乙型肝炎病毒(HBV)相关的肝细胞癌(HCC)是全球第六大最常见的癌症,病毒X蛋白(HBx)是肝癌发生发展的病因。HBx是一种高周转蛋白,但是对去泛素化酶(DUBs)在维持HBx稳态中的作用的了解非常有限。我们使用了基于74-DUB文库的酵母双杂交测定法,并确定了一种新型的DUB,含谷蛋白酶蛋白相互作用蛋白1(VCPIP1),与HBx互动。VCPIP1及其C端氨基酸863至1221上调了HBx蛋白的表达,有或没有HBV感染。机械上,VCPIP1通过不依赖泛素的途径稳定HBx蛋白,通过HBx泛素化位点突变质粒验证。免疫共沉淀试验证明了VCPIP1在招募26S蛋白酶体调节亚基6A(PSMC3)并通过相互作用与HBx形成三元复合物方面的效力。体外,纯化的His标记的PSMC3蛋白拯救了20S蛋白酶体诱导的HBx降解,和体内VCPIP1协同作用机制。功能上,与VCPIP1特异性结合的HBx通过激活NF-κB显着增强HBx的转录反式激活,AP-1和SP-1抑制肝癌细胞在Huh7和HepG2细胞中的克隆形成。此外,我们进一步证明,在HBV感染的HepG2-NTCP细胞中,VCPIP1的过表达显著影响HBV共价闭合环状DNA(cccDNA)转录。总之,我们的结果表明VCPIP1招募PSMC3与HBx结合的新机制,以不依赖泛素的方式稳定它,这可能对未来开发DUB抑制剂至关重要。重要性HBx是一种多功能的病毒癌蛋白,在病毒生命周期和肝癌发生中起着至关重要的作用。HBx降解通过泛素-蛋白酶体系统(UPS)发生。然而,UPS中的DUB的新型隔室是否也在调节HBx稳定性方面起作用尚不完全清楚。这里,第一次,我们将VCPIP1定义为一种新型DUB,用于以不依赖泛素的方式防止20S蛋白酶体对HBx的降解。PSMC3,编码26S蛋白酶体调节亚基,通过物理结合直接稳定的HBx,而不是蛋白质降解的常见方法,作为HBx上VCPIP1的关键下游效应子。因此,VCPIP1、HBx、PSMC3首次启动,这最终促进了HBx的稳定性及其功能。我们的发现通过针对UPS中的DUB为宿主病毒串扰提供了新的见解。
Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, and the viral X protein (HBx) is an etiological factor in HCC development. HBx is a high-turnover protein, but knowledge of the role of deubiquitinating enzymes (DUBs) in maintaining HBx homeostasis is very limited. We used a 74-DUB library-based yeast two-hybrid assay and determined that a novel DUB, valosin-containing protein-interacting protein 1 (VCPIP1), interacted with HBx. VCPIP1 and its C-terminal amino acids 863 to 1221 upregulated the HBx protein expression, with or without HBV infection. Mechanistically, VCPIP1 stabilized HBx protein through a ubiquitin-independent pathway, which was validated by the HBx ubiquitination site mutant plasmid. Coimmunoprecipitation assays demonstrated the potency of VCPIP1 in recruiting 26S proteasome regulatory subunit 6A (PSMC3) and forming a ternary complex with HBx through mutual interaction. In vitro, purified His-tagged PSMC3 protein rescued HBx degradation induced by the 20S proteasome, and in vivo VCPIP1 synergized the mechanism. Functionally, HBx specifically binding to VCPIP1 significantly enhanced the transcriptional transactivation of HBx by activating NF-κB, AP-1, and SP-1 and inhibited hepatoma cell clonogenicity in Huh7 and HepG2 cells. Moreover, we further demonstrated that overexpression of VCPIP1 significantly affected the HBV covalently closed circular DNA (cccDNA) transcription in HBV-infected HepG2-NTCP cells. Altogether, our results indicate a novel mechanism by which VCPIP1 recruits PSMC3 to bind with HBx, stabilizing it in a ubiquitin-independent manner, which might be critical for developing DUB inhibitors in the future. IMPORTANCE HBx is a multifunctional viral oncoprotein that plays an essential role in the viral life cycle and hepatocarcinogenesis. HBx degradation occurs through the ubiquitin-proteasome system (UPS). However, whether novel compartments of the DUBs in the UPS also act in regulating HBx stability is not fully understood. Here, for the first time, we defined VCPIP1 as a novel DUB for preventing HBx degradation by the 20S proteasome in a ubiquitin-independent manner. PSMC3, encoding the 26S proteasome regulatory subunit, directly stabilized HBx through physical binding instead of a common approach in protein degradation, serving as the key downstream effector of VCPIP1 on HBx. Therefore, the ternary binding pattern between VCPIP1, HBx, and PSMC3 is initiated for the first time, which eventually promotes HBx stability and its functions. Our findings provide novel insights into host-virus cross talk by targeting DUBs in the UPS.