INHIBITION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REPLICATION IN-VITRO BY A NOVEL COMBINATION OF ANTI-TAT SINGLE-CHAIN INTRABODIES AND NF-KAPPA-B ANTAGONISTS

Human immunodeficiency virus type 1 (HIV-1) Tat, an early regulatory p rotein that is critical for viral gene expression and replication, tra nsactivates the HIV-1 long terminal repeat (LTR) via its binding to th e transactivation response element (TAR) and, along with other cellula r factors, increases viral transcription initiation and elongation, Ta t also superactivates the HIV-1 promoter through a TAR-independent mec hanism, including tumor necrosis factor alpha-induced and protein kina se C (PKC)-dependent activation of NF-kappa B, and inhibitors of Tat a nd NF-kappa B cooperatively down-regulate this Tat-mediated LTR supera ctivation. In this study, a combined pharmacologic and genetic strateg y using two PRC (NF-kappa B) inhibitors, pentoxifylline (PTX) and Go-6 976, and a stably expressed anti-Tat single-chain intracellular antibo dy (sFv intrabody) was employed to obtain cooperative inhibition of bo th HIV-1 LTR-driven gene expression and HIV-1 replication. Treatment o f cells with PTX and Go-6976 resulted in cooperative inhibition of bot h HIV-1 LTR-driven gene expression and HIV-1 replication, In addition, the combined use of anti-Tat sFv intrabodies and tile two NF-kappa B inhibitors retained the virus in the latent state for as long as 45 da ys, The combined treatment resulted in more durable inhibition of HIV- 1 replication than was seen with the NF-kappa B inhibitors alone or th e anti-Tar sFv intrabodies alone. Together, these results suggest that in future clinical gene therapy trials, a combined pharmacologic and genetic strategy like the one reported here may improve the survival o f transduced cells and prolong clinical benefit.