Nuclear export and import of human hepatitis B virus capsid protein and particles

Hung Cheng Li, Er Yi Huang, Pei Yi Su, Szu Yao Wu, Ching Chun Yang, Young Sun Lin, Wen Chang Chang, Chiaho Shih

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

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 TAPspecific 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.

Original languageEnglish
Article numbere1001162
JournalPLoS Pathogens
Volume6
Issue number10
DOIs
Publication statusPublished - Oct 1 2010
Externally publishedYes

Fingerprint

Cell Nucleus Active Transport
Capsid Proteins
Hepatitis B virus
Arginine
Nuclear Export Signals
Proteins
Polyomavirus Transforming Antigens
Nuclear Localization Signals
Viral Tumor Antigens
Mutant Proteins
NFI Transcription Factors
Viral Core Proteins
Protein Transport
Hydrodynamics
Hepatitis B Surface Antigens
Virus Replication
Nuclear Proteins
Cell Nucleus
Fluorescence Microscopy
Immunoprecipitation

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Molecular Biology
  • Genetics
  • Virology

Cite this

Li, H. C., Huang, E. Y., Su, P. Y., Wu, S. Y., Yang, C. C., Lin, Y. S., ... Shih, C. (2010). Nuclear export and import of human hepatitis B virus capsid protein and particles. PLoS Pathogens, 6(10), [e1001162]. https://doi.org/10.1371/journal.ppat.1001162

Nuclear export and import of human hepatitis B virus capsid protein and particles. / Li, Hung Cheng; Huang, Er Yi; Su, Pei Yi; Wu, Szu Yao; Yang, Ching Chun; Lin, Young Sun; Chang, Wen Chang; Shih, Chiaho.

In: PLoS Pathogens, Vol. 6, No. 10, e1001162, 01.10.2010.

Research output: Contribution to journalArticle

Li, HC, Huang, EY, Su, PY, Wu, SY, Yang, CC, Lin, YS, Chang, WC & Shih, C 2010, 'Nuclear export and import of human hepatitis B virus capsid protein and particles', PLoS Pathogens, vol. 6, no. 10, e1001162. https://doi.org/10.1371/journal.ppat.1001162
Li, Hung Cheng ; Huang, Er Yi ; Su, Pei Yi ; Wu, Szu Yao ; Yang, Ching Chun ; Lin, Young Sun ; Chang, Wen Chang ; Shih, Chiaho. / Nuclear export and import of human hepatitis B virus capsid protein and particles. In: PLoS Pathogens. 2010 ; Vol. 6, No. 10.
@article{2131f354707440b3a0af09e468c27dca,
title = "Nuclear export and import of human hepatitis B virus capsid protein and particles",
abstract = "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 TAPspecific 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.",
author = "Li, {Hung Cheng} and Huang, {Er Yi} and Su, {Pei Yi} and Wu, {Szu Yao} and Yang, {Ching Chun} and Lin, {Young Sun} and Chang, {Wen Chang} and Chiaho Shih",
year = "2010",
month = "10",
day = "1",
doi = "10.1371/journal.ppat.1001162",
language = "English",
volume = "6",
journal = "PLoS Pathogens",
issn = "1553-7366",
publisher = "Public Library of Science",
number = "10",

}

TY - JOUR

T1 - Nuclear export and import of human hepatitis B virus capsid protein and particles

AU - Li, Hung Cheng

AU - Huang, Er Yi

AU - Su, Pei Yi

AU - Wu, Szu Yao

AU - Yang, Ching Chun

AU - Lin, Young Sun

AU - Chang, Wen Chang

AU - Shih, Chiaho

PY - 2010/10/1

Y1 - 2010/10/1

N2 - 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 TAPspecific 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.

AB - 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 TAPspecific 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.

UR - http://www.scopus.com/inward/record.url?scp=78449260869&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78449260869&partnerID=8YFLogxK

U2 - 10.1371/journal.ppat.1001162

DO - 10.1371/journal.ppat.1001162

M3 - Article

C2 - 21060813

AN - SCOPUS:78449260869

VL - 6

JO - PLoS Pathogens

JF - PLoS Pathogens

SN - 1553-7366

IS - 10

M1 - e1001162

ER -