Differential susceptibility of HIV-1 reverse transcriptase to inhibition by RNA aptamers in enzymatic reactions monitoring specific steps during genome replication.
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.
Held
Daniel M
DM
Department of Biology, Indiana University, Bloomington, Indiana 47405, USA.
Kissel
Jay D
JD
Saran
Dayal
D
Michalowski
Daniel
D
Burke
Donald H
DH
D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D016679:Genome, Viral; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000502:physiology*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D020128:Inhibitory Concentration 50; D012313:RNA / Q000737:chemistry*; D016914:Ribonuclease H / Q000737:chemistry; D013601:T-Lymphocytes / Q000821:virology
D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H