The processes mediating capsid transport during HCMV nuclear egress

HCMV 核排出过程中介导衣壳运输的过程

• 批准号: 9068655
• 负责人: Adrian Raulet Wilkie
• 金额: $ 3.11万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9068655
• 关键词: Accounting; Acquired Immunodeficiency Syndrome; Actins; Active Biological Transport; Actomyosin; Address; Adverse effects; Antiviral Agents; Binding; Biochemical; Biology; Capsid; Capsid Proteins; Cell Nucleus; Cells; Cellular biology; Complex; Congenital Abnormality; Coupled; Cytomegalovirus; Cytomegalovirus Infections; Cytoplasm; DNA; DNA Packaging; DNA biosynthesis; Data; Defect; Disease; Dominant-Negative Mutation; Ensure; F-Actin; Goals; Herpesviridae; Homologous Gene; Human; Immunocompromised Host; Individual; Intervention; Laboratories; Lamins; Lead; Life; Link; Location; MYO5A gene; Mediating; Microfilaments; Modeling; Motor; Movement; Myosin ATPase; Newborn Infant; Nuclear; Nuclear Inner Membrane; Nuclear Structure; Patients; Pharmacotherapy; Phosphotransferases; Process; Research; Site; System; Techniques; Therapeutic Intervention; Viral; Virus Replication; Work; base; clinically relevant; depolymerization; drug development; interest; live cell imaging; new therapeutic target; novel; particle; pathogen; public health relevance; research study; spatiotemporal; trafficking; viral DNA

 DESCRIPTION (provided by applicant): The process by which human cytomegalovirus (HCMV) capsids exit the nucleus (nuclear egress) is incompletely understood. Viral DNA synthesis, capsid assembly, and packaging (encapsidation) are thought to occur in sub-nuclear structures called replication compartments (RCs), after which capsids must reach the inner nuclear membrane for their release into the cytoplasm. How capsids move to the nuclear periphery is unresolved, although an actomyosin-based transport mechanism has been suggested for other herpesviruses. The HCMV nuclear egress complex (NEC) is comprised of inner nuclear membrane-bound UL50, and its soluble binding partner, UL53, both of which are essential for nuclear egress. While certain NEC functions require complex formation at the inner nuclear membrane, there is evidence that UL53 and its homologs participate in upstream processes. Our preliminary data support a model whereby UL53 links capsids to myosin Va in RCs to facilitate nuclear F-actin dependent capsid transport to the nuclear periphery. While we have made progress in our studies, we have yet to definitively show whether UL53, nuclear F-actin, and myosin Va are involved in active capsid trafficking per se, or whether UL53 directly links capsids to myosin Va to mediate this process. We are also interested in whether capsid transport initiation is coordinated with upstream processes in RCs, for which we have supporting data that we wish to explore further. In Aim 1 of this proposal we will (1) assess whether HCMV capsids undergo active transport to the nuclear periphery using live cell imaging and particle tracking analysis, and then use this system to interrogate whether deletion of UL53, depolymerization of nuclear F-actin with drug-treatment, and inhibition of myosin Va with dominant negative mutants similarly hinder capsid trafficking, and (2) use biochemical approaches to assess whether UL53 physically links capsids to myosin Va to direct this process. In Aim 2, we will (1) explore whether capsid transport is spatially coordinated with encapsidation at specific sites in RCs using TEM of cryosubstituted serial sections and immunoEM, and (2) further apply these techniques to examine whether UL53 and myosin Va engage DNA-filled "C" capsids to initiate their transport from these locations. Together, these results may illustrate that UL53 acts in a continuum during nuclear egress by directing capsid movement from RCs to the nuclear periphery, a mechanism that may be conserved among herpesviruses. Since HCMV is a clinically relevant pathogen that causes birth defects in newborns and life-threatening complications in immunocompromised individuals, there is a considerable need for additional therapeutic interventions. A better understanding of nuclear egress could not only inform HCMV drug development, but may also uncover novel features of host cell biology.

 描述（由应用提供）：未完全了解人类巨细胞病毒（HCMV）capsID的过程尚不完全理解。病毒DNA合成，衣壳组件和包装（封装）被认为发生在称为复制区室（RCS）的亚核结构中，然后衣壳必须到达内部核膜才能释放到细胞质中。尽管已经提出了针对其他疱疹病毒的基于肌动球蛋白的运输机制，但带囊膜如何转移到核周围。 HCMV核出口复合物（NEC）由内部核膜结合的UL50及其固体结合伙伴UL53组成，它们对于核出口至关重要。尽管某些NEC功能需要在内部核膜上进行复杂的形成，但有证据表明UL53及其同源物参与上游过程。我们的初步数据支持了一个模型，该模型将UL53连接到RC中的Capsids与肌球蛋白VA，以促进核F-肌动蛋白依赖的衣壳转运到核周围。尽管我们在研究方面取得了进展，但我们尚未明确地说明UL53，核F-肌动蛋白和肌球蛋白VA是否参与了活跃的衣壳运输本身，或者UL53是否将capsid与肌球蛋白VA直接连接以介导这一过程。我们还对本提案的目标1感兴趣，我们将（1）评估HCMV Capsids使用活细胞成像和粒子跟踪分析进行主动运输到核周围的运输，然后使用该系统来询问UL53的删除，核F- actin的沉积是否是否与药物验证和肌球蛋白va with Myosin va and Myosin va and Myosin and-hinderant Mytant capsing and toprant capsing and toprant capsing and hinderant capsing caps caps caps capsing capsing capsing（hinder hinder hinders capsing capsing capsing（生化方法用于评估UL53是否将衣壳与肌球蛋白VA物理联系起来以指导这一过程。在AIM 2中，我们将（1）探索Capsid Transping是否使用CryoSubsubsutted的串行区域和免疫系统的TEM在RCS中的特定地点进行空间协调，并且（2）进一步应用这些技术来检查UL53和肌球蛋白VA是否使DNA填充DNA填充的“ C” Capsids从这些位置开始运输这些位置。总之，这些结果可以说明UL53通过指导从RC到核外围的行动运动在核出口期间继续起作用，该机制可能在疱疹病毒中保守。由于HCMV是一种临床相关的病原体，会导致新生儿的先天缺陷和免疫功能低下的个体威胁生命并发症，因此需要考虑需要进行额外的治疗干预措施。更好地了解核出口不仅可以为HCMV药物开发提供信息，而且还可能揭示了宿主细胞生物学的新特征。