negatively by USP7 and positively by vIRF-2-USP7 interaction, the latter competing for USP7-TRAF association. Using depletion, depletion-complementation, and targeted mutagenesis approaches, vIRF-2 was determined to promote latent PEL cell viability, likely independently of USP7 interaction, while lytic replication was inhibited by vIRF-2, in part or in whole via USP7 interaction. Together, our data identify a new molecular determinant of USP7 recognition, TRAF3/6-specific targeting by the deubiquitinase, associated activation of these TRAFs by vIRF-2, and activities of vIRF-2 and vIRF-2-USP7 interaction in HHV-8 latent and lytic biology.IMPORTANCE Human herpesvirus 8-encoded IRF homologues were the first to be identified in a virus. Through inhibitory interactions with cellular IRFs and other mediators of antiviral signaling, the vIRFs are believed to be essential for productive replication and also for latency in particular cell types. The deubiquitinase USP7 is a regulator of key cellular pathways, modulates HHV-8 latent and lytic infection, and is targeted by vIRFs 1, 3, and 4. Here, we report that vIRF-2 also interacts with USP7, via a means distinguishable from USP7 interactions with other vIRFs and other proteins, that this interaction modulates antiviral signaling via disruption of USP7 interactions with innate immune signaling proteins TRAF3 and TRAF6, and that vIRF-2 targeting of USP7 regulates HHV-8 productive replication. The presented data are the first to identify vIRF-2 targeting of USP7 and its role in HHV-8 biology, expanding our understanding of the repertoire and importance of virus-host interactions.

Dataset Information

Contribution of Ser386 and Ser396 to activation of interferon regulatory factor 3.

Publications

Contribution of Ser386 and Ser396 to activation of interferon regulatory factor 3.

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