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
基本信息
- 批准号:10759845
- 负责人:
- 金额:$ 22.51万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:APOCEC3G geneAddressAffectAreaBiological AssayCapsidCell Culture TechniquesCell NucleusCellsCellular AssayComplementConsensusCoupledDNADNA biosynthesisDNA-Directed RNA PolymeraseDataDefense MechanismsDevelopmentDivalent CationsEnvironmentEnzymesEvolutionFutureGenetic Complementation TestGenetic VariationGenomeGoalsHIVHIV-1HealthHumanImmune responseImmunityIn VitroKineticsLeadLettersModernizationMutagenesisMutationMutation AnalysisNucleotidesPharmaceutical PreparationsPharmacotherapyPhysiologicalPolymerasePositioning AttributeProcessProductionProteinsPublicationsRNARNA Polymerase IIRNA-Directed DNA PolymeraseReactionReportingResearch PersonnelRetroviridaeReverse TranscriptionRibavirinRoleSodium ChlorideSourceSystemTimeVariantViralVirionVirusVirus ReplicationWorkds-DNAexperimental studyimprovedin vitro Assayin vitro activityin vivonext generation sequencingnoveltool
项目摘要
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.
对其他逆转录病毒RT的体外分析表明,HIV RT具有极低的保真度。比如说,
MuLV-RT在几种不同的RNA和DNA模板上显示出比HIV RT高约5倍的保真度。
不同群体的出版物,甚至在同一出版物中的相同实验。但细胞
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检测也与体外结果不一致。最近,我们的实验室和其他人发现,
当使用生理浓度的游离Mg 2+(~0.25-1 mM)时,HIV RT的保真度提高,
至通常用于保真度测定的针对体外活性优化的较高浓度(5-10 mM
体外这可能是解释HIV RT和其他病毒之间差异的一个因素,
RT。例如,MuLV和AMV RT在较低的Mg 2+和MuLV RT中没有显示出显著改善的保真度
具有与细胞复制值更一致的体外错误率。缺乏相关性
关于体外观察到的总体突变率和突变谱与细胞复制相比,
不可能确定RT对细胞中观察到的突变谱的贡献。这又使得
难以评估诱变细胞因子的作用(例如,APOBEC 3G、UNG和RNA聚合酶
(2)观察到的光谱。深入了解导致遗传多样性的因素,
艾滋病病毒将需要更好地了解艾滋病病毒逆转录酶在这一过程中的作用,并明确定义突变率
和在体外用HIV RT观察到的光谱,以及在细胞中病毒复制期间观察到的光谱。了解艾滋病毒如何
产生遗传多样性,从而促进新病毒的产生,这些病毒可以逃避免疫和药物。
治疗,一直是艾滋病研究人员的长期目标。我们相信,这一建议将使我们朝着
这个目标新的高保真下一代测序(NGS)方法,包括单链共有序列
测序(SSCS)和双链体测序,允许在一个单一的基因组中快速分析数千个突变。
实验,并带来了一个新的时代突变分析。最近的研究结果也是如此,
博士韦斯·桑德奎斯特的实验室,我们在这项工作上的合作者,证明了艾滋病毒dsDNA的完全复制,
在高效的基于无细胞病毒体的内源性逆转录(在本提议中称为“ERT”)中,
系统通过将现代NGS方法应用于体外纯化RT的DNA合成,在ERT反应中,
在病毒在细胞中复制期间,可以直接比较这些过程的突变率和谱。
值得注意的是,这将是在新的ERT系统中的第一个保真度分析。这将更清楚地界定RT的作用,
对HIV诱变的相对贡献,以及病毒体和细胞因子的作用。具体目标:目标1:使用
下一代测序(NGS)比较纯化的HIV RT和
高效内源性逆转录(ERT)测定;目的2:比较
通过纯化的RT和ERT反应中产生的突变,与在细胞中逆转录期间观察到的突变相比。
项目成果
期刊论文数量(0)
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科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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JEFFREY J DESTEFANO其他文献
JEFFREY J DESTEFANO的其他文献
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{{ truncateString('JEFFREY J DESTEFANO', 18)}}的其他基金
Development and Evaluation of Novel Aptamer-based Therapeutics Targeting SARS-CoV-2 in a Physiologically-Relevant Model of Human Airway Epithelium
在人类气道上皮生理相关模型中针对 SARS-CoV-2 的新型适体疗法的开发和评估
- 批准号:
10449392 - 财政年份:2021
- 资助金额:
$ 22.51万 - 项目类别:
Development and Evaluation of Novel Aptamer-based Therapeutics Targeting SARS-CoV-2 in a Physiologically-Relevant Model of Human Airway Epithelium
在人类气道上皮生理相关模型中针对 SARS-CoV-2 的新型适体疗法的开发和评估
- 批准号:
10287842 - 财政年份:2021
- 资助金额:
$ 22.51万 - 项目类别:
Biochemistry of HIV reverse transcriptase fidelity and inhibitor interactions
HIV逆转录酶保真度和抑制剂相互作用的生物化学
- 批准号:
9538330 - 财政年份:2016
- 资助金额:
$ 22.51万 - 项目类别:
Biochemistry of HIV reverse transcriptase fidelity and inhibitor interactions
HIV逆转录酶保真度和抑制剂相互作用的生物化学
- 批准号:
9064995 - 财政年份:2016
- 资助金额:
$ 22.51万 - 项目类别:
Development of an HIV reverse transcriptase aptamer-based detection assay
基于 HIV 逆转录酶适体的检测方法的开发
- 批准号:
8846946 - 财政年份:2014
- 资助金额:
$ 22.51万 - 项目类别:
MECHANISM OF RECOMBINATION BY HIV REVERSE TRANSCRIPTASE
HIV逆转录酶的重组机制
- 批准号:
2189459 - 财政年份:1994
- 资助金额:
$ 22.51万 - 项目类别:
MECHANISM OF RECOMBINATION BY HIV REVERSE TRANSCRIPTASE
HIV逆转录酶的重组机制
- 批准号:
2701621 - 财政年份:1994
- 资助金额:
$ 22.51万 - 项目类别:
MECHANISM OF RECOMBINATION BY HIV REVERSE TRANSCRIPTASE
HIV逆转录酶的重组机制
- 批准号:
6627193 - 财政年份:1994
- 资助金额:
$ 22.51万 - 项目类别:
Mechanism of recombination by HIV reverse transcriptase
HIV逆转录酶重组机制
- 批准号:
7028364 - 财政年份:1994
- 资助金额:
$ 22.51万 - 项目类别:
MECHANISM OF RECOMBINATION BY HIV REVERSE TRANSCRIPTASE
HIV逆转录酶的重组机制
- 批准号:
6490080 - 财政年份:1994
- 资助金额:
$ 22.51万 - 项目类别:
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