A mutant HIV-1 Tat protein termed Nullbasic has a strong antiviral activity via three independent mechanisms that disrupts; 1) reverse transcription of the viral RNA genome into a DNA copy, 2) HIV-1 Rev protein function required for unspliced and singly spliced viral mRNA transport from the nucleus and 3) HIV-1 transcription by RNA Polymerase II. The Nullbasic protein is derived from the HIV-1 subtype B strain BH10 and has only been tested against other HIV-1 subtype B strains. Using a gammaretroviral vector as a gene delivery system, fusion forms of Nullbasic, Nullbasic-mCherry and Nullbasic-ZSGreen1, were tested against representative HIV-1 strains from subtypes C, D and A/D recombinant to determine if it can inhibit their replication. The results show that Nullbasic-mCherry inhibits Tat-mediated transactivation and virus replication of all the HIV-1 strains tested in TZM-bl cells. Importantly, Nullbasic-ZSGreen1 inhibits replication of the HIV-1 strains in primary CD4+ T cells without affecting cell proliferation, cytotoxicity or level of apoptotic cells. However, long-term expression of Nullbasic protein in the cells is required to provide a strong and durable antiviral activity. Nullbasic antiviral activity in vivo has not been studied. Therefore, a humanized mouse model was established to test Nullbasic antiviral activity in vivo. This animal model harbors human CD4+ T cells for a short period, and is therefore suitable for testing Nullbasic antiviral activity against an acute HIV-1 infection. The results also indicate that Nullbasic-ZSGreen1 inhibits HIV-1 replication in vivo. Gene delivery to target cells using gammaretroviral system may raise safety concerns due to its oncogonic potential. Therefore, a safer and better vector, a lentiviral vector, was optimized to deliver Nullbasic to T cells. However, Nullbasic delivery to T cells using a lentiviral system was problematic because Nullbasic itself inhibits lentivirus replication. Therefore, we optimized the nullbasic delivery method by using viral and cellular proteins antagonistic to Nullbasic. Transduction efficiency of Jurkat T cells by Nullbasic lentiviral VLP was optimal when addition of Tat and DDX1 in combination with spinoculation method was used. Nevertheless, this method did not improve transduction of primary CD4+ T cells by Nullbasic lentiviral VLP. ii Overall, Nullbasic has antiviral activity against all strains from the HIV-1 tested in vitro and inhibits acute HIV-1 infection in vivo. Therefore, Nullbasic may have utility in a future gene therapy approach.