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

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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.