Moloney murine leukemia virua (MoMuLV)-derived retroviral vectors were engineered to express human immunodeficiency virus type 1 (HIV-1) packaging (psi) 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 psi signal sequences were expressed under control of the HSV tk promoter. Both RRE and psi 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 psi 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 RRE1- (antisense RNA containing a single copy of RRE) RNA-expressing cells. In cells expressing HIV-1 psi signal, HIV-1 production remained unaltered in psi + RNA-expressing cells, whereas it was delayed by up to 30 days in psi - RNA-expressing cells.(ABSTRACT TRUNCATED AT 250 WORDS)