Viral-based vectors can provide an efficient delivery mechanism for stable expression of antisense RNA. To enhance and propagate the antiviral effect of antisense RNA, two novel human immunodeficiency virus type 1 (HIV-1)-based vector DNAs, designated as pMAG7 and pMAG19, were constructed which contained HIV-1 cis-acting packaging elements and produced multigenic HIV-1 antisense RNA that could target the entire pol, env, vif, vpu, vpr, rev, and tat and portions of gag and nef. The two DNAs were identical except that pMAG19 had additional gag coding sequences. Cotransfection of pMAG DNA and infectious, cloned HIV-1 DNA in 293 cells inhibited virus production (81%-98% reduction in reverse transcriptase activity) of various T cell-tropic and macrophage-tropic clade B isolates, such as NL4-3, YU-2, and JR-CSF. In addition, virion-associated pMAG antisense RNA was detected in residual virus particles produced by pNL4-3 in the presence of pMAG7 DNA, and the antisense sequences were stably transferred by infection of 174 x CEM cells. The results suggest that pMAG DNA may confer broad protection against HIV-1 by reducing initial virus burden due to antisense RNA and subsequent virus spread by propagation of antisense sequences along with wild-type virus.