Abstract
This work represents the first comprehensive quantitative analysis of global histone post-translational modifications (PTMs) from a virus infection, namely human cytomegalovirus (HCMV) infection. We used a nanoLC-MS/MS platform to identify and quantify the dynamic histone H3 and H4 PTMs expressed during HCMV replication in primary fibroblasts. Specifically, we examined the changes in histone PTMs over a 96 h time course to sample the immediate early (IE), early (E), and late (L) stages of viral infection. Several changes in histone H3 and H4 PTMs were observed, including a marked increase in H3K79me2 and H3K27me3K36me2, and a decrease in H4K16ac, highlighting likely epigenetic strategies of transcriptional activation and silencing during HCMV lytic infection. Heavy methyl-SILAC (hm-SILAC) was used to further confirm the histone methylation flux (especially for H3K79) during HCMV infection. We evaluated DOT1L (the H3K79 methyltransferase) mRNA levels in mock and HCMV-infected cells over a 96 h time course, and observed a significant increase in this methyltransferase as early as 24 hpi showing that viral infection up-regulates DOT1L expression, which drives H3K79me2. We then used shRNA to create a DOT1L knockdown cell population, and found that HCMV infection of the knockdown cells resulted in a 10-fold growth defect when compared with infected control cells not subjected to knockdown. This work documents multiple histone PTMs that occur in response to HCMV infection of fibroblasts, and provides a framework for evaluation of the role of epigenetic modifications in the virus-host interaction.
Highlights
From the ‡Department of Molecular Genetics, Cleveland Clinic, Lerner Research Institute, Cleveland, Ohio 44195; §Department of Chemical Engineering, Imperial College London, London, UK, SW7 2AZ; ¶Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544; ʈEpigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104
Human cytomegalovirus (HCMV) Infection Results in the Up-regulation of H3K79 and its Methyltransferase, DOT1L—To begin to elucidate the biological consequences of the virus-induced modifications, we focused on H3K79, as there is only one known methyltransferase for modifications at this site
We observed an increase in H3K79me1 at 24 hpi, this post-translational modifications (PTMs) returned to near-baseline values for the remainder of the time course (Fig. 4C)
Summary
From the ‡Department of Molecular Genetics, Cleveland Clinic, Lerner Research Institute, Cleveland, Ohio 44195; §Department of Chemical Engineering, Imperial College London, London, UK, SW7 2AZ; ¶Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544; ʈEpigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104 When viruses infect their hosts, they modulate the intracellular environment so that it is optimized to support the viral life cycle. Concomitant with replication of the viral genome, the late (L) genes are transcribed, and their proteins include structural components of the mature particle [1]. The major immediate-early promoter (MIEP), which controls expression of mRNAs encoding IE1 and IE2, is repressed in part through the binding of heterochromatin protein 1 (HP1) during infection of peripheral blood monocytes, a model for HCMV latency [10]. Several other HCMV proteins interact with chromatin modifiers, such as the pUL29/28 interactions with the NuRD chromatin remodeling complex [11], underpinning the general importance of epigenetic regulation in the HCMV life cycle
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