The goal of the research in this application is to understand the mechanisms involved in chromatin-mediated regulation of gene expression. We will use herpes simplex virus-1 (HSV-1) to investigate these areas because its genome associates with histones during infection and the chromatin state of the HSV-1 genome is extensively regulated. During HSV-1 productive infection, multiple chromatin-remodeling proteins are recruited to viral replication compartments in the nuclei of infected host cells, which are the sites of viral DNA replication and late gene transcription. We hypothesize that chromatin-remodeling proteins play a role in HSV-1 biology by remodeling the chromatin state of the viral genome and that viral proteins regulate the activity of these cellular enzymes.
The specific aims of this application are: 1. To investigate the hypothesis that cellular chromatin-remodeling factors play a role in HSV-1 replication by remodeling the chromatin state of the viral genome, and 2. To investigate the hypothesis that viral proteins regulate the activity of cellular chromatin-remodeling complexes. To investigate Specific Aim 1, we will determine the role of chromatin-remodeling proteins in HSV-1 biology by knocking down their expression using RNAi, focusing initially on the chromatin-remodeling factor hSNF2H, and assaying the effect on viral gene expression by northern and western blotting. We will also assay whether chromatin-remodeling complexes associate with the HSV-1 genome and whether these complexes regulate the chromatin composition of the viral genome by performing chromatin immuno-precipitation assays. To investigate Specific Aim 2, we will determine the composition of chromatin-remodeling complexes during HSV-1 infection by performing tandem affinity purification assays, focusing on the interaction between ICP8 and hSNF2H. We will then investigate the importance of the associations between chromatin-remodeling factors and viral proteins, including the association between ICP8 and hSNF2H, by mapping these associations in vitro and constructing mutant viruses defective for interactions with the chromatin-remodeling protein of interest. Additionally, we will investigate whether viral proteins, including ICP8, alter the activity of chromatin-remodeling proteins by performing in vitro remodeling assays.
Defects in the cellular processes related to DNA metabolism are associated with numerous human diseases, and thus a better understanding of these processes is required to more effectively treat these disorders. Additionally, HSV-1 is a significant pathogen and therefore greater knowledge of the biology of replication of this virus can lead to the discovery and development of novel therapeutic strategies.
| Bryant, Kevin F; Yan, Zhipeng; Dreyfus, David H et al. (2012) Identification of a divalent metal cation binding site in herpes simplex virus 1 (HSV-1) ICP8 required for HSV replication. J Virol 86:6825-34 |
| Barczak, Amy K; Gomez, James E; Kaufmann, Benjamin B et al. (2012) RNA signatures allow rapid identification of pathogens and antibiotic susceptibilities. Proc Natl Acad Sci U S A 109:6217-22 |
| Bryant, Kevin F; Colgrove, Robert C; Knipe, David M (2011) Cellular SNF2H chromatin-remodeling factor promotes herpes simplex virus 1 immediate-early gene expression and replication. MBio 2:e00330-10 |
