Abstract
The hepatitis B virus (HBV) regulatory HBx protein is required for infection, and its binding to cellular damaged DNA binding protein 1 (DDB1) is critical for this function. DDB1 is an adaptor protein for the cullin 4A Really Interesting New Gene (RING) E3 ubiquitin ligase (CRL4) complex and functions by binding cellular DDB1 cullin associated factor (DCAF) receptor proteins that recruit substrates for ubiquitination and degradation. We compared the proteins found in the CRL4 complex immunoprecipitated from uninfected versus HBV-infected hepatocytes from human liver chimeric mice for insight into mechanisms by which HBV and the cell interact within the CRL4 complex. Consistent with its role as a viral DCAF, HBx was found in the HBV CRL4 complexes. In tissue culture transfection experiments, we showed that HBx expression led to decreased levels of known restriction factor structural maintenance of chromosomes protein 6 (SMC6) and putative restriction factors stromal interaction molecule 1 (STIM1, zinc finger E-box binding homeobox 2 (ZEB2), and proteasome activator subunit 4 (PSME4). Moreover, silencing of these proteins led to increased HBV replication in the HepG2-sodium taurocholate cotransporting polypeptide (NTCP) infection model. We also identified cellular DCAF receptors in CRL4 complexes from humanized mice. Increasing amounts of HBx did not reveal competitive DCAF binding to cullin4 (CUL4)-DDB1 in plasmid-transfected cells. Our results suggest a model in which HBx benefits virus replication by directly or indirectly degrading multiple cellular restriction factors.
Highlights
Chronic infection with hepatitis B virus (HBV) affects over 257 million people worldwide [1]and is a leading risk factor for the development of serious liver disease, including cirrhosis and hepatocellular carcinoma (HCC)
To determine whether an HBx–DDB1 interaction was required for the degradation of the putative restriction factors described above, we investigated a panel of HBx mutant proteins that do not bind DDB1
Our results showed that HBx does not compete with CRBN for binding to DDB1 in the CRL4 complex, which contrasts with two previous studies that concluded that HBx competed with the binding of DDB1 cullin-associated factor (DCAF) DDB2 [35]
Summary
Chronic infection with hepatitis B virus (HBV) affects over 257 million people worldwide [1]. Is a leading risk factor for the development of serious liver disease, including cirrhosis and hepatocellular carcinoma (HCC). The currently approved antiviral treatments (pegylated interferon-α; nucleos(t)ide analogs) reduce levels of viral replication (reviewed in Reference [2]). Cells 2020, 9, 834; doi:10.3390/cells9040834 www.mdpi.com/journal/cells α; nucleos(t)ide analogs) levels viral replication HBV remains elusive, in part,reduce because of anofinability to eliminate. HBV remains elusive,that in serves part, because of an inability eliminate HBV covalently. DNA, a replicative intermediate as the template for viralto transcription. DNA, a replicative intermediate that serves as the template for viral transcription
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