Viruses create cellular conditions conducive to their own replication by harnessing or inactivating cellular machinery. Since viruses possess small genomes that do not encode all the factors required for DNA replication, they rely on host cell proteins to propagate their genomes. Active recruitment of cellular proteins to viral replication compartments functions to promote virus replication. In contrast, viral early proteins target antiviral cellular DNA sensors and transcriptional repressors to prevent their access to viral genomes. Identifying host factors that facilitate or limit virus DNA replication has been hampered by the lack of technologies, and only a limited number of cellular proteins have been shown to affect virus replication. Our long-term goal is to understand how cells respond to virus genomes, and to define ways that early viral proteins overcome cellular restrictions to promote virus replication. In this application we use Adenovirus infection as a model system, and employ a novel technology to identify cellular proteins that associate with replicating viral DNA genomes. Our central hypothesis is that viral proteins selectively recruit cellular DNA replication/repair factors onto viral genomes to function with viral-encoded replication factors, while also targeting specific host antiviral factors to prevent their access to viral genomes. We propose that viral early E1b and E4 gene products overcome host responses and achieve their pleiotropic functions by influencing the cellular proteins recruited to virus genomes. These viral early proteins facilitate viral DNA replication, RNA processing, protein expression, and progeny production, but their substrates are currently poorly understood. We employ novel proteomic approaches to identify host factors that interact with these viral early proteins, and determine how they impact the landscape of host factors that recognize viral DNA. Guided by strong preliminary data, we propose three Specific Aims to identify host factors recruited or inactivated by viral early proteins and determine their impact on virus DNA replication.
Aim 1 : To determine how host factors associated with replicating viral genomes impact infection.
Aim 2 : To define cellular responses across the Ad family.
Aim 3 : To determine how E1b55K interacting proteins impact virus infection. These studies employ innovative approaches to provide a comprehensive view of cellular responses to foreign DNA genomes replicating in the host cell nucleus. We anticipate that our results will identify proteins recruited to aid viral replication, as well as wys viral proteins manipulate host responses. Identifying cellular proteins commonly exploited for viral DNA replication across different viruses will suggest potential targets for development of novel broadly acting antiviral therapeutics. Our studies provide insights into cellular sensors of foreign DNA, and these will guide development of improved gene delivery vectors, viral oncolytics, and antiviral therapies.

Public Health Relevance

Viruses elicit multifaceted responses from the cells that they infect, and these can serve to limit growth of the virus or can be exploited to aid virus DNA replication. Adenoviruses are ubiquitous viruses that have provided both important model systems for understanding fundamental cellular processes, as well as vectors for therapeutic purposes. In this proposal we will employ new technology to identify cellular proteins that associate with replicating virus DNA genomes, providing insights into virus-host interactions and suggesting targets for novel antivirals.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA097093-16
Application #
9685119
Study Section
Virology - A Study Section (VIRA)
Program Officer
Daschner, Phillip J
Project Start
2002-07-01
Project End
2020-04-30
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
16
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19146
Pancholi, Neha J; Weitzman, Matthew D (2018) Serotype-specific restriction of wild-type adenoviruses by the cellular Mre11-Rad50-Nbs1 complex. Virology 518:221-231
Dybas, Joseph M; Herrmann, Christin; Weitzman, Matthew D (2018) Ubiquitination at the interface of tumor viruses and DNA damage responses. Curr Opin Virol 32:40-47
Pancholi, Neha J; Price, Alexander M; Weitzman, Matthew D (2017) Take your PIKK: tumour viruses and DNA damage response pathways. Philos Trans R Soc Lond B Biol Sci 372:
Reyes, Emigdio D; Kulej, Katarzyna; Pancholi, Neha J et al. (2017) Identifying Host Factors Associated with DNA Replicated During Virus Infection. Mol Cell Proteomics 16:2079-2097
Avgousti, Daphne C; Della Fera, Ashley N; Otter, Clayton J et al. (2017) Adenovirus core protein VII down-regulates the DNA damage response on the host genome. J Virol :
Herrmann, Christin; Avgousti, Daphne C; Weitzman, Matthew D (2017) Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells. Bio Protoc 7:
Avgousti, Daphne C; Herrmann, Christin; Kulej, Katarzyna et al. (2016) A core viral protein binds host nucleosomes to sequester immune danger signals. Nature 535:173-7
Kulej, Katarzyna; Avgousti, Daphne C; Weitzman, Matthew D et al. (2015) Characterization of histone post-translational modifications during virus infection using mass spectrometry-based proteomics. Methods 90:8-20
Weitzman, Matthew D; Weitzman, Jonathan B (2014) What's the damage? The impact of pathogens on pathways that maintain host genome integrity. Cell Host Microbe 15:283-94
Chaurushiya, Mira S; Lilley, Caroline E; Aslanian, Aaron et al. (2012) Viral E3 ubiquitin ligase-mediated degradation of a cellular E3: viral mimicry of a cellular phosphorylation mark targets the RNF8 FHA domain. Mol Cell 46:79-90

Showing the most recent 10 out of 36 publications