Development of a human immunodeficiency virus-1 in vitro DNA synthesis system to study reverse transcriptase...

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.