An Experimental Study On Visualization Of Hepatitis B Virus In Living Cells

Production of virus tagged with green fluorescent protein (GFP) contributes tovisualizing and studying virus in living cells. But GFP tag, as a 238-amino acide largeinserted fragment, is not suitable for labeling Hepatitis B virus (HBV) that is a compactvirion with limited internal space. It was recently reported that protein of interestgenetically inserted with a smaller size tetracysteine (TC) tag could be specially labeled bya biarsenical fluorescent dye in living cells. This biarensical labeling approach offers us anew opportunity to fluorescently label HBV virion for visualizing and studying HBV inliving cells.【Objective】To generated one recombinant HBV fluorescently labeled with biarsenicaldye, which could be visualized and studied in living cells.【Methods】By using a molecular clone and PCR based site directed mutagenensis, a TCtag that could be specially bound with a biarsenical fluorescent dye was genetically insertednear the immunodominant c/el site of HBV core protein encoded by 1.3 time HBV vector.The recombinant HBV vectors were transfected into HepG2 cells to express TC-taggedcore proteins. The expression and subcellular localization of TC-tagged core proteins wereanalyzed by western blotting, immunofluorescence and biarsenical dye labeling. Furthermore, these transfected cells expressing TC-tagged core proteins were stained withbiarsenical dye, and then maintained to produce fluorescent HBV virions. Confocalmicroscopy and transmission electron microscopy (TEM) were used to analyze theproduction of fluorescent HBV virions. And the infectivity of fluorescent HBV virions toDMSO-treated HepG2 cells was investigated. Finally, the translocation of fluorescent HBVin living cells was observed by time-lapse confocal microscopy.【Results】The HBV vector encoding TC-tagged core protein were successfullyconstruted. The cells transfected with the HBV vector could express TC-tagged coreproteins and produce mature HBV virions. Moreover, TC-tagged core proteins bound withbiarsenical dye could specifically fluoresce in cells and be incorporated into nucleocapsidto form fluorescent HBV virions. The recombinant fluorescent HBV virions retained theirinfectivity as wild-type ones. The translocation of fluorescent HBV in living cells could bedirectly and conveniently observed by time-lapse confocal microscopy.【Conclusion】To the best of our knowledge, this is the first time to generate fluorescentHBV virions with biarsenical labeling and to visualize their trafficking in living cells. Thefluorescent HBV may become one highly valuable tool for further studying detaileddynamic processes of HBV life cycle and interaction of HBV with host in live-imagingapproach.