Identification and characterization of host factor VCPIP1 as a multi-functional positive regulator of hepatitis B virus

Chronic infection with hepatitis B virus (HBV) remains an important public health challenge. Viral covalently closed circular DNA (cccDNA) persists in infected hepatocytes and serves as the template for transcribing all viral RNA species. HBV regulatory protein X (HBx) interacts with other viral and cellular proteins to play diverse functions in viral life cycle, including modulation of cccDNA transcription activity. Here, we used proximity labeling coupled with proteomic analysis to screen for HBx-interacting host proteins. One of the identified candidates, deubiquitinating enzyme valosin-containing protein-interacting protein 1 (VCPIP1), directly binds HBx and stabilizes HBx by reducing its proteasomal degradation, which corroborated a recent report. VCPIP1-mediated upregulation of HBV transcription, antigen expression, and genome replication was demonstrated using a series of HBV replication and infection models. More importantly, VCPIP1 was found to upregulate HBV in the absence of HBx. Mechanistic studies revealed that VCPIP1 HBx-independently associates with HBV enhancer I/X promoter (EnI/Xp) and positively modulates both its promoter and enhancer activities, partially through promoting the binding of YY1 transcription factor to EnI/Xp. Results presented here expand the recently described role of VCPIP1 in HBV life cycle and establish it as a multi-functional positive regulator of this virus.

Importance: Hepatitis B virus (HBV) encodes the regulatory protein HBx that plays crucial roles in viral life cycle and cellular processes through interacting with viral and cellular proteins. Identifying HBx-interacting proteins may reveal novel aspects of host-virus interactions. In this work, proximity labeling coupled with proteomic analysis identified multiple HBx-interacting host factors, and among these, valosin-containing protein-interacting protein 1 (VCPIP1) was confirmed to directly bind HBx and reduce its proteasomal degradation, corroborating a recent report. In addition to upregulating HBx-expressing HBV, we showed that VCPIP1 also positively regulates mutant HBV lacking HBx expression. This novel HBx-independent function of VCPIP1 was shown to involve its association with one viral promoter/enhancer element, which upregulated the latter's promoter and enhancer activities. These results establish VCPIP1 as a positive regulator of HBV that acts through multiple, diverse mechanisms and might also contribute toward revealing novel cellular functions of VCPIP1.