Potent inhibition of human immunodeficiency virus type 1 replication by template analog reverse transcriptase inhibitors derived by SELEX (systematic evolution of ligands by exponential enrichment).
RNA aptamers derived by SELEX (systematic evolution of ligands by exponential enrichment) and specific for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bind at the template-primer cleft with high affinity and inhibit its activity. In order to determine the potential of such template analog RT inhibitors (TRTIs) to inhibit HIV-1 replication, 10 aptamers were expressed with flanking, self-cleaving ribozymes to generate aptamer RNA transcripts with minimal flanking sequences. From these, six aptamers (70.8,13, 70.15, 80.55,65, 70.28, 70.28t34, and 1.1) were selected based on binding constants (K(d)) and the degree of inhibition of RT in vitro (50% inhibitory concentration [IC(50)]). These six aptamers were each stably expressed in 293T cells followed by transfection of a molecular clone of HIV(R3B). Analysis of the virion particles revealed that the aptamers were encapsidated into the virions released and that the packaging of the viral genomic RNA or the cognate primer, tRNA(Lys)(3), was apparently unaffected. Infectivity of virions produced from 293T cell lines expressing the aptamers, as measured by infecting LuSIV reporter cells, was reduced by 90 to 99.5% compared to virions released from cells not expressing any aptamers. PCR analysis of newly made viral DNA upon infection with virions containing any of the three aptamers with the strongest binding affinities (70.8,13, 70.15, and 80.55,65) showed that all three were able to form the minus-strand strong-stop DNA. However, virions with the aptamers 70.8 and 70.15 were defective for first-strand transfer, suggesting an early block in viral reverse transcription. Jurkat T cells expressing each of the three aptamers, when infected with HIV(R3B), completely blocked the spread of HIV in culture. We found that the replication of nucleoside analog RT inhibitor-, nonnucleoside analog RT inhibitor-, and protease inhibitor-resistant viruses was strongly suppressed by the three aptamers. In addition, some of the HIV subtypes were severely inhibited (subtypes A, B, D, E, and F), while others were either moderately inhibited (subtypes C and O) or were naturally resistant to inhibition (chimeric A/D subtype). As virion-encapsidated TRTIs can predispose virions for inhibition immediately upon entry, they should prove to be efficacious agents in gene therapy approaches for AIDS.
Joshi
Pheroze
P
Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Prasad
Vinayaka R
VR
D019380:Anti-HIV Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D002460:Cell Line; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013698:Templates, Genetic; D014771:Virion / Q000502:physiology; D014779:Virus Replication / Q000187:drug effects*
D019380:Anti-HIV Agents; D008024:Ligands; D018894:Reverse Transcriptase Inhibitors; D012313:RNA; D054303:HIV Reverse Transcriptase