MECHANISM OF RECOMBINATION BY HIV REVERSE TRANSCRIPTASE

该提案将重点关注人类免疫缺陷病毒的机制 (HIV) 重组由逆转录酶 (RT) 催化。 第一链 DNA 合成过程中的重组（由 DNA 引导的合成） 逆转录病毒的RNA基因组）被推测是通过链转移发生的。 在一个模板（供体）上合成 DNA 的这一过程是 转移到另一个模板（受体）以获得额外的伸长， 在逆转录病毒复制周期中发生两次。除了这些 所需的转移发生在病毒模板的末端，其他 基因组内部区域内的转移也可能发生。一个在 旨在检查内部链转移的体外系统将用于 模型重组发生在供体的内部区域。 实验将评估与此相关的动力学参数 过程以及 RT 对特定步骤的影响（例如， 新生DNA与供体的分离及其与 受体），目标是建立重组模型。 特定病毒序列上的链转移具有可能的特征 将使用一个能够促进有效转移的系统进行检查 测量 RNA 任何区域的链转移效率。潜力 碱基错误掺入对转移效率和频率的影响 将评估转移事件期间的错误合并。这是 鉴于 HIV-RT 的 DNA 合成保真度较低，这一点尤其重要 事实上，错误掺入会导致 DNA 合成暂停 已提出促进转移。核衣壳蛋白 (NCp) 是一种 逆转录病毒病毒颗粒核心中发现的核酸结合蛋白。 这种蛋白质的特性表明它可能会影响 DNA RT 的合成和链转移催化特性。这些 将在上述系统中评估可能性。除了 重组研究中，HIV-RT 的 RNase H 切割特异性将 进行检查，实验将使用模仿结构的基材 复制过程中存在以确定 RT 如何识别和切割 此类结构并评估其空间和拓扑排列 RT 的聚合酶和 RNase H 活性位点。总体而言，研究 将深入了解逆转录病毒进化的机制之一 并逃避药物治疗和宿主免疫反应。安 了解这些过程对于设计药物疗法很重要 或其他针对独特 RT 活动的策略。

This proposal will focus on the mechanism of human immunodeficiency virus (HIV) recombination as catalyzed by reverse transcriptase (RT). Recombination during first strand DNA synthesis (synthesis directed by the RNA genome of the retrovirus) is presumed to occur by strand transfer. This process, whereby DNA synthesized on one template (donor) is transferred to another template (acceptor) for additional elongation, occurs twice during the retroviral replication cycle. In addition to these required transfers which occur at the termini of viral templates, other transfers within internal regions of the genome can also occur. An in vitro system designed to examine internal strand transfers will be used to model recombination occurring within internal regions of the donor. Experiments will evaluate the kinetic parameters associated with this process and the influence of RT on specific steps (for example, the dissociation of the nascent DNA from the donor and its association with the acceptor) with the goal of formulating a model for recombination. Strand transfer on specific viral sequences with characteristics that may promote efficient transfer will be examined using a system that can measure the strand transfer efficiency of any region of RNA. The potential effect of base misincorporations on transfer efficiency and the frequency of misincorporation during transfer events will be evaluated. This is particularly relevant given the low fidelity of DNA synthesis by HIV-RT and the fact that misincorporation leads to DNA synthesis pausing which has been proposed to promote transfer. Nucleocapsid protein (NCp) is a nucleic acid binding protein found in the core of the retroviral virion. The properties of this protein suggest that it may influence the DNA synthetic and strand transfer catalysis properties of RT. These possibilities will be evaluated in the above systems. In addition to the studies on recombination, the RNase H cleavage specificity of HIV-RT will be examined, The experiments will use substrates that mimic structures present during replication to determine how the RT recognizes and cleaves such structures and to evaluate the spacial and topological arrangement of the polymerase and RNase H active sites of the RT. Overall, the studies will offer insight into one of the mechanisms by which retroviruses evolve and escape both drug therapy and the host immune response. An understanding of these processes is important for designing drug therapies or other strategies that target unique RT activities.