- 作者: Pei-Yi Su, Shin-Chwen Bruce Yen, Ching-Chun Yang, Chih-Hsu Chang, Wen-Chang Lin & Chiaho Shih
- 作者服務機構: 1.Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, No.100, Shih-Chuan 1st Road, Sanmin, 80708, Kaohsiung, Taiwan 2.Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan 3.Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
- 中文摘要:
- 英文摘要:
Background: Hepatitis B virus (HBV) is a major human pathogen worldwide. To date, there is no curative treat‑
ment for chronic hepatitis B. The mechanism of virion secretion remains to be investigated. Previously, we found that
nuclear export of HBc particles can be facilitated via two CRM1-specifc nuclear export signals (NES) at the spike tip.
Methods: In this study, we used site-directed mutagenesis at the CRM1 NES, as well as treatment with CRM1 inhibi‑
tors at a low concentration, or CRM1-specifc shRNA knockdown, in HBV-producing cell culture, and measured the
secretion of various HBV viral and subviral particles via a native agarose gel electrophoresis assay. Separated HBV par‑
ticles were characterized by Western blot analysis, and their genomic DNA contents were measured by Southern blot
analysis. Secreted extracellular particles were compared with intracellular HBc capsids for DNA synthesis and capsid
formation. Virion secretion and the in vivo interactions among HBc capsids, CRM1 and microtubules, were examined
by proximity ligation assay, immunofuorescence microscopy, and nocodazole treatment.
Results: We report here that the tip of spike of HBV core (HBc) particles (capsids) contains a complex sensor for
secretion of both HBV virions and naked capsids. HBV virion secretion is closely associated with HBc nuclear export in
a CRM1-dependent manner. At the conformationally fexible spike tips of HBc particles, NES motifs overlap extensively
with motifs important for secretion of HBV virions and naked capsids.
Conclusions: We provided experimental evidence that virions and naked capsids can egress via two distinct, yet
overlapping, pathways. Unlike the secretion of naked capsids, HBV virion secretion is highly CRM1- and microtubuledependent. CRM1 is well known for its involvement in nuclear transport in literature. To our knowledge, this is the
frst report that CRM1 is required for virion secretion. CRM1 inhibitors could be a promising therapeutic candidate for
chronic HBV patients in clinical medicine. - 中文關鍵字:
- 英文關鍵字: Hepatitis B virus (HBV), HBV core protein (HBc), HBc capsids, CRM1 (chromosome region maintenance 1), Spike, Virion secretion, Naked capsids, Microtubule, Proximity ligation assay (PLA), Therapeutic treatment