Development of a human immunodeficiency virus-1 in vitro DNA synthesis system to study reverse transcriptase inhibitors.
A Human immunodeficiency virus type-1 endogenous reverse transcriptase reaction was developed as an in vitro assay to study the inhibition of reverse transcription by antiviral compounds. Conditions were established for producing genomic length (-) strand DNA in high yields and measuring the inhibition of this transcript as the assay endpoint. In addition to genomic length (-) strand DNA, a novel segmented (-) strand product composed of a 6.0 kb reverse transcript of the 5' 2/3 of the viral RNA genome and a 3.5 kb reverse transcript of the 3' 1/3 was observed. The most prominent (+) strand product was the size expected for plus-strong stop DNA. Additional minor (+) strand species were also observed. The triphosphate form of the nucleoside analog inhibitor 3'-azido-3'-deoxythymidine (RETROVIR, Zidovudine, AZT) and BI-RG-587 (nevirapine), a non nucleoside inhibitor, were used to demonstrate the utility of the endogenous system for the analysis of reverse transcriptase inhibitors. In a standard reaction, synthesis of genomic length DNA was 50% inhibited by 0.1 microM AZTTP and 0.1 microM nevirapine.
Borroto-Esoda
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Division of Virology, Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
Boone
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D004279:DNA, Viral / Q000096:biosynthesis* / Q000737:chemistry; D054306:Dideoxynucleotides; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D019829:Nevirapine; D011725:Pyridines / Q000494:pharmacology; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides / Q000494:pharmacology; D013997:Time Factors; D015215:Zidovudine / Q000031:analogs & derivatives / Q000494:pharmacology
D004279:DNA, Viral; D054306:Dideoxynucleotides; D011725:Pyridines; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D015215:Zidovudine; C052873:zidovudine triphosphate; D019829:Nevirapine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase