Intracellular localization of the hepatitis B virus HBx protein

National Institute for Medical Research, Division of Membrane Biology, The Ridgeway, Mill Hill, London NW7 1AA, UK 1 Department of Medicine 2 and Department of Histopathology 5 , Imperial College School of Medicine, St Mary’s Campus, South Wharf Road, London W2 INY, UK National Institutes of Health, Hepatitis Viruses Section, LID, NIAID, Building 7, Room 206, Bethesda, MD 20892, USA 3 Division of Medical and Molecular Genetics, Guy’s, King’s and St Thomas’s School of Medicine, London SE1 9RT, UK 4 Author for correspondence: Frank Henkler. Present address: Institute for Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Fax +49 711 685 7484. e-mail Frank.Henkler{at}po.uni-stuttgart.de The hepatitis B virus (HBV) X protein (HBx) was originally suggested to be a viral transcriptional activator, but its functional mechanisms are still unclear. In this study we have analysed the intracellular localization of HBx in transfected cells and demonstrate that its compartmentalization is dependent on overall expression levels. HBx was exclusively or predominantly localized in the nuclei in weakly expressing cells. However, elevated cellular levels correlated with its accumulation in the cytoplasm, suggesting that the capacity of HBx for nuclear compartmentalization might be limited. Cytoplasmic HBx was detected either as punctate granular staining or in dispersed, finely granular patterns. We have further analysed the detailed cytoplasmic compartmentalization, using confocal microscopy, and show no association with the endoplasmic reticulum, plasma membrane or lysosomes, but a substantial association of HBx with mitochondria. However, a major fraction of cytoplasmic HBx did not localize in mitochondria, indicating the presence of two distinctly compartmentalized cytoplasmic populations. Furthermore, high levels of HBx expression led to an abnormal mitochondrial distribution, involving clumping and organelle aggregation, which was not observed at lower expression levels. The data presented here provide novel insights into the compartmentalization of HBx and may prove important for future evaluations of its functions, both in the viral life-cycle and in the pathology of HBV-related liver disease.