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

项目摘要 在美国和世界各地，HSV-1会导致数百万人终身感染。自然感染 HSV-1在上皮细胞中经历了一个生产性复制的周期，随后进入神经末梢，并 在神经元中建立一个终生的病毒库。尽管需求很高，并做出了许多努力来设计一种 HSV-1疫苗，之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应对选择性压力的不完全理解 细胞和动物模型--例如在疫苗开发期间--以及在人类感染期间的体内模型。这里 我们建议使用一种实验性的进化方法来揭示HSV-1如何适应关键的细胞生态位 如成纤维细胞和神经元，以及干扰素信号水平的改变，作为宿主的关键组件 豁免权。这些研究将利用一种独特的资源，从新感染的患者中连续培养出 个人，使实验和自然进化压力之间的比较达到新的水平。 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生态位中的进化。 作为正在进行的合作的一部分，我们可以访问独一无二的顺序临床HSV-1存储库 来自有免疫能力的人的分离物，在他们感染的第一年。使用最初的病毒分离株 从这些个体出发，我们将在原代人类成纤维细胞和人类 神经元，它们代表了体内感染的两个关键细胞利基。我们会对病毒进行深度测序 在成纤维细胞、神经元或交替的实验进化中复制谱系的群体 在这两种细胞类型之间。随着时间的推移，在每个病毒种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和定向选择。观察到的变种 在这些定义的环境中的实验进化期间，然后将其与在 自然进化的过程，通过来自相同感染者的连续病毒培养。 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 与宿主干扰素反应的病毒拮抗有关，尽管这些传统上是研究的 一次一个。在目标2中，我们将使用实验进化方法来进行无偏见的基因组- 广泛筛选与单纯疱疹病毒1型干扰素拮抗相关的遗传位点。通过治疗原始人 用外源性干扰素或抗体耗尽干扰素的成纤维细胞，我们将 分别在增强或耗尽抗病毒状态下进化HSV-1。测序后复制的谱系 这些进化的病毒种群，我们将使用遗传漂移和/或正向选择的模式来识别 与干扰素拮抗相关的病毒基因。这些实验进化数据将共同提供 关于HSV-1如何适应这些不同的细胞利基的新见解，并为了解复杂的情况提供了新的线索 在自然感染中发现的选择性压力的相互作用。

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.