Using new Next Generation Sequencing (NGS) approaches to analyze the fidelity of HIV reverse transcription in Endogenous Reverse Transcription reactions (ERT)

使用新的下一代测序 (NGS) 方法来分析内源性逆转录反应 (ERT) 中 HIV 逆转录的保真度

• 批准号: 10759845
• 负责人: JEFFREY J DESTEFANO
• 金额: $ 22.51万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-20 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10759845
• 关键词: APOCEC3G gene; Address; Affect; Area; Biological Assay; Capsid; Cell Culture Techniques; Cell Nucleus; Cells; Cellular Assay; Complement; Consensus; Coupled; DNA; DNA biosynthesis; DNA-Directed RNA Polymerase; Data; Defense Mechanisms; Development; Divalent Cations; Environment; Enzymes; Evolution; Future; Genetic Complementation Test; Genetic Variation; Genome; Goals; HIV; HIV-1; Health; Human; Immune response; Immunity; In Vitro; Kinetics; Lead; Letters; Modernization; Mutagenesis; Mutation; Mutation Analysis; Nucleotides; Pharmaceutical Preparations; Pharmacotherapy; Physiological; Polymerase; Positioning Attribute; Process; Production; Proteins; Publications; RNA; RNA Polymerase II; RNA-Directed DNA Polymerase; Reaction; Reporting; Research Personnel; Retroviridae; Reverse Transcription; Ribavirin; Role; Sodium Chloride; Source; System; Time; Variant; Viral; Virion; Virus; Virus Replication; Work; ds-DNA; experimental study; improved; in vitro Assay; in vitro activity; in vivo; next generation sequencing; novel; tool

Analysis of RTs from other retroviruses in vitro suggests that HIV RT has exceptionally low fidelity. For example, MuLV-RT shows ~5-fold greater fidelity than HIV RT on both RNA and DNA templates across several publications from different groups, and even in identical experiments in the same publication. Yet cellular replication data indicates that these viruses have similar mutation rates. The mutation profile observed in cellular assay with HIV is also inconsistent with in vitro results. Recently, our lab and others have found that the in vitro fidelity of HIV RT improves when physiological concentrations of free Mg2+ (~0.25-1 mM) are used as opposed to the higher (5-10 mM) concentration optimized for in vitro activity that have typically been used in fidelity assays in vitro . This may be one factor, possibly among many, that explains differences between HIV RT and other viral RTs. MuLV and AMV RTs, for example, do not show significantly improved fidelity in lower Mg2+ and MuLV RT has an error rate in vitro that is more consistent with cellular replication values. The lack of correlation with respect to the overall mutation rate and spectrum of mutations observed in vitro vs. cellular replication has made it impossible to determine RT’s contribution to the mutation spectrum observed in cells. This in turn makes it difficult to assess the contributions of mutagenic cellular factors (e.g., APOBEC3G, UNG, and RNA polymerase II) to the observed spectrum. A thorough understanding of the factors that contribute to the genetic diversity of HIV will require a better understanding of HIV RT’s role in the process, and a clear definition of the mutation rate and spectrum observed with HIV RT in vitro, and during virus replication in cells. Understanding how HIV generates genetic diversity, which fuels the production of new viruses that can escape immunity and drug therapy, has been a long-standing goal of HIV researchers. We believe this proposal will move us forward toward this goal. New high fidelity Next Generation Sequencing (NGS) approaches, including Single Strand Consensus Sequencing (SSCS) and duplex sequencing, allow the rapid analysis of thousands of mutations in a single experiment and has brought in a new era for mutational analysis. The same can be said for recent findings from Dr. Wes Sundquist’s lab, our collaborator on this work, that demonstrate the complete replication of HIV dsDNA in a highly efficient cell-free virion-based endogenous reverse transcription (referred to as “ERT” in this proposal) system. By applying modern NGS approaches to DNA synthesis with purified RT in vitro, in ERT reactions, and during virus replication in cells, the mutation rates and spectrums of these processes can be directly compared. Notably, this will be the first fidelity analysis in the novel ERT system. This will more clearly define RT’s role and relative contribution to HIV mutagenesis, and the roles of virion and cellular factors. Specific Aims: Aim 1: To use Next Generation Sequencing (NGS) to compare the mutation rates and spectrums from purified HIV RT and highly efficient Endogenous Reverse Transcription (ERT) assays; Aim 2: To compare the rate and spectrum of mutations made by purified RT and in ERT reactions, to those observed during reverse transcription in cells.

在体外对其他逆转录病毒的RT分析表明，HIV RT的保真度异常低。例如， MULV-RT在RNA和DNA模板上均显示出比HIV RT高约5倍 来自不同群体的出版物，甚至在同一出版物中的相同实验中。尚未细胞 复制数据表明这些病毒具有相似的突变率。在细胞中观察到的突变谱 HIV的测定也与体外结果不一致。最近，我们的实验室和其他人发现体外 当使用自由Mg2+（〜0.25-1 mm）的物理浓度时，HIV RT的保真度会改善 针对通常用于保真度测定的体外活性优化的较高（5-10毫米）浓度 体外。这可能是许多因素，在许多因素中解释了HIV RT和其他病毒的差异 RTS。例如，MULV和AMV RTS在较低的Mg2+和Mulv RT中没有显着提高忠诚度 在体外具有错误率，与细胞复制值更一致。缺乏与 尊重在体外观察到的整体突变率和突变的频谱，使 不可能确定RT对细胞中观察到的突变光谱的贡献。这反过来使它 难以评估诱变细胞因子的贡献（例如，APOBEC3G，UNG和RNA聚合酶 ii）到观察到的频谱。对导致遗传多样性的因素有透彻的理解 艾滋病毒将需要更好地了解艾滋病毒在该过程中的作用，并明确地定义突变率 在体外和细胞病毒复制期间观察到HIV RT观察到的光谱。了解艾滋病毒 产生遗传多样性，这为可以逃避免疫力和药物的新病毒的产生而产生 疗法是HIV研究人员的长期目标。我们相信该建议将使我们前进 这个目标。新的高保真下一代测序（NGS）方法，包括单链共识 测序（SSC）和双链测序，允许对单个突变进行快速分析 实验并带来了一个新时代进行突变分析。最近的发现也可以说 Wes Sundquist博士实验室，我们在这项工作方面的合作者，证明了HIV DSDNA的完全复制 在高效的基于无细胞病毒的内源性逆转录中（该提案中称为“ ERT”） 系统。通过在体外纯化的RT，ERT反应和 在细胞中的病毒复制过程中，可以直接比较这些过程的突变速率和光谱。 值得注意的是，这将是新型ERT系统中的第一个保真度分析。这将更清楚地定义RT的角色， 对HIV诱变的相对贡献，以及病毒体和细胞因子的作用。具体目的：目标1：使用 下一代测序（NGS）比较纯化的HIV RT的突变率和频谱 高效的内源性逆转录（ERT）测定；目标2：比较速率和频谱 通过纯化的RT和ERT反应进行的突变与细胞逆转录过程中观察到的突变。