Human cytomegalovirus (hCMV), one of eight human herpesviruses, establishes lifelong "latent" infections in 40-100% of people worldwide. HCMV replication following primary infection or reactivation is known for causing developmental defects in human embryos and life-threatening
disease in immunocompromised individuals, but preventive and therapeutic options are still limited. One potential candidate for the development of new antiviral drugs or a vaccine is the immediate-early (IE) 1 protein of hCMV. This protein is a crucial regulator of viral and cellular gene expression and has been shown to interact with chromatin. For chromatin binding, the IE1 protein exhibits two adjacent core histone interacting regions with distinct binding specificities. One of them is the so-called "chromatin tethering domain" (CTD), a 16 amino-acid sequence
(amino acids 476-491) at the IE1 carboxy-terminus, which was recently shown to bind to the acidic patch formed by histone H2A and H2B on the nucleosomal surface to which several other viral and cellular proteins bind as well. The latency-associated nuclear antigen 1 (LANA) encoded by the Kaposi’s sarcoma-associated herpesvirus (KSHV) binds to the acidic patch in a way similar to the IE1-CTD and was shown to regulate chromatin compaction, viral genome maintenance and the cellular DNA damage response (DDR) via this interaction. Based on the similarities to LANA and the fact that nucleosome targeting by IE1 is dispensable for productive
replication of hCMV, we hypothesized that the two viral proteins may serve analogous functions during latency of their respective viruses. In this thesis, I focused on defining chromatin binding sites of IE1 and uncovering the function of nucleosome targeting by IE1 with respect to genome
maintenance and DDR. To identify chromatin binding sites of IE1 on both the viral and cellular genome, I generated primary human fibroblasts (MRC-5 cells) permissive to hCMV in which
expression of HA-tagged IE1, untagged IE1, and HA-tagged CTD-deleted IE1 (IE1₁₋₄₇₅) can be synchronously induced. Chromatin immunoprecipitation coupled to next generation sequencing (ChIP-seq) experiments using these cells revealed that IE1 broadly binds to the host genome in a CTD-dependent manner. The protein appears to be enriched at transcription end sites (TES) and excluded from the promoter regions of human genes at the transcription start sites (TSS),
which may be due to differences in the nucleosomal load at these sites. Broad binding of IE1 was also observed across the viral genome, but here four binding peaks were identified. During viral latency IE1 may use nucleosome binding as a mechanism to tether the viral genome to host chromosomes similar to what has been observed for LANA. Against all controversies regarding the presence of IE1 during non-productive stages of infection, I could identify full-length IE1mRNA and protein and IE2 protein in a latently hCMV-infected monocytic cell line (THP-1 cells). Time-course analysis of viral genome levels in these cells showed that chromatin binding by IE1 is necessary for cyclic viral DNA replication events during latency through which the virus probably ensures viral genome maintenance. Other results demonstrate that IE1 reduces the nucleosomal load on the viral and host genome in a CTD-independent manner, perhaps to reduce chromatin compaction and promote transcription. Similar to LANA, chromatin binding by IE1 also affects the DDR outcome. IE1 blocks H2A(X)K13/15 and H2A(X)K118/119
ubiquitination by binding to the nucleosomal acidic patch and thereby seems to diminish DNA double-strand break repair by non-homologous end joining. These results indicate that IE1
broadly interacts with viral and cellular chromatin via the nucleosome surface and suggest that the IE1-nucleosome interaction serves an important role in controlling viral genome maintenance and the outcome of the DDR in hCMV-infected cells
