(454b) Quantitative Proteomic Discovery of the Epigenetic Mechanisms That Control Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) is a large, double strand DNA virus that is a member of the herpesvirus family. HCMV establishes a life-long, persistent infection in its host that remains asymptomatic in healthy individuals. However, the virus can cause severe morbidity and mortality upon reactivation in immunocompromised individuals. To date, no preventative HMCV vaccinations exist and treatment options suffer from low bioavailability, toxicity, and viral resistance. Several studies have shown that HCMV viral DNA associates with host histones and undergoes chromatinization upon entering the nucleus of infected cells and that changes in chromatin structure are strongly correlated with changes in viral gene expression. Thus, an understanding of the epigenetic mechanisms driving viral gene expression and replication would benefit the design of novel therapeutics.

In this work, we have utilized liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify and quantify the dynamic histone post-translational modifications (PTMs) expressed during HCMV lytic infection (TB40/E BAC-derived clinical strain) in primary fibroblasts. Specifically, we examined the changes in histone modifications over a 96 hour time course to provide a sufficient sampling of snap shots corresponding to the immediate early (IE), early (E), and late (L) stages of viral infection.  Over 100 Bottom Up LC-MS/MS experiments were conducted and quantitative analyses of more than 60 modified histone H3 and H4 isoforms during the time course of infection revealed significant up- and down-regulation of several histone post-translational modifications, including a substantial increase in histone H3 K79 dimethylation (H3K79me2).  Previous studies have associated K79 methylation with the recruitment of DNA damage repair machinery.  Consistent with this observation, HCMV infected cells had increased levels of chromosomal damage relative to mock infected cells. Additionally, we found that the RNA levels of DOT1L, the methyltransferase specific for "writing" H3K79me2, had also increased over the course of HCMV infection.

Based on these findings, we investigated the functional role of this H3K79me2 modification on HCMV growth by shRNA knock-down of DOT1L. Two independent DOT1L knock-down stable cell lines expressing different shRNA sequences were generated in primary fibroblasts. We confirmed the knock-down of DOT1L via both RNA analysis and LC-MS/MS.  We then assessed viral replication in these DOT1L knock-down cells and found that HCMV growth was reduced.  To determine the stage in viral growth that this post-translational histone modification was predominantly impacting, we examined viral protein expression/localization as well as viral DNA replication.  Although viral proteins were expressed and localized similarly across the stable cell lines, we found that viral DNA replication in the knock-down cells was impaired when compared to the scramble knock-down cells.  Current experiments include genome-wide microarray, ChIP-Seq and heavy methyl SILAC experiments to further characterize the systems-level function of the histone PTMs during HCMV infection.