Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles

PLoS Pathogens

Wen-Chang Chang¹  Szu-Yao Wu²  Hung-Cheng Li³  Chiaho Shih⁴  Pei-Yi Su⁴  Er-Yi Huang⁴  Ching-Chun Yang⁴  Young-Sun Lin⁴ 

[1] Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan;Graduate Institute of Molecular Medicine, School of Medicine, National Taiwan University, Taipei, Taiwan;Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan;Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

关键词: SV40;    Viral packaging;    Cytoplasm;    DNA replication;    Hepatitis B virus;    Small interfering RNAs;    Cotransfection;    Subcellular localization;   

DOI  :  10.1371/journal.ppat.1001162

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

It remains unclear what determines the subcellular localization of hepatitis B virus (HBV) core protein (HBc) and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD) of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS), while ARD-II and ARD-IV behave like two independent nuclear export signals (NES). This conclusion is based on five independent lines of experimental evidence: i) Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii) These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT). iii) By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv) We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1), which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v) HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel TAP-dependent NES.

【 授权许可】

CC BY