Toxin-Based Therapeutic Approaches

Shapira, Assaf; Benhar, Itai

HIV-1, the main cause of acquired immune deficiency syndrome (AIDS), has been the most studied infectious agent in the last 30 years. The HIV retroviral genome carries six regulatory genes, including Tat, Rev, Vpr, Vif, Vpu, and Nef. Of these genes, Tat encodes a protein that plays key roles in controlling productive and processive viral gene transcription. The Tat protein binds to the specific sequences of TAR (Transactivation Response Element) located in the 5' LTR, one of two terminal repeated segments of the viral genome, and exerts its effect by increasing the rate of transcription of the nascent HIV RNA. The viral Rev protein was found to be required for expression of the viral late gene products. By binding to a secondary RNA structure, the Rev-responsive element (RRE), the Rev protein tethers partially spliced and unspliced viral RNAs encoding the late viral proteins to the cellular CRM-1-mediated nuclear-export pathway, leading to enhanced cytoplasmic levels of these RNAs and increased expression of the encoded proteins [407] [408] [409] [410] [411] [412] . Applying the knowledge about these viral molecular mechanisms for regulated gene expression, Harrison et al. have demonstrated the use of a combination of Tat and Rev cis-acting responsive sequences for achieving enhanced expression of transgenes in cells expressing both regulatory trans-acting Tat and Rev proteins; while maintaining low basal expression in naive cells [413] . Substantially impaired HIV production, following HIV proviral DNA transfection of HeLa cells containing integrated HIV-regulated (Tat-Rev responsive) DT-A gene, was shown in a subsequent study [414] . A T-Cell line (H9) that was transduced with a recombinant retroviral vector encoding HIV regulated wild-type or attenuated DT-A gene also showed substantially long-term impaired ability to produce HIV virions upon transfection with proviral DNA or infection with laboratory or clinical HIV strains [348, 415] . Using the same construct, significant protection against HIV infection (dependent both on the stock of HIV-1 used and on the dose) was also observed in the U937 cell line which exhibits many of the characteristics of tissue monocytes that serve as an important reservoir for the virus in vivo [349, 416] . It was later reported that co-transfection of the HIV regulated DT-A construct (HIV-DT-A) with an HIV proviral DNA using cationic liposome-mediated gene delivery ("lipofection") could prevent virus production in HeLa cells. However, although HIV-regulated genes were found to be expressed when transfected into chronically HIV infected cells, transfection with HIV-DT-A did not significantly reduce virus production in an already chronically or de novo HIV-infected cell population, probably due to the low percentage (~5%) of "lipofected" cells [350] .