title: DDB1 Stimulates Viral Transcription of Hepatitis B Virus via HBx-Independent Mechanisms.
abstract: HBx, a small regulatory protein of hepatitis B virus (HBV), augments viral DNA replication by stimulating viral transcription. Among numerous reported HBx-binding proteins, DDB1 has drawn attention, because DDB1 acts as a substrate receptor of the Cul4-DDB1 ubiquitin E3 ligase. Previous work reported that the DDB1-HBx interaction is indispensable for HBx-stimulated viral DNA replication, suggesting that the Cul4-DDB1 ubiquitin E3 ligase might target cellular restriction factors for ubiquitination and proteasomal degradation. To gain further insight into the DDB1-HBx interaction, we generated HBx mutants deficient for DDB1 binding (i.e., R96A, L98A, and G99A) and examined whether they support HBx-stimulated viral DNA replication. In contrast to data from previous reports, our results showed that the HBx mutants deficient for DDB1 binding supported viral DNA replication to nearly wild-type levels, revealing that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, we found that DDB1 directly stimulates viral transcription regardless of HBx expression. Through an HBV infection study, importantly, we demonstrated that DDB1 stimulates viral transcription from covalently closed circular DNA, a physiological template for viral transcription. Overall, we concluded that DDB1 stimulates viral transcription via a mechanism that does not involve an interaction with HBx.
  
  
  OBJECTIVE
  DDB1 constitutes a cullin-based ubiquitin E3 ligase, where DDB1 serves as an adaptor linking the cullin scaffold to the substrate receptor. Previous findings that the DDB1-binding ability of HBx is essential for HBx-stimulated viral DNA replication led to the hypothesis that HBx could downregulate host restriction factors that limit HBV replication through the cullin ubiquitin E3 ligase that requires the DDB1-HBx interaction. Consistent with this hypothesis, recent work identified Smc5/6 as a host restriction factor that is regulated by the viral cullin ubiquitin E3 ligase. In contrast, here we found that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, our results clearly showed that DDB1, regardless of HBx expression, enhances viral transcription. Overall, besides its role in the viral cullin ubiquitin E3 ligase, DDB1 itself stimulates viral transcription via HBx-independent mechanisms.
authors.affiliation: Department of Biochemistry, Yonsei University, Seoul, South Korea.
authors.forename: Woohyun
authors.initials: W
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, Yonsei University, Seoul, South Korea.
authors.forename: Sooyoung
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Biochemistry, Yonsei University, Seoul, South Korea.
authors.forename: Yeongnam
authors.initials: Y
authors.lastname: Son
authors.affiliation: Department of Biochemistry, Yonsei University, Seoul, South Korea.
authors.forename: Chunkyu
authors.initials: C
authors.lastname: Ko
authors.affiliation: Department of Biochemistry, Yonsei University, Seoul, South Korea wsryu@yonsei.ac.kr.
authors.forename: Wang-Shick
authors.initials: WS
authors.lastname: Ryu
mesh_terms: D045744:Cell Line, Tumor; D044842:Cullin Proteins / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology; D004270:DNA, Circular / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology; D054334:Viral Regulatory and Accessory Proteins; D014779:Virus Replication / Q000502:physiology
chemical_list: D044842:Cullin Proteins; C091435:DDB1 protein, human; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D044767:Ubiquitin-Protein Ligases