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.

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Desmosomal junctions contain two classes of desmosomal cadherin, the desmocollins and the desmogleins, each of which occurs as three distinct isoforms. To investigate the role of the "skin-type" desmosomal cadherins (desmocollin 1 and desmoglein 1) in the formation of keratinized epithelial structures, we have now cloned full-length mouse desmocollin 1 complementary deoxyribonucleic acid and examined the expression of desmocollin 1 and desmoglein 1 and messages during murine embryonic development by in situ hybridization. In the general body epidermis, desmocollin 1 and desmoglein 1 transcripts both showed considerable upregulation at 15.5 d, which is after the onset of stratification and before the start of keratinization. Before this the epidermis expressed low levels of desmocollin 1 message, although the desmoglein 1 signal was always stronger and more extensive. In the tongue, expression of desmocollin 1 message occurred several days after desmoglein 1 and coincided with the formation of the keratinizing filiform papillae. Desmoglein 1 message was also detected in epithelial tissues in which desmocollin 1 was absent, suggesting that expression of the two "skin-type" desmosomal cadherins was not tightly coupled during embryonic development. Human desmocollin 1 monoclonal antibodies that cross-reacted with mouse skin and tongue indicated that desmocollin 1 protein was first expressed in those outermost epithelial cells destined to form the keratinized layers of the stratum corneum or the papillae. The results suggest that expression of desmocollin 1 is closely associated with the keratinization of epithelial tissues during mouse development.

A third human desmocollin, designated DSC3, was identified in foreskin epidermis by reverse transcriptase-polymerase chain reaction (PCR) using degenerate desmocollin primers. cDNA clones covering the entire coding sequence of the longer DSC3 splice variant were isolated and sequenced. Sequence comparisons indicated that this new desmocollin showed greater homology (67% amino acid identity) with the original human desmocollin (now designated DSC2) than with DSC1 (52% amino acid identity) although it had a unique potential cell adhesion recognition site (YAS). DSC3 was assigned to chromosome 18 by PCR analysis of rodent-human somatic cell hybrids, where it appears to be closely linked to all the other desmosomal cadherin genes. The expression of the three human desmocollins was examined in foreskin epidermis by in situ hybridization with 3'-untranslated riboprobes and by immunofluorescence with isoform-specific anti-peptide antibodies. DSC1 was present in the upper spinous/granular layers but not in the basal/lower spinous layers of the tissue. DSC2 and DSC3 were present in most of the living layers of the epidermis. DSC1 was not detected in any of the nonkeratinizing human epithelia examined (buccal mucosa, cervix, esophagus), indicating that it is specific for the keratinizing epithelium of the epidermis. However, all these internal epithelia expressed DSC2 and DSC3, and both were present in most of the living layers of the tissues including the basal layers.