64. Characterization of the Mechanism of Action of the Anti-HIV-1 Transgene F12-Vif

Abstract

The F12-vif is a mutant isoform of the viral infectivity factor (vif) gene, derived from the natural variant of the F12-HIV proviral DNA, that we are currently testing as transgene in an anti-HIV gene therapy approach. Wild type Vif is normally required for high HIV-1 infectivity because it counteracts the action of the cellular inhibitor APOBEC3G (AP3G). AP3G is a cytidine deaminase that, in the absence of Vif, is incorporated into HIV-1 particles and induces strong G to A hypermuations in the nascent proviral DNA. Vif overcomes the action of AP3G by inducing, without the need of any other viral components, its proteasome-dependent degradation. We have previously established that T lymphocytes transduced with either retroviral or lentiviral vectors carrying F12-vif are protected from HIV-1 infection. Contrary to our hypothesis that F12-Vif functioned as a dominant negative mutant by rescuing the innate antiviral activity of AP3G, we have demonstrated that the dominant negative feature of F12-Vif does not affect the stability of AP3G. To elucidate the mechanism of F12-Vif action, we have looked at the first steps of HIV-1 life cycle in T-cells mock tansduced and transduced with F12-Vif. We have found that F12-Vif expressing cells integrate equal number of HIV-1 proviral DNA, but release HIV-1 particles few in number and with reduced infectivity compared to control cells. In addition, we have observed from 3 to 8 fold more F12-Vif into viral particles compared to wild type Vif. Finally, to reveal the protein domain of F12-Vif responsible of HIV-1 inhibition, we have constructed three chimeric proteins composed by wild type and F12-Vif domains. Results of this analysis will be presented.