Mechanism of recombination by HIV reverse transcriptase

描述（由申请人提供）：由于艾滋病毒能够迅速变异并产生能够规避免疫反应和抵抗药物治疗的遗传变异，因此，抗击艾滋病毒的努力受到阻碍。大多数突变是在基因组复制过程中产生的。重组有助于重新分类它们，进一步扩大遗传多样性。两种病毒蛋白，逆转录酶（RT）和核衣壳（NC）明确参与了这些过程。本提案的目标是回答有关重组机制的关键问题，并更好地了解RT和NC如何进化到与复制中间体相互作用。此外，将选择可能用作病毒抑制剂的紧密结合核酸分子（适体）。本文列出了三个具体目标：(1)确定体外发现的gag-pol移码区的重组热点是否也是细胞感染的热点，热点的发生是否依赖于体外测试条件；(2)确定HIV核衣壳蛋白如何抑制随机序列RNA而非多嘌呤道RNA引发DNA合成；(3)：分离出高亲和力结合RTs （MuLV、AMV和TY3）的引物模板序列，并确定其他RTs是否与HIV-RT相似，也与同源多嘌呤道序列（ppt）紧密结合。这些实验将采用体外和细胞培养相结合的方法。例如，在Aim 1中，将在这两种条件下进行重组分析，并进行比较，以验证结论。目的2建立在我们实验室的初步数据上，显示NC抑制非ppt RNA引物，但不影响ppt的使用。这可能对防止假引物第二链合成和产生具有多重不连续的原病毒具有重要意义。目的3与目的2相关，并将解决RTs进化到识别并紧密结合用于第二链DNA引物的ppt序列的可能性。这种紧密结合可能用于设计病毒难以逃脱的抑制剂。公共卫生相关性：这项工作与人类健康相关，因为它将有助于确定基于核酸的药物治疗（适体）的可能线索。此外，它将有助于确定药物干预的新特异性靶点（即ppt，重组）的潜力。这项工作与人类健康有关，因为它将有助于确定基于核酸的药物治疗（适体）的可能线索。此外，它将有助于确定新的特异性靶点（NC, ppt，重组）用于药物干预的潜力。

DESCRIPTION (provided by applicant): Attempts to combat HIV have been hampered due to the virus's ability to rapidly mutate and produce genetic variants that can circumvent the immune response and resist drug therapy. Most mutations are generated during genome replication. Recombination serves to reassort them, further amplifying genetic diversity. Two viral proteins, reverse transcriptase (RT) and nucleocapsid (NC) have been clearly implicated in these processes. The goal of this proposal is to answer key questions regarding the mechanism of recombination and to better understand how RT and NC have evolved to interact with replication intermediates. In addition, tight binding nucleic acid molecules (aptamers) that could potentially be used as viral inhibitors will be selected. Three specific aims are listed: (1) To determine if a recombination hotspot in the gag-pol frameshift region discovered in vitro is also a hotspot in cellular infections and whether occurrence of hotspots is dependent on in vitro test conditions; (2) To determine how HIV nucleocapsid protein inhibits priming of DNA synthesis by random sequence RNAs but not polypurine tract RNA; (3): To isolate primer-template sequences that bind RTs (MuLV, AMV, and TY3) with high affinity and determine if like HIV-RT, other RTs also bind their cognate polypurine tract sequences (ppt) very tightly. A combination of in vitro and cell culture approaches will be used for these experiments. For example, in Aim 1 recombination assays will be conducted under both conditions and compared in order to verify conclusions. Aim 2 builds on preliminary data from our lab showing that NC inhibits non-ppt RNA priming but does not affect the ppt usage. This could be important to preventing spurious priming 2nd strand synthesis and producing proviruses with multiple discontinuities. Aim 3 is related to aim 2 and will address the possibility that RTs evolved to recognize and bind tightly to ppt sequences that are used for second strand DNA priming. This tight binding could potentially be used to design inhibitors that may be difficult for the virus to escape from. PUBLIC HEALTH RELEVANCE: This work is relevant to human health because it will help identify possible leads for nucleic acid-based drug therapies (aptamers). In addition, it will help define the potential of new specific targets (i.e. ppt, recombination) for drug intervention. This work is relevant to human health because it will help identify possible leads for nucleic acid-based drug therapies (aptamers). In addition, it will help define the potential of new specific targets (NC, ppt, recombination) for drug intervention.