DELETION MUTAGENESIS WITHIN NONCODING SEGMENTS OF HIV1

HIV1非编码片段内的缺失突变

• 批准号: 6170819
• 负责人: MARK A. WAINBERG
• 金额: $ 19.53万
• 依托单位: SIR MORTIMER B. DAVIS JEWISH GEN HOSP
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6170819
• 关键词: AIDS vaccines; DNA replication; Macaca; RNA biosynthesis; RNA directed DNA polymerase; attenuated microorganism; gene deletion mutation; human immunodeficiency virus 1; molecular cloning; nucleic acid sequence; protein biosynthesis; recombinant virus; simian immunodeficiency virus; tissue /cell culture; vaccine development; virus genetics; virus protein; virus replication

DESCRIPTION: The long term goal of this proposal is to better understand the non-coding region of HIV so as to shed light on the contribution of this region to reverse transcription, viral gene expression, protein synthesis and viral replication. Deletion mutagenesis in this region may also help in the development of an attenuated HIV vaccine, and extensive research using the SIV model will be conducted to determine whether this may be the case. This research will provide important new information on a relatively poorly understood area of the HIV genome. Among the questions to answer are the following: 1. Whether a deletion of the 54 nt segment downstream of the primer binding site will permanently attenuate HIV replication in tissue culture, and to understand the molecular basis for reversions that involve a series of smaller deletions within the 54 nt region. To determine whether a deletion mutation at the 3' end of the 54 nt region may also compromise HIV replication capacity and interfere with the production of viral DNA, mRNA, and proteins to the same extent as the 54 nt deletion. 2. To determine whether a similar deletion in simian immunodeficiency virus (SIV), to that of the 54 nt deletion in HIV, will also impact severely on viral replication, generation of viral DNA, synthesis of viral mRNA, and production of viral protein. 3. Whether combining the 54 nt deletion with the 184V substitution in RT, previously shown to increase RT fidelity, may diminish the likelihood of reversion taking place. Also, what will the effect be of combining the 184V substitution in RT with deletion of the A-rich loop on the rapidity of reversion? 4. To determine whether combination of the large and small deletion mutations in SIV, together with the 184V substitution in RT that increases fidelity, will likewise result in a lesser likelihood of reversion to wild-type virus over extended periods. 5. To determine whether SIV variants, containing large deletions in the non-coding region, with or without the 184V substitution in RT, may serve as attenuated vaccines for purposes of stimulating anti-SIV humoral and cellular immune responsiveness in macaques. 6. To determine whether these attenuated SIV constructs may protect against subsequent challenge with virulent forms of SIV and SIV/HIV hybrids.

描述：该提案的长期目标是更好地理解 HIV的非编码区，以阐明该区域的贡献 逆转录、病毒基因表达、蛋白质合成区域 和病毒复制。 该区域的缺失突变也可能有助于 减毒艾滋病毒疫苗的开发以及使用的广泛研究 将通过 SIV 模型来确定情况是否如此。 这项研究将为相对较差的研究提供重要的新信息 了解 HIV 基因组的区域。 需要回答的问题包括 如下： 1.是否删除了下游的54nt片段 引物结合位点将永久减弱组织中的 HIV 复制 文化，并了解涉及逆转的分子基础 54 nt 区域内一系列较小的缺失。 来确定是否一个 54 nt 区域 3' 端的缺失突变也可能损害 HIV 复制能力并干扰病毒 DNA、mRNA 的产生， 和蛋白质的54 nt缺失程度相同。 2. 确定 猿猴免疫缺陷病毒（SIV）中是否存在类似的缺失 HIV中54nt的缺失，也将严重影响病毒 复制、病毒DNA的产生、病毒mRNA的合成，以及 病毒蛋白的生产。 3.是否将54nt缺失与 RT 中的 184V 替代先前已被证明可以提高 RT 保真度，可能 减少发生逆转的可能性。 另外，将会发生什么 效果是将 RT 中的 184V 取代与删除相结合 丰富的循环对回复的速度有影响吗？ 4. 判断是否 SIV 中大小缺失突变的组合，以及 RT 中的 184V 替代可提高保真度，同样会导致 长时间内恢复为野生型病毒的可能性较小。 5. 确定 SIV 变体是否包含大片段缺失 非编码区，无论 RT 中是否有 184V 取代，都可以充当 用于刺激抗 SIV 体液和免疫反应的减毒疫苗 猕猴的细胞免疫反应。 6. 判断这些是否 减毒的 SIV 构建体可以防止随后的攻击 SIV 和 SIV/HIV 杂交种的剧毒形式。