Under Pressure: Biophysical Mapping of Herpesvirus Capsid Assembly and Genome Packaging

项目摘要 疱疹病毒是双链DNA病毒，感染大多数人群。这些复杂 病毒建立终身的、休眠的感染，在某些条件下周期性地重新激活。重新激活 尤其对免疫功能低下的人有害，导致多种疾病状态，包括失明， 脑炎，癌症和死亡，但没有治愈。人类疱疹病毒有九种类型， 分为三个亚家族，但疫苗只能针对一种类型。此外，可用的抗病毒药物是 由于病毒突变而不理想。缺乏泛疱疹病毒治疗可能源于 病毒复制之间的亚家族，但某些方面，如需要适当组装衣壳 含有遗传物质的基因是保守的。因此，本研究的长期目标是制定一个 关于疱疹病毒衣壳如何组装和包装DNA的详细机制，这两者对于 所有的疱疹病毒复制。尽管多年来在理解这些方面取得了很大的进步， 虽然我们已经研究了这些过程，但我们仍然不知道这些动态和瞬态过程在分子水平上是如何发生的。 因此，本工作的科学前提是发展生物物理方法学来监测衣壳 实时组装和基因组包装。这项工作的建议利用现有的体外衣壳 我们将结合光散射和质谱分析的新技术使用组装平台 来了解单个衣壳蛋白是如何聚集在一起形成衣壳的。这不仅将提供 缺少关于这一重要过程的信息，但它也将为其他方面创造新的方法。 研究大型病毒的科学家此外，这项提案的工作将创造一种新的体外疱疹病毒， 衣壳包装分析，这在该领域还没有完成。我们将对该试验进行各种 单分子方法来了解蛋白质如何参与基因组包装，一些未知或 不完全定义的角色，协调这一过程，以实现成功的认证。所有这些 创新的研究不仅将提供有关这些基本过程的基本知识， 挑战现有的范式，从而更全面地了解疱疹病毒的复制， 用于预防和治疗方法。

Project Abstract Herpesviruses are double-stranded-DNA viruses that infect most of the human population. These complex viruses establish lifelong, dormant infections, periodically reactivating under certain conditions. Reactivation is particularly detrimental to the immunocompromised, resulting in a variety of disease states, including blindness, encephalitis, cancers, and death, yet there is no cure. There are nine types of human herpesviruses, classified into three subfamilies, yet a vaccine is only available targeting one type. Furthermore, available antivirals are suboptimal due to viral mutation. The lack of pan-herpesvirus therapeutics likely stems from the variations in viral replication between subfamilies, yet certain aspects, such as the need for properly assembled capsids containing genetic content, are conserved. Therefore, the long-term goal of this research is to formulate a detailed mechanism as to how herpesviral capsids assemble and package DNA, both of which are essential for all herpesviruses to replicate. Although great strides have been made over the years to understand these processes, we still do not know how these dynamic and transient processes occur at the molecular level. Therefore, the scientific premise of this work is to develop biophysical methodologies to monitor capsid assembly and genome packaging in real-time. The work in this proposal capitalizes on an existing in-vitro capsid assembly platform that we will use in conjunction with new technologies in light scattering and mass spectrometry to understand how individual capsid proteins come together to form the capsid shell. Not only will this provide missing information regarding this essential process but it will also create new methodologies for other researchers studying large viruses. Additionally, work in this proposal will create a novel in-vitro herpesviral capsid packaging assay, something that has yet to be done in the field. We will subject this assay to various single-molecular approaches to understand how proteins involved in genome packaging, some with unknown or incompletely defined roles, coordinate this process to achieve successful encapsidation. Together, these innovative studies will not only provide fundamental knowledge regarding these essential processes but also challenge existing paradigms, resulting in a more complete understanding of herpesviral replication that can be exploited for preventative and therapeutic approaches.