Spatiotemporal Analysis of Hepatitis B Virus X Protein in Primary Human Hepatocytes
| العنوان: | Spatiotemporal Analysis of Hepatitis B Virus X Protein in Primary Human Hepatocytes |
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| المؤلفون: | Hyock Joo Kwon, Christian Voitenleitner, Weimei Xing, Dmytro Kornyeyev, Simon P. Fletcher, Magdeleine Hung, Christine M. Livingston, Dhivya Ramakrishnan, Rudolf K. F. Beran |
| المصدر: | Journal of Virology |
| سنة النشر: | 2019 |
| مصطلحات موضوعية: | Gene Expression Regulation, Viral, Small interfering RNA, Hepatitis B virus, Viral protein, viruses, Immunology, Fluorescent Antibody Technique, Gene Expression, Enzyme-Linked Immunosorbent Assay, DDB1, medicine.disease_cause, Microbiology, 03 medical and health sciences, 0302 clinical medicine, Virology, medicine, Gene silencing, Humans, Viral Regulatory and Accessory Proteins, Amino Acid Sequence, Spotlight, 030304 developmental biology, 0303 health sciences, biology, Smc5/6 complex, Antibodies, Monoclonal, Hepatitis B, digestive system diseases, Ubiquitin ligase, Cell biology, Virus-Cell Interactions, DNA-Binding Proteins, HBx, Protein Transport, Insect Science, Host-Pathogen Interactions, biology.protein, Hepatocytes, Trans-Activators, 030211 gastroenterology & hepatology, Peptides, Nuclear localization sequence, Protein Binding |
| الوصف: | 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. 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. |
| تدمد: | 1098-5514 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::071e30dec7647049765462d07c9cc3f9 https://pubmed.ncbi.nlm.nih.gov/31167911 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....071e30dec7647049765462d07c9cc3f9 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 10985514 |
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