Abstract
We examined the suppression of HIV-1 expression by cleavage of the HIV-1 RNA gene, using a mammalian tRNA 3'processing endoribonuclease (3'tRNase) as an External Guide Sequence oligozyme (EGS) in vivo. The enzyme can also recognize and cleave a target RNA that forms a pre-tRNA like complex with a substrate RNA. We designed an EGS to specifically recognize the packaging signal region (p.s.) and the upstream portion of the gag region (g.s.) of HIV-1 mRNA, and constructed EGS expression vectors that used the tRNA(met) promoter as an expression cassette for the EGS. Their cleavage activities were assessed in cell culture, using a transient assay system. The EGS efficiencies were determined by co-transfection of the EGS expression vectors and the HIV-1 gene plasmid vector (pNL-luc) into COS cells. The assay results showed a significant inhibition of the HIV-1 gag p24 antigen expression. In addition, we constructed MLV-based VSV-G pseudotyped retrovirus vectors that express the EGS. They were co-infected into MT-4 cells with the HIV-1 virus (NL4-3). These EGS expression retrovirus vectors exhibited a similar inhibitory effect.
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