Elucidating the Role of the FACT Complex in the HSV-1 Life Cycle

阐明 FACT 复合物在 HSV-1 生命周期中的作用

• 批准号: 9188720
• 负责人: Hannah Fox
• 金额: $ 4.36万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9188720
• 关键词: Address; Affect; Binding; Biological Assay; Cell Nucleolus; Cell Nucleus; Cells; Chromatin; Complex; DNA; DNA Damage; DNA biosynthesis; Data; Development; Gene Expression; Genetic Recombination; Genetic Transcription; Genital system; Genome; Herpesvirus 1; Histones; Immediate-Early Proteins; Immunocompromised Host; Immunofluorescence Immunologic; Immunoprecipitation; Individual; Infant; Infection; Integration Host Factors; Laboratories; Lead; Life Cycle Stages; Light; Location; Lytic; Lytic Phase; Mass Spectrum Analysis; Methods; Molecular Chaperones; Mucous Membrane; Outcome; Population; Process; Proteins; Recruitment Activity; Risk; Role; Simplexvirus; Testing; Viral; Viral Genes; Viral Genome; Viral Proteins; Virus; Virus Replication; chromatin remodeling; knock-down; mutant; novel therapeutics; oral infection; repaired; research study; response; stable cell line; transcription factor; transcriptome sequencing; viral DNA

PROJECT SUMMARY Herpes Simplex Virus 1 (HSV-1) infection affects upwards of 50 percent of the population and poses an especial risk to infants and immunocompromised individuals. Identifying targets and understanding their roles during lytic infection could lead to the development of new therapeutics. Our laboratory used iPOND (Isolation of Proteins on Nascent DNA) to isolate the HSV-1 genome and associated viral and host cell proteins at different points in the HSV-1 lytic life cycle. The FACT (Facilitates Chromatin Transcription) complex was found to be associated with the HSV-1 genome both before and during viral DNA replication. The FACT complex is a heterodimer, consisting of the proteins Spt16 and SSRP1, and is involved in chromatin remodeling during transcription, DNA replication, recombination, and the DNA damage response. Preliminary data are provided showing that SSRP1 colocalized with the viral genome during viral DNA replication and that before viral DNA replication SSRP1 relocalized from the nucleolus into distinct puncta. Subsequent experiments showed that early relocalization required expression of the viral immediate early protein, ICP22, which was also found on infecting viral genomes by iPOND. We hypothesize that ICP22 alters the FACT complex's intranuclear location prior to viral DNA replication by direct interaction or through affecting other cellular proteins, and that ICP22 is involved in the association of the FACT complex with the HSV-1 genome during replication either directly or through affecting other cellular or viral proteins. We further hypothesize that the presence of the FACT complex decreases histone occupancy on the viral genome and supports processes that occur on the viral genome such as transcription. Three aims are proposed to explore how the FACT complex is recruited to HSV-1 DNA and how it might be involved in viral gene expression and replication. In Aim 1 we will define the mechanism by which SSRP1 relocalizes in the host cell nucleus prior to viral DNA replication. This includes determining if the association of the FACT complex with HSV-1 DNA prior to viral DNA replication is dependent on ICP22 and which viral and cellular proteins interact with the FACT complex prior to viral DNA replication. In Aim 2 we will explore the association of the FACT complex with the HSV- 1 genome during viral DNA replication and the role of ICP22. Immunoprecipiation experiments will allow us to determine which viral and cellular proteins interact with the FACT complex during viral DNA replication. In Aim 3 we will determine the function of the FACT complex during HSV-1 infection by observing the effects of a knockdown of Spt16 and SSRP1 on viral gene expression and replication. This will be correlated with changes in histone and transcription factor occupancy. This study will shed light on how this complex (FACT), which affects most DNA transactions, contributes to the life cycle of HSV-1.

项目概要 单纯疱疹病毒 1 (HSV-1) 感染影响了 50% 以上的人口，并且 对婴儿和免疫功能低下的个体构成特别的风险。确定目标和 了解它们在溶解性感染过程中的作用可能会导致新疗法的开发。我们的 实验室使用iPOND（新生DNA蛋白质分离）来分离HSV-1基因组并 HSV-1 裂解生命周期中不同点的相关病毒和宿主细胞蛋白。事实 （促进染色质转录）复合物被发现与 HSV-1 基因组相关 在病毒DNA复制之前和期间。 FACT 复合物是异二聚体，由蛋白质组成 Spt16 和 SSRP1，参与转录、DNA 复制过程中的染色质重塑， 重组和DNA损伤反应。提供的初步数据表明 SSRP1 在病毒 DNA 复制期间和病毒 DNA 复制之前与病毒基因组共定位 SSRP1 从核仁重新定位到不同的斑点中。随后的实验表明，早期 重新定位需要病毒立即早期蛋白 ICP22 的表达，该蛋白也在 通过 iPOND 感染病毒基因组。 我们假设 ICP22 先于病毒 DNA 改变 FACT 复合物的核内位置 通过直接相互作用或通过影响其他细胞蛋白进行复制，并且 ICP22 参与 FACT 复合物在复制过程中直接或通过 HSV-1 基因组的关联 影响其他细胞或病毒蛋白。我们进一步假设 FACT 复合体的存在 减少病毒基因组上的组蛋白占据并支持病毒上发生的过程 基因组，例如转录。提出了三个目标来探索 FACT 复合体如何被招募来 HSV-1 DNA 及其如何参与病毒基因表达和复制。在目标 1 中，我们将定义 SSRP1 在病毒 DNA 复制之前重新定位于宿主细胞核中的机制。这 包括确定 FACT 复合物与 HSV-1 DNA 的关联是否先于病毒 DNA 复制依赖于 ICP22 以及哪些病毒和细胞蛋白与 FACT 复合物相互作用 在病毒DNA复制之前。在目标 2 中，我们将探讨 FACT 复合体与 HSV 的关联 1 病毒DNA复制过程中的基因组和ICP22的作用。免疫沉淀实验将允许 我们确定哪些病毒和细胞蛋白在病毒 DNA 过程中与 FACT 复合物相互作用 复制。在目标 3 中，我们将确定 FACT 复合物在 HSV-1 感染期间的功能： 观察 Spt16 和 SSRP1 敲低对病毒基因表达和复制的影响。这 将与组蛋白和转录因子占据的变化相关。这项研究将揭示 这种影响大多数 DNA 交易的复合物 (FACT) 如何影响 HSV-1 的生命周期。