Dataset Information

Spatiotemporal Analysis of Hepatitis B Virus X Protein in Primary Human Hepatocytes.

ABSTRACT: The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the host DNA damage-binding protein 1 (DDB1) E3 ubiquitin ligase to target Smc5/6 for degradation. HBx is an attractive therapeutic target for the treatment of chronic hepatitis B (CHB), but it is challenging to study this important viral protein in the context of natural infection due to the lack of a highly specific and sensitive HBx antibody. In this study, we developed a novel monoclonal antibody that enables detection of HBx protein in HBV-infected primary human hepatocytes (PHH) by Western blotting and immunofluorescence. Confocal imaging studies with this antibody demonstrated that HBx is predominantly located in the nucleus of HBV-infected PHH, where it exhibits a diffuse staining pattern. In contrast, a DDB1-binding-deficient HBx mutant was detected in both the cytoplasm and nucleus, suggesting that the DDB1 interaction plays an important role in the nuclear localization of HBx. Our study also revealed that HBx is expressed early after infection and has a short half-life (?3?h) in HBV-infected PHH. In addition, we found that treatment with small interfering RNAs (siRNAs) that target DDB1 or HBx mRNA decreased HBx protein levels and led to the reappearance of Smc6 in the nuclei of HBV-infected PHH. Collectively, these studies provide the first spatiotemporal analysis of HBx in a natural infection system and also suggest that HBV transcriptional silencing by Smc5/6 can be restored by therapeutic targeting of HBx.IMPORTANCE Hepatitis B virus X protein (HBx) is a promising drug target since it promotes the degradation of the host structural maintenance of chromosomes 5/6 complex (Smc5/6) that inhibits HBV transcription. To date, it has not been possible to study HBx in physiologically relevant cell culture systems due to the lack of a highly specific and selective HBx antibody. In this study, we developed a novel monoclonal HBx antibody and performed a spatiotemporal analysis of HBx in a natural infection system. This revealed that HBx localizes to the nucleus of infected cells, is expressed shortly after infection, and has a short half-life. In addition, we demonstrated that inhibiting HBx expression or function promotes the reappearance of Smc6 in the nucleus of infected cells. These data provide new insights into HBx and underscore its potential as a novel target for the treatment of chronic HBV infection.

SUBMITTER: Kornyeyev D

PROVIDER: S-EPMC6675897 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Spatiotemporal Analysis of Hepatitis B Virus X Protein in Primary Human Hepatocytes.

Kornyeyev Dmytro D Ramakrishnan Dhivya D Voitenleitner Christian C Livingston Christine M CM Xing Weimei W Hung Magdeleine M Kwon Hyock Joo HJ Fletcher Simon P SP Beran Rudolf K RK

Journal of virology 20190730 16

The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the host DNA damage-binding protein 1 (DDB1) E3 ubiquitin ligase to target Smc5/6 for degradation. HBx is an attractive therapeutic target for the treatment of chronic hepatitis B (CHB), but it is challenging to study this important viral protein in the context of natural ...[more]

PMID: 31167911

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Project description:Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic, but immunocompromised patients infected with HEV genotype 3 (HEV3), HEV4, or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV), while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (oriented toward the blood) and apical (oriented toward the bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A cells. F2 cells cultured on semipermeable collagen inserts and infected basolaterally with nHEV3 released 94.6% of virus particles apically, those infected with eHEV3 released 96.8% apically, and eHEV1-infected cells released 99.3% apically. Transcytosis was not involved. Density gradient centrifugation and NP-40 treatment showed that HEV particles released both apically and basolaterally were lipid associated. The apically released HEV3 and HEV1 particles were six and nine times more infectious than those released basolaterally, respectively. Confocal microscopy indicated that the open reading frame 2 (ORF2) capsid protein colocalized apically with ORF3 virus protein, the apical marker DPP4, and the recycling endosome GTPase Rab27a. The amounts of soluble glycosylated ORF2 secreted apically and basolaterally were similar. These polarized-hepatocyte data suggest that infectious HEV particles are mainly released into bile, while the small fraction released into blood could spread HEV throughout the host.IMPORTANCE Hepatitis E virus (HEV) in stools is naked, while that in culture supernatants and patients' blood is lipid associated. Its life cycle in hepatocytes, polarized cells with a basolateral side communicating with blood and an apical side connected with bile, is incompletely understood. We have developed a polarized hepatocyte model and used the cells to analyze the supernatants bathing the apical and basolateral sides and HEV subcellular distribution. HEV particles from both sides were lipid associated, and most infectious HEV particles left the cell via its apical side. Similar amounts of the open reading frame 2 (ORF2) soluble capsid protein were secreted from both sides of the hepatocytes. This model mimicking physiological conditions should help clarify the HEV cell cycle in polarized hepatocytes.

Dataset Information

Spatiotemporal Analysis of Hepatitis B Virus X Protein in Primary Human Hepatocytes.

Publications

Spatiotemporal Analysis of Hepatitis B Virus X Protein in Primary Human Hepatocytes.

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