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In this study, we investigated an RNA (R-Ψ-sgRNA) that suppresses HIV-1 replication. This RNA is expressed by a plasmid vector (pR-Ψ-sgRNA-ter) that was constructed accidentally. To examine if this effect is caused by RNA interference, R-Ψ-sgRNA was synthesized in vitro and treated with the Dicer enzyme, an important RNase III enzyme for RNA interference. The RNA was cleaved into fragments of approximately 20 nucleotides. We then performed an HIV-1 p24 assay with the RNA fragments to evaluate their effect on HIV-1 replication. HIV-1 replication was suppressed. We are now analyzing the sequences of the RNA fragments.

In this study, we investigated an RNA (R-Ψ-sgRNA) that suppresses HIV-1 replication. This RNA is expressed by a plasmid vector (pR-Ψ-sgRNA-ter) that was constructed accidentally. To examine if this effect is caused by RNA interference, R-Ψ-sgRNA was synthesized in vitro and treated with the Dicer enzyme, an important RNase III enzyme for RNA interference. The RNA was cleaved into fragments of approximately 20 nucleotides. We then performed an HIV-1 p24 assay with the RNA fragments to evaluate their effect on HIV-1 replication. HIV-1 replication was suppressed.

We used the HIV-1 5′LTR and the Cre/IoxP system to develop an anti-gene expression system. The LTR promoter of HIV-1 has a specific activity that includes the intermediary region of gag, as shown in a previous report. We constructed the U5-region of HIV-1 as the target of the ribozyme expression vector (pCre/IoxP-Rz), with the Cre/loxP system under the control of this LTR promoter. The function of this vector is to induce the HIV-1 dependent ribozyme-mediated inhibition in a dose-responsive manner. Furthermore, ribozyme mRNA expression was detected in the presence of pNL4-3 in HeLa-CD4+ cells. These studies are expected to yield novel applications of antiviral strategies for HIV-1.

We examined the suppression effect of HIV-1 expression by cleavage of the HIV-1 RNA gene, using the catalytic RNA subunit RNase P and the 3′-half tRNA [External Guide Sequence (EGS)] <it>in vivo</it>. The vectors were designed to express an anti-HIV EGS, U5, which targets the 5′ leader sequence. We constructed an EGS expression vector, that used the tRNAmet or U6 promoter as an expression cassette for EGS. RNase P cleaves the targeted HIV-1 mRNA when they are in a complex with the EGS. To test the antiviral efficacy of these EGS vectors, we have cotransfected into COS cells with the HIV-1 proviral DNA (pNL4-3 Luc) and the plasmid expressing the EGS from the tRNAmet or U6 promoter. HIV-1 expression was inhibited by the tRNA-EGS-1 and U6-EGS-1 from the tRNAmet and U6 promoters, respectively. No difference in the inhibitory effects on HIV-1 expression between the tRNAmet and U6 promoters could be detected.

Long terminal repeats (5′-LTR) contain essential enhancer and promoter elements that act as targets for cellular and viral regulatory proteins. The interplay of these cis-acting sequences with specific transcription factors (NF κ B, Apl, and Spl) present in infected cells determines the rate of transcription initiation from the 5′-LTR promoter element, and thus strongly influences the level of viral replication. Furthermore, Tat functions to enhance transcriptional elongation by binding to the trans-activation response element (TAR). These transcriptional factors enhance the transcriptional elongation from the HIV-1 promoter. HIV-1 genomic RNA packaging is a highly specific process involving interactions between the viral Gag precursor and cis acting packaging signals in the 5′ leader sequence of HIV-1 genomic RNA. We examined the suppression effect of HIV-1 expression by the decoy RNAs targeted to these HIV-1 transcriptional and packaging regulatory regions. HIV-1 expression was inhibited by the 5′-LTR decoy RNA, and measured by the amount of HTV-1 gag p24 protein, but no inhibition of HIV-1 packaging could be detected.

Previously, we used the human methionine tRNA promoter as an expression cassette for hammerhead ribozymes. The tRNA promoter driven ribozyme was targeted against the LTR portion of the HIV-1 NL4-3 strain. We constructed VSV-G-pseudotyped MuLV-based vectors expressing the ribozyme. The ribozyme expressing retrovirus vector strongly suppressed gag p24 antigen production in freshly HIV-1 infected MT-4 cells. In this study, the potential of such a molecular genetic intervention was examined by using the Cre-IoxP recombination system. Site-specific excision of HIV-1 was achieved by using this model system with an acute infection. These studies represent one step toward the development of a novel antiviral strategy for the treatment of AIDS.