Role of IQGAP and Microtubule Motors in MLV Infection

IQGAP 和微管马达在 MLV 感染中的作用

• 批准号: 9066177
• 负责人: STEPHEN Paine GOFF
• 金额: $ 20.77万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 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/9066177
• 关键词: Acquired Immunodeficiency Syndrome; Actins; Antiviral Agents; Behavior; Binding; Bypass; Calmodulin; Cell Nucleus; Cell membrane; Cells; Cellular Morphology; Collaborations; Complex; Cytoplasm; Cytoskeleton; DCTN2 gene; DNA; Dominant-Negative Mutation; Dynein ATPase; Event; Feedback; Fluorescence; Frequencies; GTP Binding; Gammaretrovirus; Genome; Glues; Guanosine Triphosphate Phosphohydrolases; HIV-1; Image; Incidence; Infection; Insertional Activations; Instruction; Knowledge; Learning; Life Cycle Stages; Link; Location; Mediating; Membrane; Microinjections; Microtubule Stabilization; Microtubule-Organizing Center; Microtubules; Mitotic; Modeling; Modification; Moloney Leukemia Virus; Monomeric GTP-Binding Proteins; Motion; Motor; Movement; Murine leukemia virus; Mus; Mutation; Neurites; Output; Phase; Phosphorylation; Process; Protein Fragment; Protein Isoforms; Proteins; Proto-Oncogenes; Proviruses; RNA Interference; Regulation; Retroviridae; Reverse Transcription; Rodent; Role; Scaffolding Protein; Staging; Study Subject; T-Cell Leukemia; Testing; Time; Viral; Viral Proteins; Viral load measurement; Viremia; Virion; Virus; Virus Diseases; Virus Replication; Work; cell motility; dynactin; dynein light chain; gag Gene Products; gene replacement; gene therapy; human disease; inhibitor/antagonist; insight; knock-down; leukemia; live cell imaging; mutant; polymerization; polypeptide; prevent; programs; receptor; research study; tool; trafficking; viral DNA; viral RNA

Moloney murine leukemia virus (MLV) is a prototypical gammaretrovirus that replicates to high titer in neariy all mitotic rodent cells, causes a persistent viremia in infected mice, and induces a T-cell leukemia at a high incidence through insertional activation of host protooncogenes. Much of what we know about retrovirus replication was first learned through the study of the simple viruses such as the MLVs. In the eariy phases of infection, these viruses enter the cell through specific receptors, synthesize a DNA copy of the viral RNA genome by reverse transcription in the cytoplasm, direct the movement ofthe resulting preintegration complex (PIC) into the nucleus, and integrate the viral DNA into the host genome to form the provirus. In the late phases this DNA is expressed to form viral RNAs and proteins, and progeny virions are assembled at the plasma membrane and released to begin a new infectious cycle. In this project we propose to examine the early post-entry events of infection, the most pooriy characterized portion ofthe viral life cycle, focusing on a key host protein, IQGAP, microtubules, and dynein motors. We have previously identified IQGAPI as a major host protein interacting with the Moloney MuLV Gag matrix protein (MA), and have documented the critical importance of that interaction for virus replication. The IQGAPs are large cytoskeletal scaffolding proteins involved in the regulation of cell motility and morphology, and are noteworthy in binding and regulating both actin and microtubule networks. They integrate multiple inputs (especially from small GTPases) and produce multiple outputs, including stabilization of microtubules and capture of microtubule ends. We will determine the role ofthe IQGAPs in MLV infection, and the precise time and step in the life cycle at which they act. Using live-cell imaging of fluorescence-tagged virions, we will examine the trafficking of MLV mutants that do not bind IQGAP, and of wild-type virus blocked by dominant-negative fragments of IQGAP, to determine whether virions fail to be properly delivered to microtubules. We will test for the importance of phosphorylation of IQGAP, thought to be mediated by PKCe, in normal virus trafficking. Finally, we will test the key subunits of the dynein motor and dynactin for their roles in movement of the MLV PICs along both stable and dynamic microtubules. With the help of our cell biologist colleagues in this program, we hope to fill in a majorgap in our current understanding of the MLV life cycle. RELEVANCE (See instructions): Retroviruses are agents of serious human diseases, including leukemias and AIDS, and conversely hold out great promise as tools for gene therapy. Recent work has shown that these viruses are critically dependent on the cytoskeleton and microtubules (MTs) for their intracellular trafficking. In this proposal we aim to define the role of particular MT regulators and motors in eariy steps of MLV infection. Deeper understanding of these processes will provide new insights into retrovirus replication, potentially define new antiviral targets and increase our knowledge of trafficking of cargos on MTs.

莫洛尼鼠白血病病毒（Moloney murine leukemia virus，MLV）是一种典型的γ逆转录病毒，在小鼠体内复制到高滴度， 所有有丝分裂的啮齿动物细胞，在感染的小鼠中引起持续的病毒血症，并在高水平诱导T细胞白血病。 通过宿主原癌基因的插入激活而发病。我们对逆转录病毒的了解 复制最初是通过对简单病毒如MLV的研究而了解的。在早期阶段， 感染后，这些病毒通过特定的受体进入细胞，合成病毒RNA的DNA拷贝， 基因组在细胞质中通过逆转录，指导所产生的整合前的运动 复合物（PIC）进入细胞核，并将病毒DNA整合到宿主基因组中形成前病毒。在 晚期，该DNA被表达以形成病毒RNA和蛋白质，并且后代病毒体在晚期被组装。 细胞膜被释放，开始一个新的传染周期。在这个项目中，我们建议检查 病毒进入后的早期感染事件，即病毒生命周期中特征最差的部分， 关键的宿主蛋白质IQGAP微管和动力蛋白马达我们之前已经将IQGAPI确定为 与Moloney MuLV Gag基质蛋白（MA）相互作用的主要宿主蛋白，并记录了 这种相互作用对病毒复制至关重要。IQGAP是大的细胞骨架支架 参与调节细胞运动和形态的蛋白质，在结合和 调节肌动蛋白和微管网络。他们整合了多个输入（特别是来自小型 GTP酶）并产生多种输出，包括微管的稳定和微管的捕获 形接头.我们将确定IQGAP在MLV感染中的作用，以及在生命中的确切时间和步骤 他们行动的周期。使用荧光标记病毒粒子的活细胞成像，我们将检查 不结合IQGAP的MLV突变体，以及被IQGAP的显性阴性片段阻断的野生型病毒， IQGAP，以确定病毒体是否不能正确地传递到微管。我们将测试 IQGAP磷酸化的重要性，被认为是由PKCe介导的，在正常的病毒运输。 最后，我们将测试动力蛋白马达和动力蛋白的关键亚基在MLV运动中的作用 PIC沿着稳定和动态微管。在我们的细胞生物学家同事的帮助下， 计划，我们希望填补我们目前对MLV生命周期的理解中的一个主要空白。 相关性（参见说明）： 逆转录病毒是严重的人类疾病，包括白血病和艾滋病， 作为基因治疗工具的巨大希望。最近的研究表明，这些病毒严重依赖于 在细胞骨架和微管（MT）上进行细胞内运输。在本提案中，我们的目标是定义 特定的MT调节子和马达在MLV感染的早期阶段中的作用。更深入地了解 这些过程将为逆转录病毒复制提供新的见解，可能定义新的抗病毒靶点， 并增加我们对利用多边贸易系统贩运货物的了解。