Inhibition of HIV-1 Multiplication in a Human CD4 + Lymphocytic Cell Line Expressing Antisense and Sense RNA Molecules Containing HIV-1 Packaging Signal and Rev Response Element(s)

Moloney murine leukemia virua (MoMuLV)-derived retroviral vectors were engineered to express human immunodeficiency virus type 1 (HIV-1) packaging (ψ) signal and Rev response element (RRE) sequences in either sense or antisense orientation. The RRE sequences were expressed under the control of the herpes simplex virus (HSV) thymidine kinase (tk) promoter fused to the HIV-1 trans-activation-responsive (TAR) element, while the ψ signal sequences were expressed under control of the HSV tk promoter. Both RRE and ψ signal sequences were expressed as part of the 3′ untranslated region of the neomycin phosphotransferase (neo) mRNA. The constructs were used to transfect/infect packaging cell lines and the retroviral vector particles released were used to infect a human CD4⁺ lymphocyte-derived MT₄ cell line. The stable MT₄ transformants, harboring proviral vector DNA expressing one to two copies of HIV-1 RRE and ψ signal in either antisense or sense orientation, were each tested for their susceptibility to HIV-1 infection. Compared to the results obtained with the control cells lacking any of the test DNA sequences, the rate of HIV-1 production remained unaltered in RRE¹⁺ (sense RNA containing a single copy of RRE) RNA-containing cells, whereas it was delayed in cells expressing both RRE²⁺ (sense RNA containing two copies of RRE) and RRE^1- (antisense RNA containing a single copy of RRE) RNA-expressing cells. In cells expressing HIV-1 ψ signal, HIV-1 production remained unaltered in ψ⁺ RNA-expressing cells, whereas it was delayed by up to 30 days in ψ^- RNA-expressing cells. These results indicate that retroviral vectors expressing two copies of HIV-1 RRE in sense orientation, a single copy of HIV-1 RRE in antisense orientation, and particularly the HIV-1 ψ signal in antisense orientation can be used to confer HIV-1 resistance.