SV40-derived vectors provide effective transgene expression and inhibitionof HIV-1 using constitutive, conditional,and pol III promoters

Vectors based on recombinant SV40 viruses (rSV40) are highly effective in d elivering transgene expression driven by constitutive promoters. We tested here whether these vectors could be used with conditional promoters and pro moters using RNA polymerase III transcription, with inhibition of HIV-1 by Tat activation response (TAR) decoys as a functional measure of effective t ransgene delivery and activity. TAR decoys inhibit HIV-1 Tat, a trans-activ ator of HIV-1 transcription. Tat acts early in the viral replicative cycle and is essential for efficient viral replication. We evaluated rSV40 gene d elivery using two different inhibitors of Tat. One was a dual function poly TAR gene encoding 25 sequential TAR elements (TAR,,), plus an antisense tat , driven either by HIV-1 long terminal repeat (HIV-LTR) as a conditional pr omoter, or by cytomegalovirus immediate-early promoter (CMV-IEP) as a const itutive promoter. The other inhibitor was a single TAR decoy, driven by the U6 small nuclear RNA promoter (U6-P). These decoys were delivered to unsel ected cells in two different human T lymphocyte lines and to unstimulated p rimary human peripheral blood mononuclear cells (pbmc). Gene delivery was c onfirmed by PCR, and expression by FIT-PCR. By in situ hybridization analys is, > 95% of cells were transduced. These transgene constructs protected al l cell types tested from HIV-1, as measured by syncytia formation and p24 a ntigen release. Somewhat better inhibition of HIV-1 replication was achieve d with HIV-1 long terminal repeat (HIV-1 LTR) as a conditional promoter tha n with the constitutive CMV-IEP. The U6-P was also very effective, driving a TAR, transcript. Cell viability was not detectably affected by TAR decoy expression. Thus, rSV40 vectors effectively deliver HIV-1-inhibitory RNAs u sing either constitutive or conditional pol II promoters, or using a pol II I promoter. The versatility of this gene delivery system may prove to be us eful in anti-HIV-l therapeutics.