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

Abstract

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...

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

“…NVP and efavirenz (EFV) were purchased from Toronto Research Chemicals, Inc. (Toronto, Canada). RNA aptamers 70.5, 70.8, and 80.55 were previously identified by in vitro selection from random pools (5), and their complete sequences are presented elsewhere (26). The RNA sequence of aptamer T1.1 (62) used here is identical to that of the variant used previously for cocrystallization with HIV-1 M/B RT (31).…”

“…Biochemical and crystallographic analyses of RNA aptamers to HIV-1 RT established that they bind in the same cleft normally occupied by the primer-template, where they competitively inhibit primertemplate binding (26,31,66); hence, they are sometimes denoted "primer-template analog RT inhibitors" (16). Many of the RNA aptamers are capable of forming bent pseudoknot structures (5, 62), which are divided into two families (5).…”

“…The family 1 pseudoknots conform closely to a well-defined sequence definition derived from analysis of the canonical T1.1 aptamer (5,22), while the family 2 pseudoknots are defined by not conforming to that sequence definition. The most potent of the RNA aptamers from both families inhibit both DNA polymerase and RNase H activities in vitro, with IC 50 values (concentration of inhibitor at which half-maximal inhibition is observed) in the low nanomolar to high picomolar range (5,26,37,62). Importantly, when these RNA aptamers were expressed in cultured lymphocytes, they suppressed the replication of multiple viral isolates by 1 to 2 orders of magnitude (6,33,35), and at high multiplicities of infection they offered more antiviral protection than did expressed small interfering RNA (35).…”

Novel Aptamer Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase

“…RNA aptamers first isolated for HIV-RT were usually pseudoknot-type structures (Tuerk et al, 1992). RNA pseudoknot aptamers have been shown to interfere with primer-template binding and are potent inhibitors of reverse transcription (Chen and Gold, 1994;Jaeger et al, 1998;Held et al, 2006b). Singlestranded DNA aptamers with similar properties have also been selected (Schneider et al, 1995;Mosing et al, 2005).…”

Therapeutic applications of aptamers

“…All these steps, viz. DNA-and RNA-primed extensions on either DNA-or RNA-templates, strand displacement as well as RNase H-catalyzed RNA cleavage, have been checked for their response to aptamer binding [90] . The aptamers were found to inhibit polymerase as well as RNase H activities.…”