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J. Biol. Chem., Vol. 281, Issue 35, 25712-25722, September 1, 2006

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Differential Susceptibility of HIV-1 Reverse Transcriptase to Inhibition by RNA Aptamers in Enzymatic Reactions Monitoring Specific Steps during Genome Replication^*

Daniel M. Held, Jay D. Kissel, Dayal Saran^¶, Daniel Michalowski, and Donald H. Burke¹

From the Department of Biology, Indiana University, Bloomington, Indiana 47405, the ^¶Department of Chemistry, Indiana University, Bloomington, Indiana 47405, and the Department of Molecular Microbiology & Immunology and Department of Biochemistry, University of Missouri School of Medicine, Columbia, Missouri 65211

Nucleic acid aptamers to HIV-1 reverse transcriptase (RT) are potent inhibitors of DNA polymerase function in vitro, and they have been shown to inhibit viral replication when expressed in cultured T-lymphoid lines. We monitored RT inhibition by five RNA pseudoknot RNA aptamers in a series of biochemical assays designed to mimic discrete steps of viral reverse transcription. Our results demonstrate potent aptamer inhibition (IC₅₀ values in the low nanomolar range) of all RT functions assayed, including RNA- and DNA-primed DNA polymerization, strand displacement synthesis, and polymerase-independent RNase H activity. Additionally, we observe differences in the time dependence of aptamer inhibition. Polymerase-independent RNase H activity is the most resistant to long term aptamer suppression, and RNA-dependent DNA polymerization is the most susceptible. Finally, when DNA polymerization was monitored in the presence of an RNA aptamer in combination with each of four different small molecule inhibitors, significant synergy was observed between the aptamer and the two nucleoside analog RT inhibitors (azidothymidine triphosphate or ddCTP), whereas two non-nucleoside analog RT inhibitors showed either weak synergy (efavirenz) or antagonism (nevirapine). Together, these results support a model wherein aptamers suppress viral replication by cumulative inhibition of RT at every stage of genome replication.

Received for publication, May 10, 2006 , and in revised form, June 22, 2006.

^* This work was supported by National Institutes of Health Grant AI62513 (to D. H. B.) and by a Milton Taylor Graduate Fellowship in virology (to D. M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and additional references.

¹ To whom correspondence should be addressed: 471h Life Sciences Center, 1201 E. Rollins Rd., Columbia, MO 65211. Tel.: 573-884-1316; Fax: 573-884-9676; E-mail: burkedh{at}missouri.edu.

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