The tRNA 3′-processing endoribonuclease (tRNase Z or 3′ tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3′ trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression.
We examined the suppression of virus expression by cleaveage of the HIV-1 RNA gene using a mammalian tRNA 3' processing endoribonuclease and an External Guide Sequence Oligozyme (EGS) in vivo. We constructed an EGS expression vector that used the tRNA(met) promoter as an expression cassette for EGS. The EGS expression vector was targeted to the upstream region of gag, region. The EGS expression vector was co-transfected into COS cells with the HIV-1 gene plasmid vector. As compared with the EGS non-expressing cells and the EGS expressing cells, the EGS expressing cells with the targeted gag start codon had a clearly decreased amount of the HIV-1 gag p24 protein. The EGS expressing cells with the targeted gag start codon showed effective suppression of HIV-1 gene expression. Thus, these studies describe novel gene targeting agents for the inhibition of gene expression and antiviral activity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.