Genome stability maintenance factors in early HIV-1 infection

早期HIV-1感染中的基因组稳定性维持因素

• 批准号: 7846591
• 负责人: LUBBERTUS C MULDER
• 金额: $ 1.53万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-05 至 2009-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7846591
• 关键词: Adenoviruses; Affect; Anti-Retroviral Agents; Biology; Cell Line; Cells; Complementary DNA; DNA; DNA Repair; DNA Repair Inhibition; DNA Repair Pathway; Eukaryota; Event; Fungal Genome; Gene Expression; Generations; Genes; Genetic; Genome Stability; Goals; HIV-1; HIV-1 integrase; Homologous Gene; Human; Immunity; Impact evaluation; Indium; Infection; Integrase; Integration Host Factors; Invaded; Knowledge; Lead; Life Cycle Stages; Maintenance; Measurement; Mediating; Methodology; Mutation; Orthologous Gene; Pharmaceutical Preparations; Phase; Play; Predisposition; Process; Proteins; RNA-Directed DNA Polymerase; RTH-1 Nuclease; Recruitment Activity; Reporting; Research; Retrotransposition; Retrotransposon; Retroviridae; Reverse Transcription; Role; System; Tertiary Protein Structure; Testing; Titrations; Translating; Variant; Viral; Viral Proteins; Virus; Virus Diseases; Work; Yeasts; base; domain mapping; ds-DNA; insight; man; member; mutant; novel; overexpression; small hairpin RNA; therapy development

DESCRIPTION (provided by applicant): HIV-1 infection is influenced by several host factors, including components of DNA repair pathways. One example is Rad18, a member of the post-replication DNA repair system and also an important element in the maintenance of genome stability. Rad18 interacts with and stabilizes HIV-1 integrase. Moreover, Rad18 has a negative influence on HIV-1 infection, an effect exerted at a step between reverse transcription and integration. Rad18 also has a negative effect on infection by MLV, and adenovirus, suggesting that double stranded DNA is the ultimate target of the inhibitory activity. We hypothesize that HIV-1 integrase serves as a bridge to allow Rad18 to access viral cDNA, where it might recruit other effectors from DNA repair pathways, leading to cDNA degradation. We propose to determine whether Rad18 interaction with integrase is responsible for the inhibition of HIV-1 infection by Rad18. This will be accomplished by assessing the difference of cDNA synthesis between wild type viruses and viruses lacking integrase, in Rad18-/- and Rad18+/+ cells. Furthermore we plan to map the domains and residues that participate in the association of the two molecules. Mutation of the integrase residues involved in the interaction with Rad18 will yield protein variants that cannot associate with Rad18, and therefore viruses that are not inhibited by Rad18. Conversely, Rad18 mutants that cannot interact with integrase will not have an inhibitory effect on HIV-1 infection, in contrast to what observed with wild-type Rad18. We will evaluate all mutants in the context of viral infection. Furthermore, we will also examine the effect of overexpression of the interacting domains by themselves on viral infection, to assess whether they can suppress the Rad18 inhibition by titration of the host protein (in the case of integrase domains) or by competition with cellular Rad18 (in the case of Rad18 domains). We also aim to investigate the functional conservation, in HIV-1 infection, of human homologues of yeast genome stability factors that inhibit Ty retrotransposition. We will initially analyze two genes, FEN-1 and WRN, whose orthologs in yeast are known to influence Ty1 activity. Knockdown cell lines will be obtained using shRNA methodology. These cell lines will be tested for HIV-1 infection, and we will characterize which phase of the viral life cycle is affected. The proposed research will elucidate the role of Rad18 in inhibiting HIV-1 infection and reveal new host factors that modulate viral infection. The results will open perspectives for new anti-retroviral therapy development. 7. Project Narrative. The goal of this proposal is to elucidate the mechanism of HIV-1 inhibition by DNA repair molecule Rad18 and identify novel cellular suppressors of HIV-1, by taking advantage of their potential conservation from yeast to man. The identification and understanding of these proteins may lead to the ability to harness multiple levels of inhibition against which viruses would unlikely have the capacity to adapt. This can then translate not only in the precious expansion of our knowledge about the viral biology but possibly in the discovery of amenable anti- retroviral targets.

描述(由申请人提供)：HIV-1感染受到几种宿主因素的影响，包括DNA修复途径的组成部分。一个例子是Rad18，它是复制后DNA修复系统的成员，也是维持基因组稳定性的重要因素。RAD18与HIV-1整合酶相互作用并稳定。此外，Rad18对HIV-1感染有负面影响，这种影响是在逆转录和整合之间的一个阶段发挥的。RAD18对MLV和腺病毒的感染也有负性作用，提示双链DNA是其抑制活性的最终靶点。我们推测，HIV-1整合酶作为一座桥梁，允许Rad18访问病毒的cDNA，在那里它可能从DNA修复途径招募其他效应者，导致DNA降解。我们建议确定Rad18与整合酶的相互作用是否与Rad18抑制HIV-1感染有关。这将通过评估野生型病毒和缺乏整合酶的病毒在Rad18-/-和Rad18+/+细胞中合成的差异来实现。此外，我们计划绘制参与这两个分子结合的结构域和残基的图谱。参与与Rad18相互作用的整合酶残基的突变将产生不能与Rad18相关联的蛋白质变体，从而产生不被Rad18抑制的病毒。相反，与野生型Rad18相比，不能与整合酶相互作用的Rad18突变体将不会对HIV-1感染产生抑制作用。我们将在病毒感染的背景下评估所有突变体。此外，我们还将检查相互作用结构域的过度表达对病毒感染的影响，以评估它们是否可以通过滴定宿主蛋白(在整合酶域的情况下)或通过与细胞Rad18的竞争(在Rad18结构域的情况下)来抑制Rad18的抑制。我们还旨在研究在HIV-1感染中，抑制Ty逆转录转座的酵母基因组稳定因子的人类同源物的功能保守性。我们将首先分析两个基因，fen-1和wrn，它们在酵母中的同源基因已知会影响Ty1的活性。将使用shRNA方法获得击倒的细胞系。这些细胞株将接受HIV-1感染测试，我们将确定病毒生命周期的哪个阶段受到影响。这项拟议的研究将阐明Rad18在抑制HIV-1感染方面的作用，并揭示调节病毒感染的新宿主因素。这一结果将为新的抗逆转录病毒疗法的开发开辟前景。7.项目叙事。这项建议的目的是阐明DNA修复分子Rad18抑制HIV-1的机制，并通过利用它们从酵母到人类的潜在保守性来识别HIV-1的新的细胞抑制因子。对这些蛋白质的识别和理解可能导致利用多种水平的抑制，而病毒不太可能有能力对其进行适应。然后，这不仅可以转化为我们关于病毒生物学知识的宝贵扩展，而且可能转化为发现顺从的抗逆转录病毒靶标。

项目成果

Moderate influence of human APOBEC3F on HIV-1 replication in primary lymphocytes.

人 APOBEC3F 对原代淋巴细胞中 HIV-1 复制的中等影响。

• DOI: 10.1128/jvi.02630-09
• 发表时间: 2010
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Mulder,LubbertusCF;Ooms,Marcel;Majdak,Susan;Smedresman,Jordan;Linscheid,Caitlin;Harari,Ariana;Kunz,Andrea;Simon,Viviana
• 通讯作者: Simon,Viviana