Jeeninga R E, Huthoff H T, Gultyaev A P, Berkhout B
Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, PO Box 22700,1100 DE Amsterdam, The Netherlands.
Nucleic Acids Res. 1998 Dec 1;26(23):5472-9. doi: 10.1093/nar/26.23.5472.
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.
利用RNA模板和1型人类免疫缺陷病毒（HIV-1）的逆转录酶（RT）在体外分析了逆转录机制。具体而言，我们分析了放线菌素D（ActD）介导的对链转移步骤的抑制机制，在该步骤中，新合成的cDNA，即（-）链强终止子或（-）ssDNA，从供体RNA转移到受体RNA上。这种链转移反应在体外是一个效率相当低的过程。我们发现这部分是由于存在过量的供体RNA，通过减少供体RNA的量可实现高效的链转移。我们认为，由于碱基对互补性更高，（-）ssDNA与过量供体RNA退火优先于与受体RNA的有效结合。在这个优化的检测系统中，ActD仍然是链转移的有效抑制剂。我们未检测到ActD对逆转录延伸或RT酶的RNase H活性有影响。相反，我们提供的证据表明，ActD通过与（-）ssDNA直接相互作用发挥作用，从而阻断该分子的碱基配对能力。还讨论了单链DNA结合分子作为抗逆转录病毒药物的可能用途。
