Inhibition of human immunodeficiency virus type I by Tat/Rev-regulated expression of cytosine deaminase, interferon alpha 2, or diphtheria toxin compared with inhibition by transdominant Rev

A retroviral vector was designed to express toxic proteins only in the pres ence of the HIV-1 Rev and/or Tat protein(s). The design of this vector inco rporates an HIV-specific expression cassette that consists of three element s: the U3R region of the HIV-1 IIIB LTR provides the promoter and Tat-respo nsive element, a modified intron derived from the human c-src gene facilita tes the splicing of inserted genes, and the HIV-1 RRE region enhances the t ransport of unspliced mRNAs. To further limit potential readthrough transcr iption, the expression cassette was inserted in the reverse transcriptional orientation relative to the retroviral vector LTR. Three different genes, interferon alpha 2, diphtheria toxin (DT-A), and cytosine deaminase, were i nserted into this vector. Tat and Rev inducibility was demonstrated directl y by a >300-fold induction of interferon production and functionally by a d ecrease in colony-forming units when a Tat and Rev expression vector was ti tered on HeLa cells harboring the inducible DT-A cassette. The Tat-inducibl e cytosine deaminase gene was tested in the Sup-T1 T cell line and shown to inhibit HIV-1 production only when engineered cells were grown in the pres ence of 5-fluorocytosine. To test the ability of this system to inhibit HIV -1 infection in bulk PBL cultures, a series of transduction and challenge e xperiments was initiated with both the interferon and DT-A vectors. Protect ion against infection was documented against three HIV strains in PBLs. Las t, the interferon and DT-A vectors were compared with a vector encoding a t ransdominant Rev protein and were shown to mediate equal or greater inhibit ion of HIV-1.