Antisense RNA sequences targeting the 5' leader packaging signal region of human immunodeficiency virus type-1 inhibits viral replication at post-transcriptional stages of the life cycle

Abstract

Antisense RNA has proven a potent inhibitor of gene expression and has the potential to inhibit retroviral replication at a number of stages in the virus life cycle by targeting both viral and cellular RNA sequences. Antisense RNA complementary to three target regions in the 5' leader/LTR of human immunodeficiency virus type-1 (HIV-1), the TAR region, the primer binding site and the splice donor (SD)-packaging signal (ψ) region were stably expressed from the CMV IE promoter in Jurkat cells, and expression confirmed by RT-PCR. When challenged with HIV-1, cell lines expressing antisense RNA targeting the SD/ψ region showed significant inhibition of replication (at up to 106 TCID 50/ml). These sequences were also expressed in lymphocytes after transduction using recombinant retroviruses and one sequence complementary to the SD/ψ region inhibited replication of HIV-1. A co-transfection assay using COS-1 cells was also developed both to confirm the antiviral potential of these sequences, and to determine the predominant site of action of these molecules. Antisense RNAs targeting the ψ region and one sequence complementary to the TAR region inhibited expression of viral protein; furthermore, analyses of relative levels of cellular and virion RNA from these assays suggest each of these antisense molecules exerts its effect at an early stage in the transcription-translation pathway, while the longer of the sequences also inhibited packaging of virion RNA. These results suggest that the packaging signal (ψ) of HIV-1 represents an attractive target for antisense RNA-based gene therapy, although the main mode of action of such molecules may well be through antisense effects at an earlier stage of replication than packaging.