Our previous results have demonstrated that T-cell lines and primary T lymphocytes transduced with a Tat-dependent HIV-based lentiviral vectors expressing the mutant isoform of the vif gene, F12-vif, are protected from HIV-1 infection. F12-Vif is a 192-aa natural variant polypeptide owing 14 unique amino acid substitutions. The substitutions are randomly scattered along the entire sequence with the exception of a 5-aa cluster located at positions 127, 128, and 130|[ndash]|132. None of the 14 aa substitutions is present in the SOCS box that recruits the E3 ubiquitin ligase responsible of APOBEC3G (AP3G) degradation during HIV infection. In line with this notion, we have shown that the antiviral function of F12-Vif is not due to a dominant negative feature of the mutant in regards to the Vif-mediated degradation of AP3G rather to some other unknown means. Therefore, in the effort to elucidate the F12-Vif mechanism of action, we started to identify the protein domain of F12-Vif responsible of HIV-1 inhibition. To this end, we have constructed three chimeric genes (Chim1, Chim2 and Chim3) composed by wild-type and F12-vif regions. T cell lines and cord blood derived CD4+T lymphocytes were transduced with the lentiviral vectors expressing the chimeric genes and then challenged with both X4 and R5 HIV-1 strains. We show that 45 amino acids in the C-terminal domain of the F12-Vif mutant are sufficient to exert anti-viral effect in transduced cells. In contrast to F12-Vif, Chim3 does not allow the rescue of the replication of a vif-deficient HIV-1 in the context of either X4 or R5 tropism in non permissive cells. This specific feature renders Chim3 a truly dominant negative protein more suitable than F12-Vif for an anti-HIV gene therapy approach
