Manipulation of the DNA damage response and the proteome on viral genomes during Herpes Simplex Virus (HSV) infection

Children'S Hosp Of Philadelphia, Philadelphia PA

Investigators

Abstract

PROJECT SUMMARY Viruses harness host cellular functions to complete their infectious cycles. DNA virus infection introduces foreign viral genomes into the host cell nucleus which can activate kinase signaling pathways that are part of the cellular DNA damage response (DDR). Viruses have also evolved strategies to manipulate DDR signaling to promote infection, and this can be achieved through viral-encoded ubiquitin ligases which target cellular substrates with degradative and non-proteolytic outcomes. Herpes simplex virus type 1 (HSV-1) is a DNA virus whose infection is enhanced by both activation and manipulation of cellular DDR pathways. Previous studies have established that HSV-1 infection activates signaling through the ATM kinase pathway which benefits virus replication. The viral immediate early protein ICP0 is a multifunctional protein important for stimulating gene expression and overcoming host defenses during lytic HSV-1 infection. Among the known substrates for the ICP0 ubiquitin ligase are regulators of the DDR network. Our understanding of the interface between HSV-1, ICP0, and the host DDR pathways, has been constrained by prior knowledge from studies of DDR functions in response to damage of cellular genomes. We propose that the viral responses will be better informed by unbiased global approaches that define signaling networks and proteome changes specifically activated by virus infection. In this application we bring together multi-modal proteomics techniques in an integrative approach that will define ways that changes to the proteome by ATM-activated signaling and ICP0-mediated ubiquitination alter recruitment of proteins onto viral genomes to promote expression and replication. In extensive preliminary data, our global proteomic analysis of HSV-1 infected cells quantifies the landscape of phosphorylation within DDR pathways, identifies ICP0-dependent ubiquitination events induced, and informs on ways that these modify the set of host proteins associated with replicating HSV-1 viral genomes. Our data converge on a number of protein targets and complexes which we propose function as key regulators of viral gene expression and genome replication. Our complementary aims elucidate mechanisms by which phosphorylation and ubiquitination combine to promote viral replication. In Aim 1 we will define the intersection of phosphorylation and ubiquitination and determine their combined impact on the DDR during HSV-1 infection. In Aim 2 we will determine ways that ubiquitination and ATM-signaling promote HSV-1 infection by examining the proteins associated with viral gnomes to promote gene expression and replication. Integrating these global proteomic approaches will highlight mechanisms leveraged by HSV-1 to overcome repression of gene expression and facilitate productive infection. Understanding the cellular systems which control viral infection will ultimately advance translational research towards developing antiviral therapies and interventions to prevent infection.