Preliminary Study On HBV And HIV Pseudovirus Vectors

Construction of infectious Hepatitis B virus plasmids Partially repeated Hepatitis B virus (HBV) adw2 genome was cloned into plasmid pUC19 and pcDNA3.1 respectively, resulting in recombinant plasmids pUV-HBVxm (harbouring 1.6x HBV genome) and pCDNA-HBVxm (harbouring 1.4x HBV genome). Both plasmids could produce fall length HBV pregenomic RNA from endogenous HBV promoter. After both plasmids was transfected into human hepatoma cell line HepG2, the HBV antigens were detected in the conditioned media. These plasmids served as positive control and basis of other constructs in the subsequent study.Establishment of pseudovirus paskaging cell lines A helper plasmid HelperHBV was constructed which contains partially repeated HBV adw2 genome with functional X,C,P,S genes, but no packaging signalε. This helper plasmid was transfected into HepG2 and G418 was used to select stable transfectant cell lines expressing HBV surface and e antigens. Two cell lines highly expressing HBsAg and HBeAg were obtained.Construction of a pseudovirus vector A pseudovirus vector PseudoHBV was made based on the HBV genome, in which the HBV X,C,P genes were disabled by introduction of either frameshift or nonsense mutations and the S gene was replaced by a reporter gene enhanced fluorescence green protain (EGFP). This vector retains partially repeated (1.4x) HBV genome with functional packaging signalεand other HBV cis-acting element such as endogenous pregenomic promoter and the single polyadenylation site, thus enable it packable and replicable under certain situation to produce a pseudovirus with 3.2 kb genome harbouring no functional HBV genes but a reporter gene egfp. After transfection of PseudoHBV into the above packaging cell line, green fluorescrence can be observed in 24 hours. When PseudoHBV and the helper plasmid HelperHBV were cotransfected into HepG2 cells, the same result was obtained. In selecting with G418, we generated 12 cell lines expressing HBsAg, HBeAg and EGFP.Characterization of pseudovirus The above derived pseudovirus was characterized by different means including electron microscope and quantitative polymerase chain reaction (Q-PCR). The electron microscope examination revealed 42nm particles in the conditioned medium of the HepG2 cell lines stably transfected with the pseudovirus plasmid and helperHBV plasmid. To evaluate the efficiency of virus secretion from the above stable HepG2 cell lines, we developed a real-time quantitative PCR to determine the HBV genome copy number. In this test, the HBV DNA copy number was found to be about 10~3/ml in the conditioned media of pseudovirus cell lines, while no HBV DNA was detected in the conditioned media of helper cell lines.Construction of pseudotyped Human Immunodeficiency Virus-1 (HIV-1) Similar strategy was adopted to develop a pseudotyped HIV-1 virus vector. HIV-1 NL4-3 infectious clone was used to construct the pseudotyped lentivirus vector by replacing the HIV-1 env gene with a reporter gene egfp, and this recombinant genome was packed by HIV gag protein and secreted with vesicular stomatitis virus glycoprotein (VSV-G) as its membrane protein. The viral vector and the VSV-G expressing plasmids were cotransfected into 293FT cells, and 24 hours later green fluorescence could be observed. The conditioned medium harvested 72 hours after cotransfection contained adequate amount of virus which can infect naive 293 FT cells efficiently and caused fluorescence expression. When anti-HIV drug AZT was applied in the cell culture after cotransfection, the fluorescence after infection reduced drastically. So the current system can serve as a safe, high-throughout anti-HIV drug screening-tool.