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.

项目摘要 HSV-1会引起美国和世界各地数百万人的终生感染。自然感染 HSV-1遵循上皮细胞中生产性复制的循环，然后转移到神经末端，然后在神经元中建立终身病毒储存库。尽管需求很高，并且为设计一个 HSV-1疫苗，在动物模型中表现出希望后，先前的候选人在人类试验中失败了。这功效差距反映了我们对该病毒对选择性压力的反应的不完全理解细胞和动物模型 - 例如在疫苗发育过程中 - 人体感染期间的体内。这里我们建议使用一种实验进化方法来揭示HSV-1如何适应关键细胞壁ni 例如成纤维细胞和神经元，并改变干扰素信号的水平，作为主机的关键组成部分免疫。这些研究将利用新感染的连续培养隔离株的独特资源个体，可以在实验和自然进化压力之间进行新的比较。在AIM 1中，我们将探索人类成纤维细胞和神经元不同细胞壁细胞壁细胞壁细胞的进化。作为正在进行的合作的一部分，我们可以使用连续临床HSV-1的独特存储库在感染的第一年，从免疫能力的个体中分离出来。使用初始病毒分离株从这些人那里，我们将在原代人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壁细分市场。我们将深入序列病毒在成纤维细胞，神经元或替代性的实验进化中复制谱系的种群在两种单元格之间。随着时间的推移，每个病毒群中出现的遗传变异将是使用统计模型分析以区分遗传漂移与方向选择。观察到的变体然后，在这些定义的环境中的实验演化过程中，将被比较自然进化的过程，通过来自同一感染个体的顺序病毒培养物。干扰素反应是宿主免疫控制感染的关键方面。多个HSV-1基因具有在宿主干扰素反应的病毒拮抗作用中暗示，尽管这些传统上是研究一次一个。在AIM 2中，我们将使用实验进化方法进行无偏的基因组 - HSV-1参与干扰素拮抗作用的遗传位置的宽屏。通过治疗初级人具有外源干扰素或复制干扰素产生的抗体的成纤维细胞，我们将分别以增强或分离的抗病毒状态进化HSV-1。在测序重复谱系之后这些进化的病毒种群，我们将使用遗传漂移和/或阳性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共同提供关于HSV-1如何适应这些独特的蜂窝壁ni的新见解，并为复合物提供了新的灯光自然感染中发现的选择性压力的相互作用。