Towards gene therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections, we tested the potency of several antiviral constructs in transient HIV-1 production assays. Whereas little effect was obtained with antisense- and TAR decoy-constructs, we measured efficient inhibition of HIV-1 mRNA translation and virion production in the presence of HIV-1 leader-containing transcripts. The infectivity of these virions was also reduced by this sense inhibitor RNA. These results suggest that leader-encoded functions, like the dimer-linkage structure, can be used to specifically inhibit HIV expression in trans.