Inhibition of Human Immunodeficiency Virus Type 1 by Tat/Rev-Regulated Expression of Cytosine Deaminase,Interferon alpha2,or Diphtheria Toxin Compared with Inhibition by Transdominant Rev

Abstract

A retroviral vector was designed to express toxic proteins only in the presence of the HIV-1 Rev and/or Tat protein(s). The design of this vector incorporates an HIV-specific expression cassette that consists of three elements: the U3R region of the HIV-1 IIIB LTR provides the promoter and Tat-responsive element, a modified intron derived from the human c-src gene facilitates the splicing of inserted genes, and the HIV-1 RRE region enhances the transport of unspliced mRNAs. To further limit potential readthrough transcription, 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 inserted into this vector. Tat and Rev inducibility was demonstrated directly by a > 300-fold induction of interferon production and functionally by a decrease in colony-forming units when a Tat and Rev expression vector was titered on HeLa cells harboring the inducible DT-A cassette. The Tat-inducible 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 presence 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 experiments was initiated with both the interferon and DT-A vectors. Protection against infection was documented against three HIV strains in PBLs. Last, the interferon and DT-A vectors were compared with a vector encoding a transdominant Rev protein and were shown to mediate equal or greater inhibition of HIV-1.