Picornavirus Genome Replication

描述(申请人提供)：这是一项研究微小核糖核酸病毒基因组复制的拨款续期申请。所有正链RNA病毒都劫持和/或重塑宿主膜，以创建一个细胞器，作为基因组复制的地点。脊髓灰质炎病毒已成为阐明病毒因子、宿主因子以及复制细胞器生物发生所需的相应相互作用的重要模型系统。从历史上看，我们的实验室一直对基因组复制过程和P3编码蛋白在这一过程中的作用的阐明感兴趣。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，并且是该细胞器有效地将复制的基因组从合成地点转移到衣壳中所必需的。有趣的是，3CD在复制细胞器形成中的作用是浓度依赖的，并且可以在反式中补充，这表明3CD与宿主因子相互作用。最近，Altan-Bonnet实验室发现，在小核糖核酸病毒感染过程中，磷脂酰肌醇-4-磷酸(PI4P)含量增加，并定位于复制细胞器。脂类如何对复制细胞器的形成和/或功能起作用仍有待阐明。然而，我们现在已经证明，异位表达3CD足以改变细胞中PI4P脂类的定位，甚至可能增加其丰度。此外，我们的生化、生物物理和计算机实验支持PV3CD是一种磷脂酰肌醇结合蛋白的结论。这些观察放在Belov和Ehrenfeld的开创性工作的背景下，表明PV 3CD与膜相互作用，并足以激活Arf1，Arf1是一种小G蛋白，参与磷脂酰肌醇代谢和膜上的其他交易，导致了推动本应用目的的中心假说。我们认为，3CD与膜的结合至少部分是由该蛋白与PI4P以及可能其他磷脂酰肌醇的相互作用所控制的。因此，被劫持用于掺入的膜可能随着感染后的时间而变化，其方式取决于3CD-磷脂酰肌醇相互作用的强度和3CD的浓度。不同的磷脂酰肌醇会产生不同的效应器，从而改变复制细胞器的组成、形式和/或功能。功能上的时间变化将包括从复制到封装的转变。Arf1和/或其同系物在不同膜上的激活可能有助于在感染期间改变复制细胞器上表达的肌醇磷脂池，从而可能对依赖于这些脂类的“正常”水平和定位的途径产生负面影响。在下一个资助期，我们将通过以下具体目标来测试这一模型：(1)阐明3CD诱导PI4P的机制和功能；(2)阐明3CD与磷脂酰肌醇结合的机制和功能；(3)阐明在光伏感染过程中诱导其他磷脂酶的机制和功能。

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.