Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.
Development of a tRNA based transcription system to render cells resistant to viral replication.
Diss Abstr Int [B]; 52(7):3444 1992. Unique Identifier : AIDSLINE ICDB/93682490 Sullenger BA; Cornell Univ. Medical Center
Abstract:
Inhibition of gene expression via antisense RNA requires the synthesis of a considerable excess of antisense RNA over sense RNA, and with a few exceptions, RNA polymerase II-based transcription units are inadequate to generate such an excess of antisense RNA. Therefore, we have explored the use of RNA polymerase III-based transcription units to express high levels of Moloney murine leukemia virus (MoMLV)-specific antisense RNA. A mutant human tRNA(i)met gene was fused to MoMLV sequences generating chimeric tRNA-antisense genes. These tRNA fusion genes were introduced into NIH 3T3 cells via retroviral vectors. High steady state levels of chimeric tRNA-antisense transcripts were obtained in the transduced cells which, on a molar basis, correspond to 15-25% of the polyA RNA content of the cell. After viral challenge, MoMLV replication was inhibited up to 97% in cells expressing the tRNA-antisense transcripts. Efficient expression of the chimeric tRNA-antisense template and inhibition of MoMLV replication are dependent upon the use of a particular retroviral vector design, the double copy vector, which places one copy of the chimeric tRNA template outside of the vector's LTR-initiated transcription unit. This RNA synthesis system was employed to generate high intracellular levels of chimeric tRNA-TAR transcripts in an attempt to squelch tat-mediated transactivation of HIV gene expression. Human lymphocyte cell lines overexpressing TAR sequences, 'TAR decoys', were challenged with HIV-1. Viral replication was inhibited by 99% or more in TAR decoy-expressing CEM SS cells, and HIV's cytopathic effects were totally eliminated. Furthermore, replication of the related but evolutionarily distinct virus, SIV, was greatly inhibited in HIV-1 TAR decoy-expressing cell lines. Thus, TAR decoy-mediated inhibition of viral replication may be applicable to a wide range of naturally occurring HIV isolates, and as such represents an attractive intracellular immunization approach for the treatment of AIDS. (Full text available from University Microfilms International, Ann Arbor, MI, as Order No. AAD91-35421).
Keywords: Animal Gene Expression Genes, Viral Genetic Vectors HIV/GENETICS/*PHYSIOLOGY Leukemia, Experimental/*GENETICS Mice Moloney Leukemia Virus/*GENETICS/PHYSIOLOGY Retroviridae/GENETICS RNA RNA Polymerase III/*GENETICS RNA, Antisense/*GENETICS RNA, Transfer/ANALYSIS/*GENETICS Transcription Factors/ANALYSIS Transfection *Virus Replication 3T3 Cells THESIS 930228
M9320861
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Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.
