Hepatitis B Virus X Protein Promotes Degradation of SMC5/6 to Enhance HBV Replication

Christopher M Murphy,Yanping Xu,Feng Li,Kouki Nio,Natalia Reszka-Blanco,Xiaodong Li,Yaxu Wu,Yanbao Yu,Yue Xiong,Lishan Su

doi:10.1016/j.celrep.2016.08.026

Christopher M Murphy, Yanping Xu + Show 8 more

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https://doi.org/10.1016/j.celrep.2016.08.026

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Abstract

The hepatitis B virus (HBV) regulatory protein X (HBx) activates gene expression from the HBV covalently closed circular DNA (cccDNA) genome. Interaction of HBx with the DDB1-CUL4-ROC1 (CRL4) E3 ligase is critical for this function. Using substrate-trapping proteomics, we identified the structural maintenance of chromosomes (SMC) complex proteins SMC5 and SMC6 as CRL4(HBx) substrates. HBx expression and HBV infection degraded the SMC5/6 complex in human hepatocytes invitro and in humanized mice invivo. HBx targets SMC5/6 for ubiquitylation by the CRL4(HBx) E3 ligase and subsequent degradation by the proteasome. Using a minicircle HBV (mcHBV) reporter system with HBx-dependent activity, we demonstrate that SMC5/6 knockdown, or inhibition with a dominant-negative SMC6, enhance HBx nullmcHBV-Gluc gene expression. Furthermore, SMC5/6 knockdown rescued HBx-deficient HBV replication in human hepatocytes. These results indicate that aprimary function of HBx is to degrade SMC5/6, which restricts HBV replication by inhibiting HBV gene expression.

Highlights

Hepatitis B virus (HBV) infection causes chronic hepatitis B in an estimated 350 million people worldwide, putting these people at high risk for developing liver cirrhosis and, eventually, hepatocellular carcinoma (HCC) (Dienstag, 2008; Revill et al, 2016; Scaglione and Lok, 2012)
Identification of hepatitis B virus X protein (HBx) Substrates by Substrate-Trapping Proteomics To identify the substrate of CRL4HBx, we performed tandem affinity purification (TAP) of HBx from a stable HepG2 cell line that inducibly expresses a biologically active HBx with N-terminal FLAG and streptavidin-binding peptide (SBP) tags (Figure 1A; Figures S1A–S1C)
HBx Degrades structural maintenance of chromosomes 5/6 (SMC5/6) in a CRL4HBx- and ProteasomeDependent Manner To determine whether HBx regulates SMC5/6 degradation via the CRL4HBx E3 ligase, we examined the effect of knocking down DDB1, CUL4A, or CUL4B on HBx-induced SMC5/6 degradation (Figure 2C)

Summary

Introduction

Hepatitis B virus (HBV) infection causes chronic hepatitis B in an estimated 350 million people worldwide, putting these people at high risk for developing liver cirrhosis and, eventually, hepatocellular carcinoma (HCC) (Dienstag, 2008; Revill et al, 2016; Scaglione and Lok, 2012). The HBV-encoded regulatory protein hepatitis B virus X protein (HBx) stimulates HBV gene expression from the cccDNA template, but the mechanism by which HBx facilitates HBV replication remains unclear (Keasler et al, 2007; Leupin et al, 2005; Slagle and Bouchard, 2016; Tang et al, 2005). The interaction between HBx and DDB1 is conserved among the HBx proteins from all mammalian hepadnaviruses and woodchuck hepatitis virus (WHV) X protein (Sitterlin et al, 1997). This binding is essential for HBV replication (Hodgson et al, 2012; Leupin et al, 2005). The mechanism and functional significance of HBx-DDB1 interaction during infection remain elusive

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