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: 8979939
• 负责人: Adrian Raulet Wilkie
• 金额: $ 3.06万
• 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/8979939
• 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; Drug Targeting; Ensure; F-Actin; Goals; Herpesviridae; Homologous Gene; 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; Pharmaceutical Preparations; Phosphotransferases; Process; Research; Site; System; Techniques; Therapeutic Intervention; Viral; Work; base; cellular imaging; clinically relevant; depolymerization; drug development; interest; 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（由申请人提供）： 人类巨细胞病毒 (HCMV) 衣壳离开细胞核（核出口）的过程尚不完全清楚。病毒 DNA 合成、衣壳组装和包装（衣壳化）被认为发生在称为亚核结构中。复制区室 (RC)，之后衣壳必须到达内核膜才能释放到细胞质中。尽管其他疱疹病毒已提出基于肌动球蛋白的转运机制，但 HCMV 核出口复合物 (NEC) 由内核膜结合的 UL50 及其可溶性结合伴侣 UL53 组成，两者对于核出口都是必需的。虽然某些 NEC 功能需要在内核膜上形成复杂的结构，但有证据表明 UL53 及其同系物参与上游过程，我们的初步数据支持 UL53 链接的模型。虽然我们的研究取得了进展，但我们尚未明确表明 UL53、核 F-肌动蛋白和肌球蛋白 Va 是否参与活性。衣壳运输本身，或者 UL53 是否直接将衣壳连接到肌球蛋白 Va 来介导这一过程，我们还对衣壳运输启动是否与上游过程协调感兴趣。 RC，我们有希望进一步探索的支持数据，在该提案的目标 1 中，我们将 (1) 使用活细胞成像和粒子跟踪分析评估 HCMV 衣壳是否主动运输到核外围，然后使用它。系统询问 UL53 的缺失、药物治疗核 F-肌动蛋白的解聚以及显性失活突变体对肌球蛋白 Va 的抑制是否同样阻碍衣壳运输，以及 (2) 使用生化方法评估 UL53 是否将衣壳与肌球蛋白 Va 物理连接以指导这一过程。在目标 2 中，我们将 (1) 使用冷冻替代连续切片的 TEM 和免疫电镜探索衣壳运输是否与 RC 中特定位点的衣壳化在空间上协调。进一步应用这些技术来检查 UL53 和肌球蛋白 Va 是否与充满 DNA 的“C”衣壳结合以启动它们从这些位置的运输，这些结果可能说明： UL53 在核排出过程中通过引导衣壳从 RC 运动到核外围连续发挥作用，这种机制可能在疱疹病毒中保守，因为 HCMV 是一种临床相关病原体，可导致新生儿出生缺陷和免疫功能低下个体的危及生命的并发症。迫切需要额外的治疗干预措施，更好地了解核排出不仅可以为 HCMV 药物开发提供信息，还可以揭示宿主细胞生物学的新特征。