Human Herpesvirus 8 Interferon Regulatory Factors 1 and 3 Mediate Replication and Latency Activities via Interactions with USP7 Deubiquitinase

Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRF-1 to -4) that likely function to suppress innate immune and cellular stress responses through inhibitory interactions with various cellular proteins involved in these activities. It is notable that vIRF-1 and -4 have been reported to interact with the deubiquitinase ubiquitin-specific protease 7 (USP7), substrates of which include p53 and the p53-targeting and -destabilizing ubiquitin E3 ligase MDM2. Structural studies of vIRF-1 and vIRF-4 USP7 binding sequences in association with USP7 have been reported; both involve interactions with N-terminal-domain residues of USP7 via EGPS and ASTS motifs in vIRF-1 and vIRF-4, respectively, but vIRF-4 residues also contact the catalytic site. However, the biological activities of vIRF-1 and vIRF-4 via USP7 interactions are unknown. Here, we report that vIRF-3, which is latently, as well as lytically, expressed in HHV-8-infected primary effusion lymphoma (PEL) cells, also interacts with USP7-via duplicated EGPS motifs-and that this interaction is important for PEL cell growth and viability. The interaction also contributes to suppression of productive virus replication by vIRF-3, which we identify here. We further show that vIRF-1, which is expressed at low levels in PEL latency, promotes latent PEL cell viability and that this activity and vIRF-1-promoted productive replication (reported previously) involve EGPS motif-mediated USP7 targeting by vIRF-1. This study is the first to identify latent and lytic functions of vIRF-1 and vIRF-3, respectively, and to address the biological activities of these vIRFs through their interactions with USP7. IMPORTANCE HHV-8 is associated with Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease; both latent and lytic viral functions are believed to contribute. Viral interferon regulatory factors specified by HHV-8 are thought to be critically important for successful productive replication through suppression of innate immune and stress responses triggered by the lytic cycle. Latently expressed vIRF-3 contributes significantly to PEL cell survival. Here, we identify ubiquitin-specific protease 7 (USP7) deubiquitinase targeting by vIRF-3 (in addition to previously reported USP7 binding by vIRF-1 and vIRF-4); the importance of vIRF-1 and vIRF-3 interactions with USP7 for latent PEL cell growth and viability; and the positive and negative contributions, respectively, of USP7 targeting by vIRF-1 and vIRF-3 to HHV-8 productive replication. This is the first report of the biological importance of vIRF-1 in PEL cell latency, the modulation of productive replication by vIRF-3, and the contributions of vIRF-USP7 interactions to HHV-8 biology.