Actinomycin D inhibition of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase and nucleocapsid protein.
Actinomycin D was found to be a potent inhibitor of HIV-1 reverse transcriptase catalyzed DNA strand transfer reactions. Using an oligonucleotide model system, actinomycin D inhibition of DNA strand transfer was examined to elucidate the mechanism of inhibition and further define the mechanism of DNA strand transfer. Our results show that actinomycin D inhibits HIV-1 reverse transcriptase catalyzed DNA strand transfer without inhibiting RNA-dependent or DNA-dependent DNA polymerase activity. Actinomycin D was found to strongly inhibit annealing of a primary DNA product to the DNA acceptor template, preventing the formation of a key reaction intermediate. The HIV-1 nucleocapsid protein has been shown to participate in catalytic events during reverse transcription including DNA strand transfer. Recombinant nucleocapsid protein was used in conjunction with actinomycin D in this model system to investigate how NC may participate in the mechanism of inhibition by actinomycin D and in DNA strand transfer. The inclusion of nucleocapsid protein was found to partially relieve both DNA annealing and strand transfer inhibition caused by actinomycin D. This study suggests a potential new mechanism for inhibiting retroviral replication by preventing the formation of replication intermediates.
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA.
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Davis
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Gabbara
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Hupe
J A
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Peliska
D002384:Catalysis / Q000187:drug effects; D004279:DNA, Viral / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D019590:Nucleocapsid Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D016914:Ribonuclease H / Q000037:antagonists & inhibitors / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects
D004279:DNA, Viral; D019590:Nucleocapsid Proteins; D018894:Reverse Transcriptase Inhibitors; D003609:Dactinomycin; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H