Neurotropic herpesvirus envelopment and microtubule-mediated transport

嗜神经性疱疹病毒包膜和微管介导的运输

• 批准号: 10585954
• 负责人: Gregory Allan Smith
• 金额: $ 67.07万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-15 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10585954
• 关键词: Address; Afferent Neurons; Alphaherpesvirinae; Axon; Axonal Transport; Bacterial Artificial Chromosomes; Binding; Biochemistry; Blindness; Capsid; Cell Line; Cell Nucleus; Cells; Cellular biology; Complex; Congenital Abnormality; Cytoplasmic Organelle; DNA biosynthesis; Development; Disease; Episome; Epithelium; Event; Family suidae; Fluorescence Microscopy; Gene Expression; Goals; Herpes Labialis; Herpesviridae; Herpesvirus 1; Human; Human Herpesvirus 2; Image; In Vitro; Induced pluripotent stem cell derived neurons; Infection; Kinesin; Laboratories; Mediating; Membrane Proteins; Microtubules; Molecular; Motor; Mucous Membrane; Nerve; Nervous System; Neurons; Nose; Oral; Organelles; Oropharyngeal; Pathogenesis; Pathway interactions; Periodicals; Principal Investigator; Process; Proteins; Role; Simplexvirus; Structure; Structure of trigeminal ganglion; Surface; Testing; Tissues; Travel; Viral; Viral Genes; Viral Genome; Virion; Virus; Virus Replication; anterograde transport; ds-DNA; experience; human disease; in vivo; induced pluripotent stem cell; innovation; interest; mad itch virus; neuronal cell body; neuronal transport; neurotropic; novel; novel therapeutics; particle; pathogen; protein complex; reactivation from latency; recruit; trafficking; transmission process

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.

α疱疹病毒亚科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）和猪病毒伪狂犬病病毒（PRV）。初始感染通常发生在粘膜上皮，如HSV的口腔和肛门生殖器粘膜，PRV的鼻和口咽粘膜。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细胞体。病毒基因组最终被递送到细胞核，并在人类（HSV-1）和猪（PRV）的三叉神经节中随后的潜伏期期间作为环状dsDNA附加体持续存在。潜伏期的周期性再激活之后是病毒基因表达、DNA复制和新衣壳的组装。这些与病毒基因组一起包装，从细胞核中出现并芽入细胞质细胞器，在细胞器腔中产生有包膜的感染性病毒颗粒。这些转运和转运事件如何以及在何处发生在α疱疹病毒感染的神经元中尚不清楚。成熟的α疱疹病毒颗粒从神经元细胞体通过微管定向的顺行运输使用驱动蛋白马达运输到轴突中并沿着轴突。感染性成熟病毒颗粒积聚在神经末梢，然后被释放以感染邻近的粘膜上皮，导致随后的病毒复制和传播。在每个阶段使用的驱动蛋白马达的身份，用于招募驱动蛋白运输病毒粒子的机器，甚至是沿着轴突运输的病毒颗粒的结构，无论是无包膜衣壳还是在神经元细胞体中获得包膜的衣壳，都是我们在此应用中解决的关键问题。在具体目标1中，我们研究了gE/gI-US 9 p膜蛋白复合物在驱动蛋白马达KIF 1A和KIF 5向HSV-1和PRV募集中的功能。我们还测试了一个创新的假设，关于大被膜蛋白UL 36 p在组装KIF 5到运输病毒粒子上的作用。在《特定目标2》中，我们使用一种新型的“包膜陷阱”来解决有争议的问题：HSV-1在哪里获得其包膜，无论是在一系列神经元细胞系、人类iPSC中的细胞体还是神经末梢（分别为“已婚”和“分离”机制）。衍生的三叉神经节和外植的感觉神经元。我们还测试了关于HSV-1和PRV通过已婚和分离途径外出期间驱动蛋白募集机制的关键问题。因此，该提案的重点是潜伏期重新激活后α疱疹病毒从神经系统传播到粘膜表面的三个主要事件：衣壳包裹、微管定向运输和顺行轴突运输。具体目标利用两个主要研究者的互补的体外和体内专业知识，以及我们对gE/gI-US 9 p复合物和UL 36 p的共同兴趣和经验，以剖析支持这些病毒引起的复发性疾病的分子机制。