Neurotropic herpesvirus envelopment and microtubule-mediated transport

甲型疱疹病毒包括神经系统的病原体，如单纯疱疹病毒1型和2型(HSV-1和HSV-2)以及猪病毒伪狂犬病病毒(PRV)。最初的感染通常发生在粘膜上皮细胞，如口腔和肛门粘膜对于HSV，鼻腔和口咽粘膜对于PRV。在这些组织中复制后，后代病毒颗粒被释放并感染邻近感觉神经元的末端。然后，它们通过微管引导的逆行交通，沿着轴突到达神经元胞体。病毒基因组最终被送入细胞核，并在随后的人(HSV-1)和猪(PRV)三叉神经节潜伏期间以环状dsDNA Episome的形式持续存在。潜伏期的周期性重新激活之后是病毒基因的表达、DNA复制和新衣壳的组装。这些病毒与病毒基因组一起包装，从核和芽中冒出，进入细胞质细胞器，在细胞器管腔中产生被包裹的、有感染性的病毒颗粒。这些转运和包膜事件在甲型疱疹病毒感染的神经元中如何以及在哪里发生，目前还知之甚少。成熟的甲型疱疹病毒颗粒通过使用动蛋白马达的微管定向顺行运输从神经元细胞体进入轴突并沿着轴突运输。具有感染性的成熟病毒颗粒聚集在神经末梢，然后被释放，感染邻近的粘膜上皮，导致随后的几轮病毒复制和传播。在每个阶段使用的动蛋白马达的身份，用来招募动蛋白来运输病毒粒子的机制，甚至是沿着轴突运输的病毒颗粒的结构，无论是无包膜的衣壳还是在神经元细胞体中获得包膜的衣壳，都是我们在这一应用中解决的关键问题。在特定目的1中，我们研究了GE/GI-US9P膜蛋白复合体在运动蛋白发动机KIF1A和KIF5对HSV-1和PRV募集中的作用。我们还测试了一个创新的假设，即大被膜蛋白UL36P在KIF5组装到贩运病毒粒子上的作用。在特定目标2中，我们使用一种新的“包膜陷阱”来解决有争议的问题，即HSV-1在一系列神经元细胞系、人IPSC来源的三叉神经节和移植的感觉神经元中从哪里获得包膜，无论是在细胞体还是在神经末梢(分别是结婚机制和分离机制)。我们还测试了有关HSV-1和PRV通过结婚和分离途径外出时运动蛋白募集机制的关键问题。因此，这一建议集中在甲型疱疹病毒从潜伏期重新激活后从神经系统传播到粘膜表面的三个主要事件：衣壳被膜、微管导向的运输和顺行轴突运输。具体目的是利用两位主要研究人员在体外和体内的互补专业知识，以及我们对GE/GI-US9p复合体和UL36p的共同兴趣和经验，剖析这些病毒导致复发疾病的分子机制。

The Alphaherpesvirinae include pathogens of the nervous system such as herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and the swine virus pseudorabies virus (PRV). Initial infection is commonly at mucosal epithelia such as the oral and anogenital mucosa for HSV, and nasal and oropharyngeal mucosa for PRV. Following replication in these tissues, progeny viral particles are released and infect the termini of adjacent sensory neurons. They then travel by microtubule-directed retrograde traffic along the axon to the neuronal cell body. The viral genome is ultimately delivered to the nucleus and persists as a circular dsDNA episome during ensuing latency in the trigeminal ganglia of humans (HSV-1) and swine (PRV). Periodic reactivation from latency is followed by viral gene expression, DNA replication and assembly of new capsids. These become packaged with the viral genome, emerge from the nucleus and bud into cytoplasmic organelles to generate enveloped, infectious viral particles in the organellar lumen. How and where these transport and envelopment events occur in alphaherpesvirus-infected neurons is poorly understood. Maturing alphaherpesvirus particles are transported from the neuronal cell body into and along the axon by microtubule-directed anterograde transport using kinesin motors. Infectious mature viral particles accumulate at the nerve terminal then are released to infect adjacent mucosal epithelia, leading to subsequent rounds of viral replication and spread. The identity of the kinesin motors utilized at each stage, the machinery used to recruit kinesins to trafficking virions and even the structure of the viral particle that traffics down the axon, whether non-enveloped capsids or capsids that have acquired envelopes in the neuronal cell body, are key questions that we address in this application. In Specific Aim 1 we investigate the function of the gE/gI-US9p membrane protein complex in recruitment of the kinesin motors KIF1A and KIF5 to HSV-1 and PRV. We also test an innovative hypothesis concerning the role of the large tegument protein UL36p in assembly of KIF5 onto trafficking virions. In Specific Aim 2 we use a novel “envelopment trap” to address the controversial question of where HSV-1 acquires its envelope, whether in the cell body or nerve terminal (the Married and Separate mechanisms, respectively) in a range of neuronal cell lines, human iPSC-derived Trigeminal Ganglia and explanted sensory neurons. We also test key questions concerning the mechanisms of kinesin recruitment during egress of HSV-1 and PRV via the Married and Separate pathways. This proposal is therefore focused on the three major events that underlie alphaherpesvirus transmission from the nervous system to mucosal surfaces following reactivation from latency: capsid envelopment, microtubule-directed trafficking, and anterograde axonal transport. The specific aims exploit the complementary in vitro and in vivo expertise of the two principal investigators, and our common interests and experience with the gE/gI-US9p complex and UL36p, to dissect the molecular mechanisms supporting recrudescent disease caused by these viruses.