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dc.contributor.authorDuclair, Sonald
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 71-05, Section: B, page: 2880.;Advisors: Vinayaka R. Prasad.
dc.description.abstractReplication of the human immunodeficiency virus type 1 (HIV-1) requires the virally-encoded aspartyl protease (PR) to process the Gag and Gag-Pol polyprotein precursors into their functional constituents during viral maturation. This process is absolutely required for the virus to become infectious. As a result, PR has been established to be an effective target for therapeutic intervention of HIV infections. However, the use of protease inhibitors (PIs) in combination therapy has been associated with a number of cytotoxicities. Moreover, PI-resistant mutant viruses continue to emerge, fueling the search for alternative means to inhibit viral maturation. In the present work, we report the isolation and characterization of the first RNA aptamers against HIV-1 protease.;SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was employed to isolate RNA aptamers against HIV-1 PR. Four unique families of aptamers with moderate Kds ranging from 92-140nM were initially isolated. They competed with each other for PR binding and inhibited PR activity noncompetitively, with Kis ranging from 138-647nM. Second generation RNA aptamers with Kds ranging from 2.18-22.2nM were selected from doped pools based on the previously selected aptamer sequences. These new aptamers inhibited PR with greater efficiency with Kis ranging from 31.5-48.8nM. They did not inhibit the enzymatic activity of the eukaryotic aspartyl protease, cathepsin D, nor did they bind to the closely related HIV-2 aspartyl protease, suggesting a high specificity for their target.;In order to test the effects of anti-PR PR RNA aptamers on viral replication, pseudotyped viruses were generated from 293T cells expressing first- and second-generation aptamers. Aptamers predominantly localized to the cytoplasm and were incorporated into the budding virions. However, they had minimal effect on viral production. In some instances, expression of the aptamers resulted in measurable polyprotein processing defects in the viruses released, but the observed defects were far milder compared to that of viruses produced in the presence of PIs. Moreover, the infectivities of viruses produced in the presence of the various aptamers were not affected. Thus, these aptamers may inhibit polyprotein processing to some extent but the level of inhibition achieved is insufficient to affect infectivity.
dc.publisherProQuest Dissertations & Theses
dc.titleIsolation and characterization of RNA aptamers targeted to the aspartyl protease of the human immunodeficiency virus type 1 (HIV -1)

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