Non-permissive cell production for the study of HIV-1 vif

APOBEC3G (Apolipoprotein B mRNA Editing enzyme, Catalytic polypeptide-like 3G) is an anti-HIV-1 vif cellular factor that modifies minus strand of viral DNA during reverse transcription. To assess the interaction of this cellular factor with HIV-1 vif, natural non-permissive cells, such as H9 and PBMC cells, are used. Since the transfection of these cells with test DNAs is very inefficient, we are to use non-permissive cells which can stably produce high levels of APOBEC3G. Sort of non-permissive cells for this purpose were 293 T cells. 293T cells were used for producing non-permissive cells. This was carried out by transfection of 293T cells using PcDNA-APO3G molecular clone. Then the APOBEC3G expression was checked by western blotting. Also the HIV-1 replication and infectivity were assessed using RT and MAGI assay, respectively. A number of 293T cell lines that express a human antiHIV-1 factor, APOBEC3G, were stablished. Immunoblotting analysis proved that 4 out of 7 examined cell clones readily express APOBEC3G. In particular, two clones (i.e. A3G-C1 and -C4) were found to produce much higher levels of functional APOBEC3G relative to that by pooled cell clones. The transfection efficiency of all cell clones was similar to that of the parental cells, with a potential of producing a comparable level of virions upon transfection of wild type and vif-minus proviral DNA clones. Furthermore, the expression level of APOBEC3G in the best cell line (A3G-C1) was much higher than that of APOBEC3G-positive lymphocyte cell line and peripheral blood mononuclear cells. Finally, the incorporation of APOBEC3G into virions produced in A3G-C1 was monitored. Upon transfection of vif-minus proviral clone, but not of wild type, APOBEC3G was easily detected in progeny viral particles. These Results indicated that our new A3G-C1 cell line is eminently useful for studying various study on the interactions of human APOBEC3G and HIV-1 vif