USP7-Dependent Regulation of TRAF Activation and Signaling by a Viral Interferon Regulatory Factor Homologue

Abstract

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.

P22077 inhibits LPS-induced inflammatory response by promoting K48-linked ubiquitination and degradation of TRAF6

“…In our study, P22077 did not appear to impact the K63linked ubiquitination of TRAF6. But Xiang et al reported that USP7 regulated the K63-linked polyubiquitination of TRAF3 and TRAF6 [42], indicated that USP7 regulating polyubiquitination of TRAF6 maybe difference in different stimulus. Furthermore, our data suggest that small molecule inhibitor of DUBs maybe an excellent antiinflammatory drug with potential clinical application.…”

The Role of Deubiquitinating Enzymes in Acute Lung Injury and Acute Respiratory Distress Syndrome

“…NLRP3 [38] Regulates NLRP3 inflammasome activation [38] NF-κB [39], NEMO [33] Regulates NF-κB signaling [33,39] VP24 [49] Involves in virus replication [49] Tat [50] Involves in virus production [50] TRAF3/TRAF6 [51] Modulates antiviral signaling [51] TRAF6/IKKγ [34] Regulates TLR signaling [34] USP-10 CFTR [37,52] Epithelial mucosal clearance [37,52] NICD1 [53] Regulates Notch signaling [53] USP-11 E2F1 [54] Regulates lung epithelia proliferation and wound healing [54] LPA1 [36] Enhances inflammation [36] USP-13 IL-1R8/Sigirr [40] Suppresses lung inflammation [40] PTEN [41] Regulates cell apoptosis [41] MCL1 [42] Regulates transformation of fibroblasts [42] STAT1 [55] Regulates IFN Signaling [55] STING [56] Negatively regulates antiviral responses [56] USP-14 I-kB [31] Increases cytokine release [31] CBP [32] Lung inflammation [32] USP-15 IκBα [57] NF-κB activation [57] USP-17 HDAC2 [58] Reverses glucocorticoid resistance [58] TRAF2/TRAF3 [59] Lung inflammation [59] [92] Inhibits type I IFN signaling and antiviral response [92] POH1 pro-IL-1β [93] Negatively regulates the immune response [93] BRCC3 NLRP3…”

“…USP7 (HAUSP)is originally identified as a viral binding protein that preferentially cleaves K11-, K63-and K48-linked ubiquitin chains [119,120]. USP7 is involved in viral infection by targeting virus related protein to modulate virus replication and production [49][50][51]. USP7 is reported to deubiquitinate and stabilize NF-κB to increase its transcriptional activity in TLR-induced inflammatory gene expression [39].…”

STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

“…Nonetheless, the actual functions of the vIRFs and particular vIRF-protein interactions in HHV-8 de novo infection, latency, and productive replication are still poorly understood. However, it is known that in PEL cells, vIRFs 1, 2 and 3, which are expressed at least to some degree in the latent phase, support PEL cell viability, and in lytic replication, vIRF-1 has been shown to promote virus production from PEL cells and iSLK epithelial cells (inducible for immediate-early RTA expression and used experimentally to host genetically modified HHV-8) and viral lytic gene expression in the former [8][9][10][11]. Counterintuitively, vIRF-2 and vIRF-3 inhibit productive replication in PEL and iSLK cells, as does vIRF-2 in HuARLT endothelial cells [9,10,12].…”

“…However, it is known that in PEL cells, vIRFs 1, 2 and 3, which are expressed at least to some degree in the latent phase, support PEL cell viability, and in lytic replication, vIRF-1 has been shown to promote virus production from PEL cells and iSLK epithelial cells (inducible for immediate-early RTA expression and used experimentally to host genetically modified HHV-8) and viral lytic gene expression in the former [8][9][10][11]. Counterintuitively, vIRF-2 and vIRF-3 inhibit productive replication in PEL and iSLK cells, as does vIRF-2 in HuARLT endothelial cells [9,10,12]. Indeed, identified activities of vIRF-2 include the activation of innate immune signaling leading to interferon induction, direct induction of interferon-stimulated genes, and specific repression of early viral genes [10,12].…”