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.The reverse transcriptases (RTs) of the human and simian immunodeficiency viruses (HIVs and SIVs, respectively) are encoded by the viral pol gene and are expressed from viral mRNA as part of the multifunctional Gag-Pol polyprotein. Mature RT is the product of proteolytic processing of the polyprotein, first into an asymmetric homodimer and then into the mature heterodimer (21). Due to its central role in HIV type 1 (HIV-1) replication and the early clinical availability of anti-RT compounds, RT has long been an established therapeutic target. Of the 22 anti-HIV compounds currently approved by the U.S. Food and Drug Administration, 15 target the viral RT (60). The nucleoside analogue RT inhibitors (NRTIs) are deoxynucleoside triphosphate analogues that result in chain termination when incorporated by RT into a growing DNA strand. The nonnucleoside RT inhibitors (NNRTIs) bind RT near the active site and disrupt enzymatic activity by allosteric inhibition (38, 59). The search for new anti-RT compounds is fueled by cytotoxicity and clinically selected resistance-resulting from drug exclusion or from postincorporation excision (45)-which are persistent problems for both classes of RT inhibitors.Nucleic acid aptamers, ribozymes, antisense RNA, and small interfering RNA exhibit potent antiviral effects and are under development as potential gene therapy adjuvants to smallmolecule therapeutics (25,34), and several of these have recently entered into clinical trials (1,39,4...

Nucleic acid aptamers can potentially be developed as broad-spectrum antiviral agents. Single-stranded DNA (ssDNA) aptamer RT1t49 inhibits reverse transcriptases (RT) from HIV-1 and diverse lentiviral subtypes with low nanomolar values of Kd and IC50. To dissect the structural requirements for inhibition, RT-catalyzed DNA polymerization was measured in the presence of RT1t49 variants. Three structural domains were found to be essential for RT inhibition by RT1t49: a 5′ stem (stem I), a connector and a 3′ stem (stem II) capable of forming multiple secondary structures. Stem I tolerates considerable sequence plasticity, suggesting that it is recognized by RT more by structure than by sequence-specific contacts. Truncating five nucleotides from the 3′ end prevents formation of the most stable stem II structure, yet has little effect on IC50 across diverse HIV-1, HIV-2 and SIVCPZ RT. When bound to wild-type RT or an RNase H active site mutant, site-specifically generated hydroxyl radicals cleave after nucleotide A32. Cleavage is eliminated by either of two polymerase (pol)-active site mutants, strongly suggesting that A32 lies within the RT pol-active site. These data suggest a model of ssDNA aptamer–RT interactions and provide an improved molecular understanding of a potent, broad-spectrum ssDNA aptamer.

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 (IC 50 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. The reverse transcriptase (RT)2 of HIV-1 catalyzes the multistep conversion of the single-stranded RNA viral genome into a double-stranded DNA copy for insertion into the host chromosome by the viral integrase. RT possesses both DNA-and RNA-dependent DNA polymerase functions, as well as RNase H activity to degrade the RNA strand in DNA/RNA heteroduplex replication intermediates. The enzyme is an asymmetric heterodimer composed of 66-kDa (p66) and 51-kDa (p51) subunits. The subunits associate initially as a p66 homodimer prior to removal of the C terminus of one of the two subunits by the viral protease (1, 2). The catalytic sites for both polymerase and RNase H function reside in the large subunit, whereas the small subunit contributes to primer/template binding and provides structural support for the large subunit (3, 4). Its essential role in viral replication and the early availability of anti-RT drugs have made RT a featured target for clinical treatment of HIV infection. Eleven of the twenty FDA-approved anti-HIV compounds are RT inhibitors, which are themselves grouped into two classes, nucleoside analog RT inhibitors (NRTIs) and nonnucleoside analog RT inhibitors (NNRTIs), based on their mode of action. The eight approved NRTIs are chain terminators that prevent the completion of viral genome replication following their incorporation by RT during DNA polymerization. The three NNRTIs, on the other hand, inhibit catalytic function directly by binding to RT near the polymerase active site and disrupting the local enzyme geometry (5-7).Administration of combinations of two NRTIs along with an NNRTI or protease inhib...