Using experimental evolution to probe herpesvirus adaptation to neurons, fibroblasts, & interferon signaling

利用实验进化来探究疱疹病毒对神经元、成纤维细胞的适应，

• 批准号: 10373624
• 负责人: MORIAH SZPARA
• 金额: $ 23.64万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-06 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373624
• 关键词: Animal Model; Antibodies; Antiviral Agents; Area; Cell model; Cells; Clinical; Collaborations; Complex; Consensus; DNA Viruses; Data; Disease; Encephalitis; Environment; Epithelial; Epithelial Cells; Evolution; Exhibits; Failure; Fibroblasts; Frequencies; Genes; Genetic; Genetic Drift; Genetic Variation; Genetic study; Genome; Genotype; Herpes Simplex Infections; Herpesviridae; Herpesvirus 1; Human; Image; Immune; Immunity; Immunocompetent; Individual; Infection; Infection Control; Interferon-beta; Interferons; Keratitis; Laboratories; Lesion; Life; Light; Link; Minor; Modeling; Monitor; Mucous Membrane; Mutation; Neonatal; Nerve Endings; Neurons; Outcome; Pattern; Persons; Pilot Projects; Population; Production; Public Health; Publishing; Recurrence; Resources; Route; Severity of illness; Signal Transduction; Simplexvirus; Statistical Models; Testing; Time; Vaccines; Variant; Vero Cells; Viral; Viral Genes; Viral reservoir; Virus; Virus Diseases; antagonist; antiviral drug development; cell type; comparative genomics; design; fitness; flexibility; forward genetics; genetic variant; genital herpes; genome wide screen; genomic locus; in vivo; insight; interferon antagonist; pressure; repository; response; reverse genetics; vaccine candidate; vaccine development; virus genetics

Project Summary HSV-1 causes lifelong infections in millions of people in the USA and around the world. Natural infections of HSV-1 follow a cycle of productive replication in epithelial cells, followed by transit into nerve endings, and establishment of a lifelong viral reservoir in neurons. Despite high demand and many efforts to design a HSV-1 vaccine, prior candidates have failed in human trials after showing promise in animal models. This efficacy gap reflects our incomplete understanding of how this virus responds to selective pressures in cellular and animal models – e.g. during vaccine development – and in vivo during human infections. Here we propose to use an experimental evolution approach to reveal how HSV-1 adapts to key cellular niches such as fibroblasts and neurons, and to altered levels of interferon signaling, as a key component of host immunity. These studies will utilize a unique resource of sequential cultured isolates from newly-infected individuals, to enable a new level of comparison between experimental and natural evolutionary pressures. In Aim 1, we will explore HSV-1 evolution in the distinct cellular niches of human fibroblasts and neurons. As part of an ongoing collaboration, we have access to a unique repository of sequential clinical HSV-1 isolates from immunocompetent individuals in the first year of their infection. Using the initial virus isolates from these individuals, we will carry out experimental evolution in primary human fibroblasts and human neurons, which represent the two key cellular niches of infection in vivo. We will deep sequence the viral populations of replicate lineages from experimental evolution in either fibroblasts, neurons, or alternating between the two cell types. The genetic variations that arise in each viral population over time will be analyzed using statistical models to differentiate genetic drift from directional selection. Variants observed during experimental evolution in these defined environments will then be compared to those detected in the course of natural evolution, via sequential viral cultures from the same infected individuals. The interferon response is a critical aspect of host immune control of infection. Multiple HSV-1 genes have been implicated in viral antagonism of the host interferon response, although these are traditionally studied one at a time. In Aim 2, we will use the experimental evolution approach to conduct an unbiased, genome- wide screen for genetic loci involved in interferon antagonism by HSV-1. By treating primary human fibroblasts with either exogenous interferon, or with antibodies to deplete interferon production, we will evolve HSV-1 in a heightened or depleted antiviral state, respectively. After sequencing replicate lineages of these evolved viral populations, we will use patterns of genetic drift and/or positive selection to identify viral genes associated with interferon antagonism. Together these experimental evolution data will provide new insights on how HSV-1 adapts to these distinct cellular niches, and shed new light on the complex interplay of selective pressures found in natural infections.

项目摘要 HSV-1导致美国和世界各地数百万人终身感染。自然感染 HSV-1在上皮细胞中进行生产性复制，然后进入神经末梢， 在神经元中建立终身病毒库。尽管有很高的需求和许多努力来设计一个 HSV-1疫苗，先前的候选疫苗在动物模型中显示出希望后，在人体试验中失败了。这 疗效差距反映了我们对这种病毒如何对选择性压力作出反应的不完全理解， 细胞和动物模型-例如在疫苗开发期间-以及在人体感染期间的体内。这里 我们建议使用实验性进化方法来揭示HSV-1如何适应关键的细胞小生境 例如成纤维细胞和神经元，以及干扰素信号传导水平的改变，作为宿主细胞的关键组分， 免疫力这些研究将利用一个独特的资源，连续培养分离，从新感染的 个体，使实验和自然进化压力之间的比较达到一个新的水平。 在目标1中，我们将探索HSV-1在人类成纤维细胞和神经元的不同细胞小生境中的进化。 作为正在进行的合作的一部分，我们可以访问一个独特的库，连续临床HSV-1 在感染的第一年从免疫活性个体分离。使用最初的病毒分离物 从这些个体中，我们将在原代人成纤维细胞和人成纤维细胞中进行实验进化。 神经元，其代表体内感染的两个关键细胞小生境。我们会对病毒进行深度测序 来自成纤维细胞、神经元或交替细胞中实验进化的复制谱系群体， 两种细胞类型之间的差异。随着时间的推移，每个病毒群体中出现的遗传变异将是 使用统计模型分析，以区分遗传漂变和定向选择。观察到的变体 在这些定义的环境中的实验进化过程中，然后将与在这些环境中检测到的那些进行比较。 自然进化的过程，通过连续的病毒培养，从相同的感染个体。 干扰素应答是宿主免疫控制感染的关键方面。多个HSV-1基因具有 与宿主干扰素应答的病毒拮抗作用有关，尽管这些传统上是研究 一次一个。在目标2中，我们将使用实验进化方法进行无偏见的基因组- 通过HSV-1广泛筛选干扰素拮抗作用相关的遗传位点。通过治疗初级人类 成纤维细胞与外源性干扰素，或与抗体耗尽干扰素的生产，我们将 发展HSV-1，分别处于增强或耗尽的抗病毒状态。在测序后， 这些进化的病毒群体，我们将使用遗传漂变和/或正选择的模式来识别 与干扰素拮抗作用相关的病毒基因。这些实验进化数据将提供 关于HSV-1如何适应这些不同的细胞小生境的新见解，并为复杂的 在自然感染中发现的选择性压力的相互作用。