关键词: HIV-1, 病毒感染因子, 载脂蛋白B mRNA编辑酶催化多肽样蛋白3G

Abstract: Objective To study the restriction of HIV-1 and the Vif-deficient strain.
Methods Viruses of HIV-1 wild type(BH10 WT) or the Vif-deficient strain(BH10 ΔVif) produced from transfected 293T cells were used to infect various cell lines, including MT4 and H9. The results were determined by reverse transcriptase(RTase) assay. Molecular-cloning technique was used to construct expression plasmid pEGFP-3G which express APOBEC3G with a C-terminal GFP tag. The virus strains of BH10 WT or BH10 ΔVif cotransfection with different dose of pEGFP-3G in 293T cells, and the expression of APOBEC3G-GFP was observed by live cell fluorescence microscopy. The infectivity of virus was determined by multinuclear activation of galactosidase indicator(MAGI) assay and RTase assay.
Results BH10 WT replication in MT4 cells showed much faster replication kinetics than that in H9 cells, with peak RT values being observed as early as 4 days post-infection. RT activity of BH10 ΔVif in H9 cells was suppressed almost to the level of negative control and that in MT4 cells was observed on the 12th day. GFP-APOBEC3G and a GFP-only control localized to the cytoplasmic and cell-wide, respectively. The titer of BH10 ΔVif by MAGI assay is about 2.75×10⁴ U/mL. The titer of viruses, produced in 293T cells by cotransfection with increasing amounts of APOBEC3G, was significantly reduced from 1.48×10³ U/mL to 0.33×10³ U/mL. The infectivity of BH10 ΔVif produced in the presence of 0.2 μg of co-transfected pEGFP-3G was twenty fold less infectious than BH10 ΔVif produced in the absence of APOBEC3G.
Conclusion The anti-HIV activity of APOBEC3G was dose-dependent, and HIV-1 Vif has the essential role in the virus replication. Based on the results we would construct a new platform to accurately and promptly select efficacious drugs.

Key words: HIV-1, viral infectivity factor, apolipoprotein B mRNA editing enzyme catalytic polypeptide like 3G