The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is a multifunctional enzyme, capable of several discrete activities required for viral replication . These essential activities include DNA- and RNA-dependent DNA polymerase (DDDP and RDDP), ribonuclease H (RNase H), strand transfer and strand displacement activities. Native HIV-1 RT is a heterodimer of p66 and p51 subunits, of which the p66 subunit contains both the polymerase and RNase H domains. The p51 subunit is derived by proteolytic cleavage of the p66 subunit and is thought to play both an architectural role in the context of the p66/p51 heterodimer as well as facilitate template·primer binding .
Due to its essential role in synthesizing the double-stranded proviral DNA from single-stranded HIV-1 RNA genome, the HIV-1 RT is a major target of current antiviral therapies directed against HIV-1. Current anti-HIV drug regimens, termed highly active antiretroviral therapy (HAART), typically consist of a combination of at least three antiretroviral drugs, with two or more nucleotide reverse transcriptase inhibitors (NRTIs) being a staple of most regimens [3, 4]. In addition to NRTIs, which are both competitive inhibitors and chain-terminators, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) consist of structurally dissimilar hydrophobic compounds that bind to a hydrophobic pocket on the RT adjacent to, but distinct from, the active site, which accommodates dNTPs and NRTIs. While HAART regimens have decreased both the mortality and morbidity of HIV-infected individuals, several factors contribute to drug failure. The highly error-prone nature of HIV-1 RT [5, 6] combined with a robust rate of viral replication [7, 8] provides the virus with an ideal context for the emergence of resistant variants. In addition, the significant toxicity associated with the current crop of anti-HIV drugs often leads to noncompliance, which in turn results in treatment failure . For these reasons, there is a high level of interest in the development of more potent anti-HIV inhibitors that are both less likely to lead to drug-resistant variants and display less toxicity in patients.
Among a number of anti-HIV agents being developed for potential use in the treatment of AIDS are nucleic acid-based inhibitors that can serve as useful complementary therapies . Of these, three nucleic acid-based approaches have recently been shown to have potent influence on HIV replication. In one, using a long antisense env RNA approach, strong inhibition of HIV replication was observed in cultured T cells . This approach combined with a lentiviral vector completed the phase I clinical trials and is about to enter phase II trials . The second approach, RNA interference (RNAi), uses a natural cellular pathway for gene silencing via small interfering RNAs [13–16]. The third approach is based on DNA and RNA aptamers that are derived by the iterative process of SELEX, to bind to specific protein targets  and has been recently shown to be effective in blocking HIV replication [18–20].
Tuerk and Gold first reported the isolation of RNA aptamers targeting HIV-1 RT using an iterative selection process of binding, washing and eluting the RNAs from a random library of RNA sequences . Subsequent reports showed that both DNA and RNA aptamers generated against HIV-1 RT [22, 23] are highly specific (do not bind to FIV or MuLV RTs), bind tightly to HIV-1 RT (Kd in the range of 0.05 to 50 nM) and competitively inhibit its polymerase activity. The crystal structure of an HIV-1 RT complexed with an anti-RT aptamer confirmed that the aptamer RNA is bound by the template·primer cleft of HIV RT . Since these aptamers compete with template·primer for the template-binding cleft, they have been termed template analog RT inhibitors (TRTIs) . In order to test the utility of anti-RT aptamers as inhibitors of HIV replication, we previously expressed RNA aptamers specific to HIV-1 RT in Jurkat T cells and showed that the tightest binding aptamers were able to potently block the infection and the subsequent spread of HIV-1 in cell culture . In addition, five of the nine different clades of HIV-1 tested and all of the RTI and PI-resistant isolates tested were also severely inhibited . The block was found to be in the early steps of reverse transcription. A subsequent report, using single cycle infection experiments involving one RNA aptamer (1.1), has confirmed the strong inhibition of HIV-1 replication by anti-RT aptamers .
It has been suggested that resistance to aptamers in vivo may be difficult due to the presumed need for multiple mutations required to disengage the interactions via the large interface between the inhibitor and HIV-1 RT . In order to address this notion, we previously used a phenotypic screen based on the in situ detection of RNA-dependent DNA polymerase activity of HIV-1 RT expressed within bacterial colonies, and isolated two variants of recombinant HIV-1 RT bearing the substitutions N255D or N265D, both of which displayed in vitro resistance to the DNA aptamer RT1t49 . The mechanism of resistance to these aptamers appeared to be based on the loss of affinity to the aptamer and the level of resistance increased from a range of 2- to 11-fold for single mutations to ~150-fold when the two mutations were combined. When the mutant RT sequences were incorporated into molecular clones of HIV-1, the resulting HIV virions were compromised for infectivity in single cycle infection assays and for virus replication in multi-day cell culture replication experiments . Thus, despite the biochemically robust enzymatic activity that allows one to measure drug-susceptibility levels of the mutant RTs, it appeared that the aptamer-resistance mutations tend to target biologically crucial sites. In support of this view, we have further demonstrated that all three mutants (the N255D, N265D and the double mutant (Dbl) RTs containing both mutations) are defective for processive DNA-dependent DNA polymerase activity (DDDP), although N265D retained processive polymerization activity on RNA templates .
The data available demonstrate the utility of aptamers in inhibiting HIV-1 replication. In addition to their exquisite specificity, high level of resistance to anti-RT aptamers appears to require multiple mutations, which affect the polymerase activity of the enzyme. Although resistant virus particles could be produced from molecular clones with mutant RTs, the mutant viruses displayed reduced replication competence and thus lacked a competitive edge in the presence of a large complexity of virus population. It is important to know whether the aptamer-resistant RTs retain their sensitivity to other classes of anti-RT drugs. In the present communication, we have further evaluated the enzymatic properties of the aptamer-resistant RTs. First, we measured the breadth of cross-resistance to other anti-RT inhibitors, including several standard NRTIs and NNRTIs and otherDNA and RNA aptamers specific to HIV-1 RT. Second, we have investigated biochemical defects that may be responsible for their reduced replication fitness. These are important questions concerning the potential of anti-RT aptamers as a viable treatment option. We find that these mutants are resistant to several additional DNA aptamers, thus suggesting a common contact point on HIV-1 RT to this new class of nucleic acid-based anti-RT inhibitors. Importantly, we find that the aptamer-resistant mutations retain wild-type susceptibilities to all NRTIs and NNRTIs tested. Furthermore, amongst a series of NRTI-resistant HIV-1 RT variants, only the K65R RT mutant displayed a significant (5-fold) level of resistance to RT1t49. Our results, combined with previous reports, demonstrate that mutations conferring resistance to the DNA aptamer, RT1t49 in vitro affect the RNase H domain in addition to previously shown effect on polymerase domain, both of which are essential for efficient viral DNA replication.