Morphological Identification And Live-cell Observation Of Fluorescent Hepatitis B Virus Particles

【Objective】To generate recombinant HBV fluorescently labeled with biarsenical dye and analyze its morphological charateristics,and to observe its biological behavior in living cells.【Methods】A human hepatoblastoma cell line was transfected by liposome with pTHBV1.3-mTC1,a vector that has been successfully prepared by insertion of a TC-tag into plasmid 1.3-overlength HBV.By using PCR based site directed mutagenensis, a TC-tag that could be specifically bound with a biarsencal fluorescent dye was inserted near the immunodominant c/e site of HBV core protein. 48 hours post transfection,HBsAg expressed in the cytoplasma of transfected cells could be detected by indirect immuofluorescence for its distribution.Medium from transfected cells was also measured by using enzyme linked immunosorbent assay kit.In the meanwhile, culture medium from the transfected ones was collected and then seperated and purified by discontinuous surcrose density gradient centrifugation.Subsequently, purified products were incubated with an equal volume of diluted goat anti-HBsAg.The resulting precipitates were examined by immunoelectronic microscope as described previoursly. Samples conditioned by growing HepG2 and 2.2.15 cells were served as negative and positive control,respectively for comparative purposes. In live cell studies,we employed Oregon Green ? 488 Taxol to label microtubles.The process of cytoplamic trafficking was visualized under time-lapse confocal microscope in living cells.【Results】Indirect immunofluorescence suggested that the intensity of red fluorescence from TC-tagged core protein expressed in pTHBV1.3-mTC1-thansfected cells was greater than in pTHBV1.3-thransfected cells which was predominantly distributated in cytoplasma and nucleoplasma.The electron micrographs revealed that in pTHBV1.3-mTC1-thansfected cells HBsAg could be expressed and eventually self-assembly to be 12-nanometer spherical paritcles,whereas no HBV-like particles were obtained in negative and pTHBV1.3-mTC1-transfected groups.Fluorescent HBV particles began to attach and entry the HepG2 cells at appoximately 3 h p.i. by time-lapse confocal microscope. The fluorescent signals we have observed were aggregated at the cytoplasmic area , minority of which had reached the nuclear periphery.We also displayed that the transport of fluorescent HBV particles is occurring in a microtuble-dependent manner.These data preliminarily demonstrated that the recombinant HBV retained the antigenicity and infectivity.【Conclusion】TC-tagged core protein could specifically fluorescence in pTHBV1.3-mTC1-transfected cells. Furthermore, the insertion of TC tag is unable to affect the expression of the suface antigen and core protein.Biarsenical compouds, a novel fluorescent labeling technique, facilitate the observation of HBV tracking in living cells. It could be served as a highly valuable tool for studying dynamic trafficking of fluorescent HBV particles as well as the interaction with the host cells.