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.

人类疱疹病毒8（HHV-8）编码四种病毒干扰素调节因子（vIRF-1至-4），它们可能通过与参与这些活动的各种细胞蛋白发生抑制性相互作用来抑制先天性免疫和细胞应激反应。值得注意的是，据报道vIRF-1和-4与去泛素酶泛素特异性蛋白酶7（USP7）相互作用，其底物包括p53以及靶向p53和破坏稳定泛素E3的连接酶MDM2。已经报道了vIRF-1和vIRF-4 USP7与USP7结合序列的结构研究。两者都涉及分别通过vIRF-1和vIRF-4中的EGPS和ASTS基序与USP7的N末端域残基相互作用，但vIRF-4残基也接触催化位点。然而，通过USP7相互作用的vIRF-1和vIRF-4的生物学活性是未知的。在这里，我们报道了在HHV-8感染的原发渗出性淋巴瘤（PEL）细胞中潜在和裂解表达的vIRF-3，也通过重复的EGPS图案与USP7相互作用，并且这种相互作用对PEL细胞的生长和活力。相互作用还有助于通过vIRF-3抑制生产性病毒复制，我们在这里确定了这一点。我们进一步表明，在PEL潜伏期中以低水平表达的vIRF-1促进了潜在的PEL细胞活力，并且该活性和vIRF-1促进的生产性复制（先前报道）涉及由vIRF-1靶向的EGPS基序介导的USP7 。这项研究是首次确定vIRF-1和vIRF-3的潜在功能和裂解功能，

重要性HHV-8与卡波济氏肉瘤，原发性渗出性淋巴瘤（PEL）和多中心Castleman病有关。潜伏和裂解病毒功能均被认为起作用。通过抑制由裂解周期触发的先天免疫和应激反应，HHV-8规定的病毒干扰素调节因子被认为对于成功的生产复制至关重要。潜在表达的vIRF-3对PEL细胞存活有重要贡献。在这里，我们确定了由vIRF-3靶向的泛素特异性蛋白酶7（USP7）去泛素酶（除了先前报道的由vIRF-1和vIRF-4结合的USP7）；vIRF-1和vIRF-3与USP7相互作用对于潜在PEL细胞生长和活力的重要性；以及正面和负面的贡献，vIRF-1和vIRF-3对HHV-8生产性复制的USP7定位。这是关于vIRF-1在PEL细胞潜伏期中的生物学重要性，vIRF-3对生产性复制的调控以及vIRF-USP7相互作用对HHV-8生物学的贡献的首次报道。