Retroviral Replication and Vector Design

逆转录病毒复制和载体设计

基本信息

批准号：
7733048
负责人：
stephen h hughes
金额：
$ 115.76万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7733048
关键词：
Amino Acids Animals Biochemical Biological Models Cells Chickens Complex Cultured Cells Decompression Sickness Development Effectiveness Embryo Engraftment Enzymes Gene Expression Genes Genome Goals HIV HIV Drug Resistance Program HIV-1 HIV-1 Reverse Transcriptase HIV-1 integrase Hematopoietic stem cells In Vitro Infection Control Integrase Laboratories Learning Methods Modification Molecular Monitor Murine leukemia virus Mus Mutate Mutation Nucleic Acids Nucleosides Pathogenesis Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacology Phenotype Polymerase Positioning Attribute Predisposition Process Property Proteins RNA-Directed DNA Polymerase Reporting Resistance Retroviral Vector Reverse Transcription Ribonuclease H Role Shuttle Vectors Site Visit Specificity Structure System Viral Work base design human disease in vivo interest mutant non-nucleoside reverse transcriptase inhibitors promoter research study tool vector viral DNA

项目摘要

Given the importance of HIV-1 in human disease and the wealth of information about the structure and function of the HIV-1 proteins, including reverse transcriptase (RT), we want to understand HIV-1 replication in more detail. We are particularly interested in correlating the structure of HIV-1 RT, and the biochemical properties of wild-type and mutant RTs, with the process of reverse transcription in cells infected with a one-round HIV-1 vector. In the experiments with HIV-1 vectors, we can monitor several of the important steps in reverse transcription and the specificity of RNase H cleavage. Fortunately, many of the lessons we have learned in developing the RCAS vectors are useful in planning the HIV-1 vector experiments, and some of the tools we have developed (in particular, the shuttle cassette from the RCAS-derived RSVP vectors) can, with some modifications, be used in the HIV-1 system. We believe that the interactions between HIV-1 RT and the different types of nucleic acid substrates it uses are important for the ability of the enzyme to constrain, bend, and properly position its nucleic acid substrates and that this positioning is critical for both the RNase H and polymerase activities. As a consequence, we are particularly interested in mutating amino acids that contact the nucleic acid substrates to determine their roles in reverse transcription. We plan to select pseudorevertants (compensatory mutations) for some of the mutations that have interesting in vivo phenotypes. We are also interested in determining why the process of reverse transcription is significantly more efficient in an infected cell than it is in an in vitro system comprised of purified components. We will try to develop methods to identify and isolate reverse transcription complexes (RTCs) and preintegration complexes (PICs) from infected cells with the goal of identifying the viral and host proteins present in RTCs and PICs. We have also developed HIV-1 vectors that carry many of the common mutations associated with resistance to nucleoside RT inibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). These vectors are being used to evaluate candidate compounds; the long-term goal of our work with HIV-1 RT and the HIV-1 vectors is the development of more effective drugs. We are using the HIV-1 vectors to help Dr. Terrence Burke (Laboratory of Medicinal Chemistry, NCI) and Dr. Yves Pommier (Laboratory of Molecular Pharmacology, NCI) evaluate the effectiveness of compounds that target HIV-1 integrase. We are continuing to use the RCAS retroviral vector system to study viral replication. These vectors are replication competent, grow to high titer, and are safe and easy to use, both in cultured cells and in animals. Although we have worked with the RCAS vectors both in chickens and in mice for many years, we have made some difficult decisions about which experiments to pursue, and have eliminated all of the work with the RCAS vectors in chickens and in chicken embryos. We are continuing to do mouse experiments with the RCAS vectors, but have reduced the scope of the work, focusing on determining the ability of the RCAS vectors to sustain the expression of a foreign gene in experiments involving the ex vivo treatment of hematopoietic stem cells which are engrafted into mice. We are doing control infection/engraftment experiments in mice with mouse leukemia virus (MLV) and HIV-1 vectors that carry the same promoters and marker genes. We have developed an RCAS shuttle vector and have used it to show that linear viral DNAs with one defective end can be inserted into the host genome efficiently, although the defective end does not appear to be inserted by integrase (IN). [Corresponds to Hughes Project 2 in the April 2007 site visit report of the HIV Drug Resistance Program]

鉴于HIV-1在人类疾病中的重要性以及有关HIV-1蛋白的结构和功能的丰富信息，包括逆转录酶（RT），我们希望更详细地了解HIV-1的复制。我们对将HIV-1 RT的结构以及野生型和突变体RT的生化特性与感染一轮HIV-1载体感染的细胞中的逆转录过程相关联。在使用HIV-1载体的实验中，我们可以监视逆转录和RNase H裂解的特异性的几个重要步骤。幸运的是，我们在开发RCAS向量方面学到的许多课程对于计划HIV-1矢量实验很有用，我们开发的某些工具（尤其是来自RCAS衍生的RSVP矢量的梭子盒）可以在HIV-1系统中使用一些修改。我们认为，它使用的HIV-1 RT与不同类型的核酸底物之间的相互作用对于酶约束，弯曲和正确定位其核酸底物的能力很重要，并且这种定位对于RNase H和聚合酶活性至关重要。因此，我们特别有兴趣突变与核酸底物接触以确定其在逆转录中的作用的氨基酸。我们计划为某些具有有趣体内表型的突变选择伪逆想者（补偿性突变）。我们还有兴趣确定为什么在感染细胞中逆转录的过程比在由纯化成分组成的体外系统中明显高得多。我们将尝试开发从感染细胞中识别和隔离逆转录复合物（RTC）和前整合复合物（PIC）的方法，其目的是识别RTC和PICS中存在的病毒蛋白和宿主蛋白。我们还开发了HIV-1载体，这些向量携带了许多与抗核苷RT抗抑制剂（NRTIS）和非核苷RT RT抑制剂（NNRTIS）相关的常见突变。这些向量用于评估候选化合物。我们使用HIV-1 RT和HIV-1载体工作的长期目标是开发更有效的药物。我们正在使用HIV-1载体来帮助Terrence Burke博士（NCI药物化学实验室）和Yves Pommier博士（NCI分子药理学实验室）评估靶向HIV-1积分酶的化合物的有效性。我们正在继续使用RCAS逆转录病毒载体系统来研究病毒复制。这些向量具有复制能力，在培养的细胞和动物中都可以安全且易于使用。尽管我们已经与鸡和小鼠的RCAS载体合作了很多年，但我们已经对要进行哪些实验做出了一些艰难的决定，并消除了与鸡和鸡胚胎中的RCAS载体一起使用的所有工作。我们正在继续使用RCAS载体进行小鼠实验，但已降低了工作范围，重点是确定RCAS矢量在涉及植入小鼠的造血干细胞的实验治疗中维持外国基因表达的能力。我们正在用小鼠白血病病毒（MLV）和HIV-1载体进行控制感染/植入实验，这些载体和HIV-1载体携带相同的启动子和标记基因。我们已经开发了一个RCAS班车向量，并使用它表明，具有有缺陷端的线性病毒DNA可以有效地插入宿主基因组中，尽管有缺陷的末端似乎并未被综合酶（IN）插入。 [对应于Hughes Project 2在2007年4月的现场访问艾滋病毒耐药计划报告中]

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Combining mutations in HIV-1 reverse transcriptase with mutations in the HIV-1 polypurine tract affects RNase H cleavages involved in PPT utilization.

HIV-1 逆转录酶的突变与 HIV-1 多嘌呤束的突变相结合，会影响 PPT 利用中涉及的 RNase H 裂解。

DOI：
10.1016/j.virol.2005.12.042
发表时间：
2006
期刊：
Virology
影响因子：
3.7
作者：
McWilliams,MaryJane;Julias,JohnG;Sarafianos,StefanG;Alvord,WGregory;Arnold,Eddy;Hughes,StephenH
通讯作者：
Hughes,StephenH

Mutations of a residue within the polyproline-rich region of Env alter the replication rate and level of cytopathic effects in chimeric avian retroviral vectors.

Env富含多聚脯氨酸的区域内残基的突变改变了嵌合禽类逆转录病毒载体的复制速率和细胞病变效应水平。