Picornavirus Genome Replication

描述（由申请人提供）：这是一份研究小核糖核酸病毒基因组复制的资助续期申请。所有的正链RNA病毒都会劫持和/或改造宿主细胞膜，以产生一个细胞器，作为基因组复制的位点。脊髓灰质炎病毒作为一种重要的模型系统，用于阐明病毒因子、宿主因子和复制细胞器生物发生所需的相应相互作用。从历史上看，我们的实验室一直对基因组复制过程和阐明P3编码蛋白在此过程中的作用感兴趣。在之前的资助期间，我们意外地观察到PV蛋白酶和RNA结合蛋白3CD有助于复制细胞器的形成，并且该细胞器需要有效地将复制的基因组从合成位点转移到衣壳中。有趣的是，3CD在复制细胞器形成中的作用是浓度依赖性的，并且可以反式互补，这表明3CD与宿主因子的相互作用。最近，Altan-Bonnet实验室表明，磷脂酰肌醇-4-磷酸（PI 4P）在小核糖核酸病毒感染期间大量增加，并定位于复制细胞器。脂质如何有助于复制细胞器的形成和/或功能仍有待阐明。然而，我们现在已经表明，异位表达的3CD是足以改变定位，甚至可能增加细胞中的PI 4P脂质的丰度。此外，我们的生物化学，生物物理和计算实验支持的结论，PV 3CD是一个磷酸肌醇结合蛋白。这些观察结果，置于Belov和Ehrenfeld的开创性工作的背景下，显示PV 3CD与膜相互作用，足以激活Arf 1，Arf 1是一种参与磷酸肌醇代谢和膜上其他交易的小G蛋白，导致驱动本申请目的的中心假设。我们建议，3CD结合到膜是受控制的，至少部分地，通过这种蛋白质与PI 4P和其他磷酸肌醇的相互作用。因此，被劫持用于掺入的膜可能以依赖于3CD-磷酸肌醇相互作用的强度和3CD浓度的方式作为感染后时间的函数而改变。不同的磷酸肌醇会产生不同的效应物，这些效应物可能会改变复制细胞器的组成、形式和/或功能。功能上的时间性改变包括从复制到降解的转变。Arf 1和/或其同源物在不同膜上的激活可能有助于改变在感染期间在复制细胞器上表达的磷酸肌醇池，对依赖于这些脂质的“正常”水平和定位的途径具有潜在的负面后果。在下一个资助期内，我们将通过追求以下特定目标来测试该模型：（1）阐明3CD诱导PI 4P的机制和功能;（2）阐明磷酸肌醇与3CD结合的机制和功能;（3）阐明PV感染期间诱导其他磷酸肌醇的机制和功能。

DESCRIPTION (provided by applicant): This is an application for renewal of a grant to study picornavirus genome replication. All positive-strand RNA viruses hijack and/or remodel host membranes to create an organelle that serves as the site of genome replication. Poliovirus has served as an important model system for elucidation of viral factors, host factors and corresponding interactions required for biogenesis of the replication organelle. Historically, our laboratory has been interested in the process of genome replication and elucidation of the roles of P3-encoded proteins in this process. During the previous funding period, we made the unexpected observation that the PV protease and RNA-binding protein, 3CD, contributes to formation of the replication organelle and is needed for this organelle to efficiently transfer replicated genomes from the site of synthesis into capsids. Interestingly, the role of 3CD in formation of the replication organelle is concentration dependent and can be complemented in trans, suggesting an interaction of 3CD with a host factor. Recently, the Altan-Bonnet lab showed that phosphatidylinositol-4-phosphate (PI4P) increases in abundance during picornavirus infection and localizes to the replication organelle. How a lipid contributes to formation and/or function of the replication organelle remains to be elucidated. However, we have now shown that ectopic expression of 3CD is sufficient to alter the localization and perhaps even increase the abundance of PI4P lipids in cells. In addition, our biochemical, biophysical and computational experiments support the conclusion that PV 3CD is a phosphoinositide-binding protein. These observations, placed into the context of the pioneering work of Belov and Ehrenfeld showing that PV 3CD interacts with membranes and is sufficient for activation of Arf1, a small G protein involved in phosphoinositide metabolism and other transactions of/on membranes, leads to the central hypothesis driving the aims of this application. We propose that 3CD binding to membranes is governed, at least in part, by the interaction of this protein with PI4P and perhaps other phosphoinositides. The membranes hijacked for incorporation may therefore change as a function of time post-infection in a manner dependent on both the strength of the 3CD-phosphoinositide interaction and concentration of 3CD. With different phosphoinositides come different effectors that may change the composition, form and/or function of the replication organelle. Temporal alterations in function would include a transition from replication to encapsidation. Activation of Arf1 and/or its homologues on the different membranes may contribute to changes to the phoshphoinositide pools expressed on the replication organelle during infection, with the potential for negative consequences on pathways that rely on "normal" levels and localization of the these lipids. During the next funding period, we will test this model by pursuing the following specific aims: (1) Elucidation of the mechanism and function for induction of PI4P by 3CD; (2) Elucidation of the mechanism and function for phosphoinositide binding to 3CD; (3) Elucidation of the mechanism and function for induction of other phosphoinositides during PV infection.