Human herpesvirus-8 encoded Kaposin: subcellular localization using immunofluorescence and biochemical approaches.
Human herpesvirus-8 (HHV-8) has been causally linked to the development of Kaposi's sarcoma (KS). DNA sequence analysis of the viral genome revealed a total of 81 open reading frames (ORF). Interestingly, only a small subset of these ORFs has been shown to be transcribed in cells latently infected with HHV-8 and in cells of the KS lesions. Among the genes active during latency, kaposin, is noted for its abundance and ability to transform cells in culture, thus implicating a potential role in KS pathogenesis. This has prompted us to undertake an investigation on elucidating the mechanism(s) by which Kaposin brings about transformation of cells. Towards this goal, we have generated an eukaryotic expression plasmid encoding Kaposin (Kap). As Kaposin is predicted to be a type II membrane protein, several strategies were utilized to address this, including the generation of Kaposin with the Flag (FL) epitope (DYKDDDDK) at the C-terminus of the protein (Kap-C-FL). Antibodies specific for Kaposin (kap-2), recognized both Kaposin and Kaposin-Flag, while antibodies against the Flag epitope recognized only Kaposin-Flag. Transfection of Kap and Kap-C-FL expression plasmid DNA into NIH3T3 cells resulted in cellular clones that exhibited a phenotypic property of transformation by forming large, multiclustered cells, when grown on soft agar. Because there is controversial data regarding the localization of Kaposin in cells, we examined the subcellular localization of Kaposin using confocal microscopy. We observed that Kaposin and Kaposin-Flag showed an intense staining surrounding the nucleus. Although there was no staining at the cell membrane of transfected cells, FACS analysis using kap-2 or Flag antibodies, under nonpermeable conditions, showed positivity. Cell fractionation studies further showed that the majority of Kaposin was detected in the nuclear fraction by Western blot analysis. The cytoplasmic and detergent soluble membrane fractions did not show Kaposin protein; however, a small amount was detected in the detergent insoluble membrane fraction. Taken together, these results suggest that Kaposin exhibits multicompartmental localization in cells.
Tomkowicz
Brian
B
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Singh
Satya P
SP
Cartas
Maria
M
Srinivasan
Alagarsamy
A
D016475:3T3 Cells; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D002455:Cell Division; D002467:Cell Nucleus / Q000737:chemistry; D002472:Cell Transformation, Viral; D002999:Clone Cells; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D019288:Herpesvirus 8, Human / Q000472:pathogenicity; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry; D051379:Mice; D018613:Microscopy, Confocal; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology
D000914:Antibodies, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C120136:kaposin B protein, Human herpesvirus 8