Roda Ricardo H, Balakrishnan Mini, Kim Jin K, Roques Bernard P, Fay Philip J, Bambara Robert A
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
J Biol Chem. 2002 Dec 6;277(49):46900-11. doi: 10.1074/jbc.M208638200. Epub 2002 Oct 4.
Recombination promotes retrovirus evolution. It involves transferring a growing DNA primer from one genomic RNA template in the virus to the other. Strand transfer results in vitro suggested that pausing of the reverse transcriptase during synthesis allows enhanced RNase H cleavage of the initial, or donor, RNA template that facilitates primer interaction with the acceptor template. Hairpins are common structures in retrovirus RNAs that induce pausing. Analyzing primer transfers in hairpins by base substitution markers showed transfer sites well beyond the site of pausing. We developed methods to distinguish the initial site of primer-acceptor template interaction from the site of primer terminus transfer. The strand transfer mechanism was confirmed to involve two steps. In the first, the acceptor template invades the primer-donor complex. However, the primer terminus continues elongation on the donor RNA. The interacting primer and acceptor strands then propagate by branch migration to catch the advancing primer terminus. Some distance downstream of the invasion site the primer terminus transfers, marking the genetic shift from donor to acceptor. Nucleocapsid protein (NC) is known to influence primer elongation and strand exchange. The presence of NC increased the efficiency of transfers but did not appear to alter the fundamental transfer mechanism.
重组促进逆转录病毒进化。它涉及将正在延伸的DNA引物从病毒中的一个基因组RNA模板转移到另一个模板。体外链转移结果表明，逆转录酶在合成过程中的暂停会增强对初始（或供体）RNA模板的核糖核酸酶H切割，这有助于引物与受体模板相互作用。发夹结构是逆转录病毒RNA中常见的诱导暂停的结构。通过碱基替代标记分析发夹结构中的引物转移，结果显示转移位点远在暂停位点之外。我们开发了一些方法来区分引物 - 受体模板相互作用的起始位点和引物末端转移位点。链转移机制被证实涉及两个步骤。第一步，受体模板侵入引物 - 供体复合物。然而，引物末端继续在供体RNA上延伸。然后，相互作用的引物链和受体链通过分支迁移延伸，以赶上前进的引物末端。在侵入位点下游的一定距离处，引物末端发生转移，标志着遗传信息从供体转移到受体。已知核衣壳蛋白（NC）会影响引物延伸和链交换。NC的存在提高了转移效率，但似乎并未改变基本的转移机制。
