Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.
A structural and functional characterization of the 2-5A synthetase/RNase L antiviral pathway and its role in HIV-1 regulation: the pivotal role of 2-5A.
Abstract:
The mechanism of 2-5A synthesis and the binding and activation processes of RNase L were characterized using structurally and stereochemically modified analogs of ATP and 2-5A. It was established that the mechanism of 2-5A synthesis proceeds by inversion of configuration and that the 2',5'-phosphorothioate dimer can activate RNase L; however, activation of RNase L is dependent on the 3'-hydroxyl group of the second adenosylyl moiety. Utilizing 2',5'-phosphorothioates, these studies revealed that RNase L is a functionally stereoselective enzyme, in which each internucleotide linkage of 2-5A functions independently. Most importantly, these 2',5'-phosphorothioates may be utilized as highly stable 2-5A agonists or antagonists for dissecting the biological significance of the 2-5A system. The specific interactions occurring in the nucleotide binding domains of 2-5A synthetase and RNase L were approached by employing the technique of photoaffinity labeling. Utilizing 8-N3ATP, the 2-5A synthetase from human placenta was characterized with respect to (i) the mechanism of the ATP binding process, (ii) the structural requirements of nucleotides for binding, and (iii) the influence of dsRNA on the binding of ATP. Further, the 8-azido 2-5A photoprobes were synthesized and characterized and procedures have been developed for the in vitro synthesis of highly photolabile RNA utilizing 8-azido ATP. These will eventually be used to photolabel the ssRNA and dsRNA binding sites of RNase L, 2-5A synthetase and the dsRNA-dependent protein kinase. Finally, it is demonstrated that the 2-5A analogs inhibit HIV-1 reverse transcriptase. In order to establish the mechanism of 2-5A-mediated inhibition of HIV-1 RT, the nature of the RT-primer complex formation was characterized with the primer analogs oligo d(T)8 and oligo d(T)16. Through UV and laser cross-linking studies, it is shown that oligo d(T)(n) binds to the kinetically significant primer binding site of the recombinant HIV-1 RT and that this primer site is localized to a 12-kD subdomain within the enzyme. Finally, these studies establish that authentic 2-5A and phosphorothioate-substituted 2-5A inhibit HIV-1 RT via the inhibition of template-primer binding. In conclusion, the research presented implicates 2-5A as a critical component in anti-HIV regulation and suggests that this pivotal role of 2-5A mediates at least some of the anti-HIV activity of interferon. (Full text available from University Microfilms International, Ann Arbor, MI, as Order No. AAD91-35011).
Keywords: Adenosine Triphosphate/METABOLISM Endoribonucleases/*METABOLISM Human HIV-1/ENZYMOLOGY/*METABOLISM Placenta/METABOLISM RNA-Directed DNA Polymerase 2',5'-Oligoadenylate Synthetase/*BIOSYNTHESIS/METABOLISM THESIS 930228
M9320860
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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.
