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，

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 HSV-1 导致美国和世界各地数百万人终身感染。 HSV-1遵循上皮细胞中的高效复制循环，然后转运到神经末梢，并且尽管设计一个病毒库的需求很高并且付出了很多努力，但在神经元中建立了终生的病毒库。 HSV-1 疫苗在动物模型中显示出希望，但在人体试验中却失败了。功效差距反映了我们对这种病毒如何应对选择性压力的不完全理解细胞和动物模型 - 例如疫苗开发期间 - 以及人类感染期间的体内模型。我们建议使用实验进化方法来揭示 HSV-1 如何适应关键的细胞生态位例如成纤维细胞和神经元，以及干扰素信号传导水平，作为宿主的关键组成部分这些研究将利用从新感染者中连续培养的分离株的独特资源。个体，以实现实验和自然进化压力之间的新水平比较。在目标 1 中，我们将探索人类成纤维细胞和神经元的独特细胞生态位中的 HSV-1 进化。作为持续合作的一部分，我们可以访问序列临床 HSV-1 的独特存储库使用最初的病毒分离株从感染第一年的免疫功能正常的个体中分离出来。从这些个体中，我们将在原代人类成纤维细胞和人类中进行实验进化神经元，代表体内感染的两个关键细胞生态位，我们将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随着时间的推移，两种细胞类型之间出现的遗传变异将是使用统计模型进行分析，以区分遗传漂变和观察到的变异。然后，在这些定义的环境中进行实验进化期间，将与在自然进化的过程，通过来自同一感染个体的连续病毒培养。干扰素反应是宿主免疫控制感染的一个重要方面。多个 HSV-1 基因具有这种作用。与宿主干扰素反应的病毒拮抗作用有关，尽管这些是传统上研究的在目标 2 中，我们将使用实验进化方法进行无偏见的基因组研究。通过治疗原代人类，广泛筛选参与 HSV-1 干扰素拮抗作用的基因位点。成纤维细胞用外源干扰素或用抗体来消耗干扰素产生，我们将在对的复制谱系进行测序后，分别将 HSV-1 进化为氮或耗尽的抗病毒状态。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这些实验进化数据将共同提供与干扰素拮抗相关的病毒基因。关于 HSV-1 如何适应这些不同的细胞生态位的新见解，并为复杂的复杂环境提供了新的线索自然感染中发现的选择性压力的相互作用。