Involvement of host cellular multivesicular body functions in hepatitis B virus budding

doi:10.1073/pnas.0704000104

Involvement of host cellular multivesicular body functions in hepatitis B virus budding

*Institute for Molecular Virology,
^¶McArdle Laboratory for Cancer Research, and
^§Howard Hughes Medical Institute, University of Wisconsin, Madison, WI 53706

Contributed by Paul Ahlquist, April 30, 2007 (received for review March 23, 2007)

Abstract

Hepatitis B virus (HBV) is a major human pathogen that chronically infects ≈350 million people, causing liver disease and liver cancer. HBV virions bud into an endoplasmic reticulum (ER)-associated intracellular compartment, but the mechanisms of HBV assembly, budding, and release remain poorly understood. Budding of retroviruses and some other enveloped RNA viruses from plasma membranes requires host functions involved in protein sorting into late endosomal multivesicular bodies (MVBs). To determine whether budding of DNA-containing HBV virions at intracellular membranes also involves MVB functions, we used immunofluorescence to show that, in human hepatoma cells, HBV envelope protein colocalizes with MVB proteins AIP1/ALIX and VPS4B. We also found that a dominant negative (DN) AIP1 mutant inhibited production and/or release of enveloped virions without significant effects on intracellular nucleocapsid formation, whereas DN VPS4B inhibited both nucleocapsid production and budding. By contrast, DN AIP1 and VPS4 had no effect on the efficiency of release of enveloped, nucleocapsid-lacking HBV subviral particles, which are produced in vast excess over virions, and dramatically increased the release of unenveloped, naked nucleocapsids by an apparently nonlytic route. Thus, host MVB functions are required for efficient budding and release of enveloped HBV virions and may be a valuable target for HBV control. Moreover, HBV enveloped virions, enveloped subviral particles, and unenveloped nucleocapsids are all released by distinct pathways with separate host factor requirements.

Footnotes

^‖To whom correspondence should be addressed at:
Institute for Molecular Virology, University of Wisconsin, 1525 Linden Drive, Madison, WI 53706.
E-mail: ahlquist{at}wisc.edu
Author contributions: T.W. and P.A. designed research; T.W., E.M.S., A.N., and M.S. performed research; T.W., E.M.S., A.N., M.S., S.K., and P.A. analyzed data; and T.W. and P.A. wrote the paper.
↵ ^†Present address: School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706.
↵ ^‡Present address: School of Medicine and Public Health, University of Wisconsin, Madison, WI 53706.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0704000104/DC1.
Abbreviations:

DN,

dominant negative;

ER,

endoplasmic reticulum;

EPR,

endogenous viral polymerase reaction;

ESCRT,

endosomal sorting complexes required for transport;

HBcAg,

HBV core antigen;

HBsAg,

HBV envelope protein surface antigen;

HBV,

hepatitis B virus;

MVB,

multivesicular body;

NC,

nucleocapsid;

pgRNA,

pregenomic RNA;

SVP,

subviral particle;

VPS,

vacuolar protein sorting.