title: The mechanism of actinomycin D-mediated inhibition of HIV-1 reverse transcription.
abstract: The mechanism of reverse transcription was analyzed in vitro with RNA templates and the reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1). In particular, we analyzed the mechanism of actinomycin D (ActD) mediated inhibition of the strand transfer step, in which the newly synthesized cDNA, termed the (-) strand strong stop or (-)ssDNA, is transferred from the donor RNA onto the acceptor RNA. This strand transfer reaction is a rather inefficient process in vitro. We found that this is in part due to the presence of an excess donor RNA, and highly efficient strand transfer was achieved by reducing the amount of donor RNA. We suggest that annealing of the (-)ssDNA to the excess donor RNA is preferred over productive binding to the acceptor RNA because of a higher basepair complementarity. ActD remains a potent inhibitor of strand transfer in this optimized assay system. We measured no effect of ActD on the elongation of reverse transcription or the RNase H action of the RT enzyme. Instead, we provide evidence that ActD acts through direct interaction with the (-)ssDNA, thereby blocking the basepairing capacity of this molecule. The possible use of single-stranded DNA binding molecules as antiretroviral agents is discussed.
authors.affiliation: Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, PO Box 22700,1100 DE Amsterdam, The Netherlands.
authors.forename: R E
authors.initials: RE
authors.lastname: Jeeninga
authors.forename: H T
authors.initials: HT
authors.lastname: Huthoff
authors.forename: A P
authors.initials: AP
authors.lastname: Gultyaev
authors.forename: B
authors.initials: B
authors.lastname: Berkhout
mesh_terms: D019380:Anti-HIV Agents / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000037:antagonists & inhibitors / Q000378:metabolism; D003609:Dactinomycin / Q000378:metabolism / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000037:antagonists & inhibitors / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D019380:Anti-HIV Agents; D004277:DNA, Single-Stranded; D012367:RNA, Viral; D003609:Dactinomycin; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H