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Spatiotemporal Analysis of Hepatitis B Virus X Protein in Primary Human Hepatocytes
Dmytro Kornyeyev, Dhivya Ramakrishnan, Christian Voitenleitner, Christine M. Livingston, Weimei Xing, Magdeleine Hung, Hyock Joo Kwon, Simon P. Fletcher, Rudolf K. Beran
Journal of Virology Jun 2019, JVI.00248-19; DOI: 10.1128/JVI.00248-19.
ABSTRACT
The structural maintenance of chromosomes5/6 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 DDB1E3 ubiquitin ligase to
target Smc5/6 for degradation. HBx is an attractive therapeutic targetfor the treatmentof
chronic hepatitis B (CHB), but it is challenging to study this importantviral 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 blot and immunofluorescence. Confocal imaging studies with this antibody demonstratedthat
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 infectionand has a short half-life (~3 hours) in HBV-infected PHH.
In addition, we found that treatment with 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. |
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发表于 2019-6-14 13:03