Replication of human immunodeficiency virus type 1 (HIV-1) is regulated at reverse transcription. Cellular topoisomerase I has been reported to be carried into HIV-1 virions and enhance cDNA synthesis in vitro, suggesting that topoisomerase I expressed in virus producer cells regulates reverse transcription. Here, by employing both indicator cell assay and endogenous reverse transcription (ERT) assay, we show that topoisomerase I and adenosine triphosphate (ATP) enhanced cDNA synthesis of HIV-1. In addition, topoisomerase I mutants, R488A and K532A, lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1, suggesting that the activity of topoisomerase I lacking in these mutants is indispensable for the cDNA synthesis in the HIV-1 replication process. Furthermore, ATP could dissociate topoisomerase I from the topoisomerase I-RNA complex and enhance cDNA synthesis in vitro. These findings suggest that cellular topoisomerase I and ATP play a pivotal role in the synthesis of cDNA of HIV-1. (C) 2002 Elsevier Science (USA). All rights reserved.