Role of IQGAP and Microtubule Motors in MLV Infection

莫洛尼小鼠白血病病毒(Moloney小鼠白血病病毒，MLV)是一种典型的伽玛逆转录病毒，可在体内复制并获得高滴度 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，并诱导T细胞白血病 通过宿主原癌基因的插入激活而发生。我们对逆转录病毒的了解很多 复制首先是通过研究简单的病毒，如MLV。在…的早期阶段 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副本 基因组通过在细胞质中反转录，指导由此产生的预整合的运动 复合体(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 晚期这种DNA被表达以形成病毒RNA和蛋白质，后代病毒粒子组装在 并释放质膜，开始新的侵染循环。在这个项目中，我们建议检查 进入感染后的早期事件，是病毒生命周期中最贫穷的特征部分，重点是 在关键宿主蛋白、IQGAP、微管和动力蛋白马达上。我们之前已经将IQGAPI标识为 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是大型细胞骨架支架 参与调节细胞运动和形态的蛋白质，在结合和 同时调节肌动蛋白和微管网络。它们集成了多个输入(特别是从小的 GTP酶)并产生多种产出，包括稳定微管和捕获微管 结束了。我们将确定IQGAP在MLV感染中的作用，以及生命中确切的时间和步骤 他们采取行动的周期。利用荧光标记的病毒粒子的活细胞成像，我们将检查 不结合IQGAP的MLV突变体和被显性负性片段阻断的野生型病毒的 IQGAP，以确定病毒粒子是否未能正确地输送到微管。我们将测试 IQGAP的磷酸化在正常的病毒传播中的重要性，被认为是由PKCE介导的。 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 图片沿着稳定的和动态的微管。在我们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(请参阅说明)： 逆转录病毒是人类严重疾病的病原体，包括白血病和艾滋病，反之亦然 作为基因治疗的工具，前景看好。最近的研究表明，这些病毒严重依赖于 对细胞骨架和微管(MTS)细胞内运输的影响。在这项提案中，我们的目标是定义 特定的MT调节器和马达在MLV感染的早期步骤中的作用。更深入地了解 这些过程将提供对逆转录病毒复制的新见解，潜在地定义新的抗病毒靶点 并增加我们对MTS上贩运货物的了解。

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.