Nonintegrated circular forms of the HIV-1 provirus DNA in experimental HIV infection.

Abstract

In this study, various proviral HIV-1 DNA forms were analyzed in a chronically infected culture of human monocytes. To elucidate the role of viral RT and cellular DNA polymerases in the formation of the circular forms, we used azidothymidine (AZT) as a specific terminator of viral RT. We previously developed a method for testing the circular proviral DNA forms by means of polymerase chain reaction (PCR) [5] and specific pairs of primers located at the 3' and 5' ends of the HIV-1 DNA (in the env and gag gene regions, respectively). The primers were so oriented that both one-LTR and two-LTR circular proviral DNAs could be determined simultaneously [5]. The total HIV-1 DNA concentration was measured to prepare diluted samples with an equal amount of proviral DNA, in which the relative content of circular nonintegrated forms of viral DNA was detected by means of PCR. The primers nov1‐nov2 were used. An additional amplification with primers V1‐V2 or nov3‐nov4 was performed in samples with low two-LTR circular DNA concentrations.