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Journal of Virology, May 2007, p. 5375-5384, Vol. 81, No. 10
0022-538X/07/$08.00+0     doi:10.1128/JVI.01923-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cross-Clade Inhibition of Recombinant Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian Immunodeficiency Virus SIVcpz Reverse Transcriptases by RNA Pseudoknot Aptamers{triangledown}

Daniel M. Held,1,2,{dagger} Jay D. Kissel,2 Sarah J. Thacker,1 Daniel Michalowski,1 Dayal Saran,1,3,{ddagger} Jianfei Ji,4 Richard W. Hardy,2 John J. Rossi,5 and Donald H. Burke1*

Department of Molecular Microbiology & Immunology and Department of Biochemistry, University of Missouri School of Medicine, Columbia, Missouri 65211,1 Department of Biology, Indiana University, Bloomington, Indiana 47405,2 Department of Chemistry, Indiana University, Bloomington, Indiana 47405,3 Division of Cardiology, Cedars-Sinai Medical Center, Los Angeles, California 90048,4 Division of Molecular Medicine, Beckman Research Institute, and City of Hope National Medical Center, Duarte, California 910105

Received 4 September 2006/ Accepted 20 February 2007

Reverse transcriptase (RT) remains a primary target in therapies directed at human immunodeficiency virus type 1 (HIV-1). RNA aptamers that bind RT from HIV-1 subtype B have been shown to protect human cells from infection and to reduce viral infectivity, but little is known about the sensitivity of the inhibition to amino sequence variations of the RT target. Therefore, we assembled a panel of 10 recombinant RTs from phylogenetically diverse lentiviral isolates (including strains of HIV-1, simian immunodeficiency virus SIVcpz, and HIV-2). After validating the panel by measuring enzymatic activities and inhibition by small-molecule drugs, dose-response curves for each enzyme were established for four pseudoknot RNA aptamers representing two structural subfamilies. All four aptamers potently inhibited RTs from multiple HIV-1 subtypes. For aptamers carrying family 1 pseudoknots, natural resistance was essentially all-or-none and correlated with the identity of the amino acid at position 277. In contrast, natural resistance to aptamers carrying the family 2 pseudoknots was much more heterogeneous, both in degree (gradation of 50% inhibitory concentrations) and in distribution across clades. Site-directed and subunit-specific mutagenesis identified a common R/K polymorphism within the p66 subunit as a primary determinant of resistance to family 1, but not family 2, pseudoknot aptamers. RNA structural diversity therefore translates into a nonoverlapping spectrum of mutations that confer resistance, likely due to differences in atomic-level contacts with RT.

* Corresponding author. Mailing address: University of Missouri School of Medicine, 471h Life Sciences Center, 1201 E. Rollins St., Columbia, MO 65211. Phone: (573) 884-1316. Fax: (573) 884-9676. E-mail: burkedh{at}missouri.edu

{triangledown} Published ahead of print on 28 February 2007.

{dagger} Present address: Biosciences Division, SRI International, Menlo Park, CA 94025.

{ddagger} Present address: J. Craig Venter Institute, Rockville, MD 20850.

Journal of Virology, May 2007, p. 5375-5384, Vol. 81, No. 10
0022-538X/07/$08.00+0     doi:10.1128/JVI.01923-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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  • Michalowski, D., Chitima-Matsiga, R., Held, D. M., Burke, D. H. (2008). Novel bimodular DNA aptamers with guanosine quadruplexes inhibit phylogenetically diverse HIV-1 reverse transcriptases. Nucleic Acids Res 36: 7124-7135 [Abstract] [Full Text]  
  • Kissel, J. D., Held, D. M., Hardy, R. W., Burke, D. H. (2007). Active site binding and sequence requirements for inhibition of HIV-1 reverse transcriptase by the RT1 family of single-stranded DNA aptamers. Nucleic Acids Res 35: 5039-5050 [Abstract] [Full Text]  


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