The overall objective of this proposal is to determine the significance of epigenetic histone post-translational modifications (PTMs) during human cytomegalovirus (HCMV) infection. HCMV is the largest known human herpes virus and it is well recognized that a large percentage of the human population (>60%) is infected with HCMV. HCMV infections cause birth defects; and for immunocompromised populations, these infections are often times life threatening. Upon infection of cells and chromatinization of the virus in the host nucleus, the major immediate-early promoter (MIEP) controls the expression of the viral IE1 and IE2 proteins which interact with many nuclear proteins. These interactions ultimately modulate both viral and cellular gene expression, in part through epigenetic mechanisms. Epigenetics refers to mechanisms that can regulate gene expression patterns without involving changes in DNA sequence, and include DNA methylation, small ncRNAs and histone PTMs. The limited picture of how HCMV harnesses epigenetic mechanisms presents a significant barrier to understanding HCMV pathogenesis and developing novel antiviral therapy to target these chromatin factors. Previously, we determined that HCMV infection results in several changes to single PTM sites on histones, and that one modification site in particular H3K79me2 and its corresponding enzyme DOT1L affected HCMV growth. Here we aim to answer the following questions: How does HCMV utilize combinatorial PTMs on histones for controlling both host and viral gene expression patterns and viral replication? Are there any non- histone chromatin proteins that are needed for viral growth? As certain protein lysine methyltransferases are upregulated during HCMV infection, are there other non-histone proteins methylated during infection that are needed for viral replication? We will address these questions using a combination of biological and quantitative methods. We will develop enhanced mass spectrometry methods to quantify combinatorial histone H3 and H4 PTMs from MRC5 cells during an HCMV infection time-course. Genome-wide approaches will be employed to identify both host and viral genes harboring significant combinatorial PTMs. The effect of depleting the enzymes responsible for the most abundant combinatorial PTM changes on both host and viral gene expression, and on viral growth and replication will be assessed. Using biochemical and affinity approaches, we will isolate both global viral chromatin and MIEP specific viral chromatin to determine the chromatin factors that contribute to regulation of viral gene transcription. Lastl, we will use our novel affinity quantitative proteomics approach to identify non-histone proteins that are lysine methylated during viral infection. It is expected that these experiments will produce new fundamental insights in an understudied area of HCMV biology, and could lead to development of new epigenetic drug therapy for treatment of HCMV.

Public Health Relevance

Viruses have adapted to evade host immune defenses, and target several areas of cellular signaling to maximize replication. This research will help define the epigenetic changes that occur in response to viral infection, thus laying down the foundation for potential development of novel epigenetic drug targeted antiviral therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI118891-01
Application #
8941146
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Beisel, Christopher E
Project Start
2015-04-15
Project End
2020-03-31
Budget Start
2015-04-15
Budget End
2016-03-31
Support Year
1
Fiscal Year
2015
Total Cost
$498,000
Indirect Cost
$104,999
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Shinde, Mansi Y; Sidoli, Simone; Kulej, Katarzyna et al. (2017) Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing. J Biol Chem 292:18240-18255
Kulej, Katarzyna; Avgousti, Daphne C; Sidoli, Simone et al. (2017) Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection. Mol Cell Proteomics 16:S92-S107
Janssen, K A; Sidoli, S; Garcia, B A (2017) Recent Achievements in Characterizing the Histone Code and Approaches to Integrating Epigenomics and Systems Biology. Methods Enzymol 586:359-378
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
Coetzee, Nanika; Sidoli, Simone; van Biljon, Riƫtte et al. (2017) Quantitative chromatin proteomics reveals a dynamic histone post-translational modification landscape that defines asexual and sexual Plasmodium falciparum parasites. Sci Rep 7:607
Simithy, Johayra; Sidoli, Simone; Yuan, Zuo-Fei et al. (2017) Characterization of histone acylations links chromatin modifications with metabolism. Nat Commun 8:1141
Sidoli, Simone; Lu, Congcong; Coradin, Mariel et al. (2017) Metabolic labeling in middle-down proteomics allows for investigation of the dynamics of the histone code. Epigenetics Chromatin 10:34
Kori, Yekaterina; Sidoli, Simone; Yuan, Zuo-Fei et al. (2017) Proteome-wide acetylation dynamics in human cells. Sci Rep 7:10296
Sidoli, Simone; Kulej, Katarzyna; Garcia, Benjamin A (2017) Why proteomics is not the new genomics and the future of mass spectrometry in cell biology. J Cell Biol 216:21-24
Sidoli, Simone; Garcia, Benjamin A (2017) Characterization of Individual Histone Posttranslational Modifications and Their Combinatorial Patterns by Mass Spectrometry-Based Proteomics Strategies. Methods Mol Biol 1528:121-148

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