Modification of a Tegument Protein during the Assembly of HSV-1

描述(申请人提供)：疱疹病毒的被膜区域位于衣壳的外表面和包膜的内表面之间，由大约20种不同的蛋白质组成。最近的证据表明，被膜不是一个静态的结构，在组装的病毒粒子内，被膜蛋白之间可能发生某些动态的相互作用。我们研究计划的长期目标是确定与1型单纯疱疹病毒(HSV-1)被层组装相关的分子事件。本申请的具体目的是定义与HSV-1丰富的被膜蛋白UL46(VP11/12)的组装相关的动态事件。我们最近有证据表明，UL46经历了一次切割事件，导致UL46全长和UL46的氨基末端截断，命名为t-UL46，与组装的病毒粒子有关。我们还证明了UL46的氨基末端截断突变体运输到细胞核，可能是由于在蛋白质的羧基末端发现了保守的核定位信号。我们的工作假设是，在病毒粒子组装过程中，病毒粒子相关的UL46被切割，使从传入病毒释放的t-UL46能够调节VP16在即刻早期基因转录中的活性。相反，新合成的UL46不被切割，与膜结合留在细胞质内，并在病毒在TGN包膜期间并入病毒。如果我们的假设被证明是正确的，这将是关于特定被膜蛋白在组装过程中的动态处理如何有助于其在感染病毒的细胞中发挥作用的首批例子之一。拟议的研究将包括两个具体目标。第一个目标将集中在切割UL46(t-UL46)的特征上。我们的第一个目标是使用生化和遗传方法来鉴定卵裂位点。然后，我们将制备含有编码t-UL46基因的重组病毒和表达载体，以及其中UL46裂解位点已被去除的基因(？T-UL46)。我们的目标是使用这些病毒和表达质粒来确定这些突变对t-UL46和？TUL-46运输的影响以及它们被包装在病毒粒子中的能力。其他研究将包括确定UL46在组装过程中实际发生切割的时间。第二个目标将集中于确定与UL46切割相关的功能意义。我们将确定从传入病毒中释放的被切割的t-UL46是否运输到细胞核，以增强即刻早期启动子的转录活性。本申请中提出的研究可能为定义与其他人和动物疱疹病毒被层内可能发生的动态事件相关的分子机制提供一个新的模型。与公共卫生相关：疱疹病毒的被膜区域位于衣壳和包膜之间，包含大约20种蛋白质；然而，有新的证据表明，在病毒粒子内，被膜不是一个静态结构。在本申请中提出的使用1型单纯疱疹病毒的实验，是确定病毒颗粒成熟过程中被膜蛋白之间动态相互作用的首批研究之一。对病毒内发生的这些动态事件的了解可能会为破坏这种人类病毒的组装提供新的靶点。

DESCRIPTION (provided by applicant): The tegument region of herpesviruses is located between the outer surface of the capsid and the inner surface of the envelope and is made up of approximately 20 different proteins. Recent evidence suggests that the tegument is not a static structure and certain dynamic interactions may occur among tegument proteins within the assembled virion. The long-term objective of our research program has been to define molecular events associated with the assembly of the tegument of herpes simplex virus type 1 (HSV-1). The specific objective of this application is to define the dynamic events associated with the assembly of the HSV-1 abundant tegument protein designated UL46 (VP11/12). We have recent evidence that UL46 undergoes a cleavage event that results in both full-length UL46 and an amino-terminal truncation of UL46, designated t-UL46, being associated with assembled virions. We have also shown that amino-terminal truncated mutants of UL46 traffic to the nucleus, possibly due to the unmasking of a conserved nuclear localization signal at the carboxyl end of the protein. Our working hypothesis is that cleavage of virion-associated UL46 during the assembly of the virion, enables the t-UL46 that is released from the incoming virus to traffic to the nucleus to modulate the activity of VP16 in the transcription of the immediate-early genes. In contrast, newly synthesized UL46 is not cleaved and remains within the cytoplasm in association with membranes and is incorporated into the virus during its envelopment at the TGN. If our hypothesis proves to be correct, this will represent one of the first examples as to how the dynamic processing of a specific tegument protein during the assembly process contributes to its functional role within the virus-infected cell. The proposed studies will include two specific aims. The first aim will focus on the characterization of the cleaved UL46 (t-UL46). Our first objective is to identify the cleavage site using both biochemical and genetic approaches. We will then prepare recombinant viruses and expression plasmids that contain genes encoding t-UL46 and well as a gene in which the cleavage site of UL46 has been removed (?t-UL46). Our goal is to use these viruses and expression plasmids to determine the effect of these mutations on the trafficking of t-UL46 and ?tUL-46 as well as their ability to be packaged within virions. Other studies will include defining when the cleavage of UL46 actually occurs during the assembly process. The second aim will focus on determining the functional significance associated with the cleavage of UL46. We will determine if the cleaved t-UL46, which is released from the incoming virus, traffics to the nucleus to enhance the transcriptional activity of immediate-early promoters. The studies proposed within this application may provide a new model for defining molecular mechanisms associated with dynamic events that may occur within the tegument of other human and animal herpesviruses. PUBLIC HEALTH RELEVANCE: The tegument region of the herpesviruses is located between the capsid and envelope and contains approximately 20 proteins; however, there is new evidence that within the virion the tegument is not a static structure. The experiments proposed within this application using herpes simplex virus type 1, represent one of the first studies to define the dynamic interactions that occur among tegument proteins during the maturation of the virus particle. An understanding of these dynamic events that occur within the virus may provide insight as to new targets for disrupting the assembly of this human virus.