Kaposi肉瘤相关性疱疹病毒（KSHV）是Kaposi肉瘤（KS）、多中心Castleman病的浆细胞型形式和原发性渗出性淋巴瘤的病原体。在撒哈拉以南非洲，KS是HIV相关恶性肿瘤中最常见的一种，也是最常见的儿童癌症之一。包括HIV感染者在内的免疫抑制患者更容易患上KSHV相关疾病。KSHV编码一个从ORF36表达的病毒蛋白激酶（vPK）。 KSHV vPK有助于产生传染性的病毒后代和上调蛋白质合成。为了阐明vPK与KSHV感染细胞中的细胞蛋白的相互作用，我们使用了自下而上的蛋白质组学方法，并确定了宿主蛋白泛素特异性去肽酶9X-连接（USP9X）作为vPK的潜在相互作用物。随后，我们使用共同免疫沉淀分析验证了这种相互作用。我们报道了USP9X的泛素样和催化域对于与vPK的结合很重要。为了揭示USP9X/vPK相互作用的生物学意义，我们调查了USP9X的缺乏是否会调节病毒复活。我们的数据表明，USP9X的耗竭抑制了病毒复活和传染性病毒的产生。了解USP9X如何影响KSHV的复活将为我们提供关于细胞去泛素酶如何调节病毒激酶活性以及病毒如何利用细胞去泛素酶传播感染的见解。因此，阐明USP9X和vPK在KSHV感染期间的作用是确定可能受未来治疗的重要相互作用的第一步。

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy and one of the most common childhood cancers. Immunosuppressed patients, including HIV-infected patients, are more prone to KSHV-associated disease. KSHV encodes a viral protein kinase (vPK) that is expressed from ORF36. KSHV vPK contributes to the optimal production of infectious viral progeny and upregulation of protein synthesis. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used a bottom-up proteomics approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Subsequently, we validated this interaction using a co-immunoprecipitation assay. We report that both the ubiquitin-like and the catalytic domains of USP9X are important for association with vPK. To uncover the biological relevance of the USP9X/vPK interaction, we investigated whether the knockdown of USP9X would modulate viral reactivation. Our data suggest that depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Understanding how USP9X influences the reactivation of KSHV will provide insights into how cellular deubiquitinases regulate viral kinase activity and how viruses co-opt cellular deubiquitinases to propagate infection. Hence, characterizing the roles of USP9X and vPK during KSHV infection constitutes a first step toward identifying a potentially critical interaction that could be targeted by future therapeutics. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy. KSHV encodes a viral protein kinase (vPK) that aids viral replication. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used an affinity purification approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Overall, our data suggest a proviral role for USP9X.