Mechanism of recombination by HIV reverse transcriptase

HIV逆转录酶重组机制

• 批准号: 7848185
• 负责人: JEFFREY J DESTEFANO
• 金额: $ 29.7万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7848185
• 关键词: Acquired Immunodeficiency Syndrome; Address; Affect; Affinity; Anti-Retroviral Agents; Basic Science; Behavior; Binding; Biological Assay; Cell Culture Techniques; Cells; Cessation of life; DNA; DNA Primers; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Drug Delivery Systems; Environment; Enzymes; Evolution; Gagging; Genetic Crossing Over; Genetic Materials; Genetic Recombination; Genetic Variation; Genome; Goals; Grant; HIV; Health; Human; Immune response; In Vitro; Infection; Intervention; Left; Ligands; Light; Maps; Modeling; Mutate; Mutation; Nature; Nucleic Acid Binding; Nucleic Acids; Nucleocapsid; Nucleocapsid Proteins; Pharmaceutical Preparations; Pharmacotherapy; Polymerase; Preclinical Drug Evaluation; Process; Progress Reports; Proteins; Proviruses; RNA; RNA Sequences; RNA-Directed DNA Polymerase; Reaction; Retroviridae; Reverse Transcription; Ribonuclease H; Role; Site; Specificity; Structure; System; Testing; Time; Transcription Process; Uncertainty; Viral; Viral Proteins; Virus; Virus Inhibitors; Virus Replication; Work; aptamer; base; combat; design; fitness; genetic variant; improved; in vitro testing; inhibitor/antagonist; novel; nucleic acid inhibitor; preference; pressure; prevent; public health relevance; research study; stem; viral DNA; viral RNA

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.

描述（由申请人提供）：由于病毒能够快速突变并产生可以绕过免疫反应和抵抗药物治疗的遗传变异，因此对抗艾滋病毒的努力受到阻碍。大多数突变在基因组复制过程中产生。繁殖作用使它们重新分类，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）已明确涉及这些过程。该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如何演变为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）。列出了三个具体目的：（1）确定体外发现的gag-pol移码区中的重组热点是否也是细胞感染中的热点，以及热点的发生是否取决于体外测试条件;（2）确定HIV核衣壳蛋白如何抑制随机序列RNA而不是多嘌呤段RNA引发DNA合成;（3）：为了分离以高亲和力结合RT（MuLV、AMV和TY 3）的引物-模板序列，并确定其他RT是否与HIV-RT一样也非常紧密地结合其同源多嘌呤段序列（ppt）。这些实验将使用体外和细胞培养方法的组合。例如，在目标1中，重组试验将在两种条件下进行并进行比较，以验证结论。目标2建立在我们实验室的初步数据基础上，这些数据表明NC抑制非ppt RNA引发，但不影响ppt的使用。这对于防止虚假引发第二链合成和产生具有多个不连续性的前病毒可能是重要的。目标3与目标2相关，并将解决RT进化为识别并紧密结合用于第二链DNA引发的ppt序列的可能性。这种紧密结合可能被用来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有关，因为它将有助于确定基于核酸的药物疗法（适体）的可能线索。此外，它将有助于确定药物干预的新的特异性靶点（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疗法（适体）的可能线索。此外，它将有助于确定药物干预的新的特异性靶点（NC，PPT，重组）的潜力。