Host proteins in Vaccinia viral membrane biogenesis

描述(由申请人提供)：人们对了解牛痘病毒的复制很感兴趣，因为它代表了理解包括天花病原体天花病毒在内的天花病毒的模型系统。病毒复制的一个关键阶段涉及获得膜，因为这一事件将非传染性组装的病毒核心转变为传染性病毒粒子。积累的证据表明，该病毒从宿主的细胞器中获得了两套膜。内膜来自内质网高尔基体中间隔室(ERGIC)，形成细胞内成熟病毒(IMV)，外膜随后从反式高尔基体网络获得，形成细胞内包膜病毒。参与膜包裹的病毒蛋白已开始被识别，但宿主蛋白仍不清楚，因为膜来自宿主细胞器，因此预计在这一过程中也将发挥关键作用。在初步研究中，我们发现痘苗病毒特异性地将宿主蛋白--COPI外壳复合体的一个亚单位--浓缩在其内膜上。因此，我们建议阐明COPI外壳复合体如何在病毒生物发生中发挥作用。首先，我们将确定首次在病毒膜上检测到COPI时病毒生物发生的阶段。其次，我们将确定当COPI功能被取消时，病毒的哪个阶段可能被阻止。第三，我们将确定病毒是否篡夺任何已知的宿主调节蛋白来将COPI招募到病毒膜上。第四，我们将测试目前已知的任何调节病毒形态发生的病毒蛋白是否会影响COPI在病毒膜上的募集。这些连续的方法还提出了一种系统的方法来识别潜在的其他宿主蛋白，这些蛋白将被预测为影响IMV形成，因为这是病毒感染的第一阶段。因此，我们将筛选目前已知的在ERGIC隔间起作用的转运调节因子，以测试是否有类似的富含在病毒膜上的转运调节因子。随后，候选宿主蛋白的功能将被取消，以确定病毒的生物发生是否受到影响。这些努力不仅可能更好地了解病毒在复制过程中如何与宿主相互作用，而且还将在未来合理设计针对痘病毒的治疗干预措施中提出新的生化靶点。

DESCRIPTION (provided by applicant): Much interest exists in understanding the replication of vaccinia virus, as it represents the model system to understand poxviruses that include variola virus, the causative agent of smallpox. A key phase of viral replication involves the acquisition of membranes, as this event transforms non-infectious assembling viral cores into infectious virions. Accumulated evidence suggests that the virus acquires two set of membranes from organellar compartments of the host. The inner set of membranes is derived from the Endoplasmic Reticulum Golgi Intermediate Compartment (ERGIC) to form an intracellular mature virus (IMV), while the outer set of membranes is subsequently acquired from the trans-Golgi network to form an intracellular envelope virus. Viral proteins that participate in membrane wrapping are beginning to be identified, but host proteins that would also be predicted to play a key role during this process, as the membranes are derived from host organelles, have remained unknown. In preliminary studies, we have found that vaccinia virus specifically concentrates a host protein, a subunit of the COPI coat complex, on its inner set of membranes. Thus, we propose to elucidate how the COPI coat complex might play a role in viral biogenesis. First, we will determine the stage of viral biogenesis when COPI is first detected on viral membranes. Second, we will determine which viral stage might be arrested when COPI function is abrogated. Third, we will determine whether the virus usurps any of the known host regulatory proteins to recruit COPI onto viral membranes. Fourth, we will test whether any of the current known viral proteins that regulate viral morphogenesis affects COPI recruitment onto viral membranes. These sequential approaches also suggest a systematic way of identifying potential other host proteins that would be predicted to affect IMV formation, as it is the first stage that the virus becomes infectious. Thus, we will screen the currently known transport regulators that function at the ERGIC compartment to test whether any is similarly enriched on viral membranes. Subsequently, the function of candidate host proteins will be abrogated to determine whether viral biogenesis is affected. These efforts will likely not only provide a better understanding of how the virus interacts with its host during replication, but also suggest novel biochemical targets in the future rational design of therapeutic intervention against poxviruses.