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.