Charneau P, Mirambeau G, Roux P, Paulous S, Buc H, Clavel F
Unité d'Oncologie Virale, CNRS URA 1157, Institut Pasteur, Paris, France.
J Mol Biol. 1994 Sep 2;241(5):651-62. doi: 10.1006/jmbi.1994.1542.
During HIV-1 reverse transcription, the plus-strand of viral DNA is synthesized as two discrete segments. We show here that synthesis of the upstream segment terminates at the center of the genome after an 88 or 98 nucleotide strand displacement of the downstream segment, initiated at the central polypurine tract. Thus, the final structure of unintegrated linear HIV-1 DNA includes a central plus-strand overlap. In vitro reconstitution using only purified reverse transcriptase with appropriate DNA hybrids gave rise to efficient and accurate termination, which was dramatically amplified in the context of strand displacement. Mutation of the sequence immediately upstream of the termination sites almost completely abolished termination both in infected cells and in vitro. This mutation profoundly impaired replication of HIV-1. We conclude that proper central plus-strand termination, mediated by a novel cis-active termination sequence, is a key step in HIV-1 replication.
在HIV-1逆转录过程中，病毒DNA的正链作为两个离散片段合成。我们在此表明，上游片段的合成在下游片段从中央多聚嘌呤序列起始进行88或98个核苷酸的链置换后，在基因组中心终止。因此，未整合的线性HIV-1 DNA的最终结构包括一个中央正链重叠区。仅使用纯化的逆转录酶与合适的DNA杂交体进行体外重建，可实现高效且准确的终止，这在链置换的情况下会显著放大。终止位点紧邻上游序列的突变几乎完全消除了感染细胞和体外的终止。这种突变严重损害了HIV-1的复制。我们得出结论，由一个新的顺式活性终止序列介导的适当中央正链终止是HIV-1复制中的关键步骤。
