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

描述（由申请方提供）：疱疹病毒的被膜区位于衣壳外表面和包膜内表面之间，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和某些动态的相互作用可能会发生在组装的病毒粒子内的被膜蛋白。我们研究计划的长期目标是确定与单纯疱疹病毒1型（HSV-1）被膜组装相关的分子事件。本申请的具体目的是定义与命名为UL 46（VP 11/12）的HSV-1丰富的被膜蛋白的组装相关的动态事件。我们最近有证据表明，UL 46经历了一个切割事件，导致全长UL 46和氨基末端截短的UL 46，命名为t-UL 46，与组装的病毒粒子。我们还表明，氨基末端截短突变体的UL 46交通到细胞核，可能是由于一个保守的核定位信号在羧基端的蛋白质的揭露。我们的工作假设是，在病毒体组装期间病毒体相关的UL 46的切割使得从进入的病毒释放的t-UL 46能够运输到细胞核以调节VP 16在即刻早期基因转录中的活性。相比之下，新合成的UL 46不会被切割，而是与膜结合留在细胞质中，并在病毒包裹TGN期间被掺入病毒中。如果我们的假设被证明是正确的，这将代表第一个例子之一，在装配过程中的特定被膜蛋白的动态加工如何有助于其功能的作用，在病毒感染的细胞。拟议的研究将包括两个具体目标。第一个目标将集中在裂解的UL 46（t-UL 46）的表征上。我们的第一个目标是使用生物化学和遗传学方法来确定切割位点。然后，我们将制备含有编码t-UL 46的基因以及其中UL 46的切割位点已被去除的基因的重组病毒和表达质粒（？t-UL46）。我们的目标是使用这些病毒和表达质粒，以确定这些突变对t-UL 46和？tUL-46以及它们被包装在病毒体内的能力。其他研究将包括确定在组装过程中何时实际发生UL 46的裂解。第二个目标将集中于确定与UL 46的切割相关的功能意义。我们将确定从传入病毒释放的切割的t-UL 46是否运输到细胞核以增强立即早期启动子的转录活性。本申请中提出的研究可能提供一种新的模型，用于定义与其他人类和动物疱疹病毒被膜内可能发生的动态事件相关的分子机制。公共卫生相关性：疱疹病毒的被膜区域位于衣壳和包膜之间，含有大约20种蛋白质;然而，有新的证据表明，在病毒体中，被膜不是静态结构。本申请中提出的使用1型单纯疱疹病毒的实验代表了定义病毒颗粒成熟期间外皮蛋白之间发生的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理解可能会提供关于破坏这种人类病毒组装的新靶点的见解。

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.