*2.4. Summary*

Highly complex interactions between a multitude of cellular and viral components at the level of DNA, chromatin and upstream signalling pathways determine the initiation and magnitude of transcription from the HCMV MIEP. The highly dynamic transcription from the major IE gene is complemented by post-transcriptional processing and translational regulation, ultimately controlling the synthesis of the IE1 and IE2 families of predominantly nuclear proteins. The major IE proteins are subject to post-translational regulation and are thought to activate the viral replicative cycle during both initial infection and reactivating from latency. It is therefore believed that the eventual outcome of HCMV infection depends on the level and timing of IE1 and IE2 expression.

#### **3. Major IE Protein Function**

#### *3.1. Role in Activation and Repression of Transcription*

IE1 and IE2 were initially identified as activators of transcription in reporter assays using transiently transfected plasmids [11,13,14]. In these assays, the IE proteins were shown to activate the HCMV MIEP (positive auto-regulation) and various viral early gene promoters. In addition, IE2 turned out to be a repressor of the MIEP (negative auto-regulation). In fact, IE2 sequence-specifically binds to the crs in the core promoter (Figure 1) to block RNA polymerase II occupancy at the transcription start site. Furthermore, several heterologous viral promoters as well as cellular promoters proved to be responsive to activation by the IE proteins. The impact of IE1 and IE2 on transcription from a broad variety of promoters in transient transfection assays earned them the title "promiscuous transactivators". IE2 usually appeared as the stronger activator compared to IE1 and, depending on the reporter construct, the two proteins often acted in an additive or synergistic manner. Activation by IE1 and IE2 was mapped to both upstream elements as well as core promoter regions including the TATA-box. Accordingly, numerous specific and basic transcription factors or transcription factor complexes were reported to interact with IE1 (e.g., CEBP, E2F1-5, SP1) and IE2 (e.g., AP1, CREB1, EGR1, SP1, TAF4, TBP, TFIIB, TFIID).

Many key findings from transient transfection assays about the impact of IE1 and IE2 on HCMV transcription were later corroborated by studies involving mutant viruses and global transcriptome analyses. These findings confirmed positive autoregulation at the MIEP by IE1 [217], crs-dependent repression of the MIEP by IE2 and activation of viral early genes by IE1 and IE2 [9,218–221]. In contrast, "promiscuous transactivation" by IE1 and IE2 was not replicated in transcriptome analyses of endogenous human genes. Instead of showing broad activation of gene expression from the human genome, the differential transcript profiles from cells individually expressing IE1 or IE2 were rather distinct with little or no overlap to the genes activated by the IE proteins in assays with transfected reporter plasmids. Following expression under conditions closely mimicking the situation during productive infection, IE1 turned out to be as significant a repressor as it is an activator of host gene expression in growth-arrested human fibroblasts [222,223]. Cells induced to express IE1 exhibited global repression of interleukin 6 (IL6)- and oncostatin M-responsive signal transducer and activator of transcription (STAT) 3 target genes. This repression was followed by STAT1-dependent activation of type II IFN-stimulated genes (ISGs), normally induced by IFN-γ, many of which encode immune-stimulatory proteins including proinflammatory chemokines [222–224]. Moreover, in the presence of IFN-α or IFN-β, IE1 was found to inhibit the induction of type I ISGs by the trimeric complex of STAT1, STAT2 and IRF9 known as ISG factor 3 (ISGF3) [225,226]. The effects IE1 exerts on the human transcriptome are thought to result largely from direct physical interactions with STAT2 and STAT3 (see Section 3.4). While transcriptional regulation by IE1 appears to be dominated by pathways depending on proteins of the STAT family, IE2 has been shown to inhibit the induction of IFN and other antiviral cytokine genes via a mechanism involving NF-kB and STING (see Section 3.4). However, the transcriptome profile for IE2 in cycling human fibroblasts was dominated by genes regulating the cell cycle and DNA replication many of which are E2F-responsive [227]. This finding likely reflects the impact of IE2 on the cell cycle. IE2 promotes cell cycle progression from G0/G1 to G1/S and arrests cells at the G1/S interface, inhibiting cellular DNA synthesis, or at the G2/M interface [13,14,228]. Many human genes activated or repressed by isolated expression of IE1 or IE2 were also shown to be differentially regulated during productive HCMV infection.

### *3.2. Role in Chromatin-Based Epigenetic Regulation*

IE2 is known to bind sequence-specifically to DNA, but there is no convincing evidence for direct DNA binding by IE1 [11,108,131]. However, IE1 associates with chromatin via core histones. IE1 exhibits two physically separable histone interacting regions with differential binding specificities for H2A-H2B dimers and H3-H4 dimers or tetramers. The H2A-H2B binding region was mapped to an evolutionarily conserved nucleosome binding motif (amino acids 479–488) within the chromatin tethering domain (CTD) at the C-terminus [229,230]. This motif docks with the acidic patch formed by H2A-H2B on the nucleosome surface [229,230]. The consequences of the IE1-nucleosome interaction have not been fully elucidated, but they appear to include alterations to higher order chromatin structure [230]. Histone binding by IE1 might also be linked to the overall low nucleosome levels and temporal reorganization of nucleosomes across viral genomes observed during productive HCMV infection [9,105]. To our knowledge, transcriptional regulation via the IE1 CTD has not been experimentally addressed. Instead, it has been reported that IE1x4, a small variant form of IE1 expressed from a promoter internal to major IE exon 4, facilitates viral genome maintenance and replication during HCMV latency via a CTD-dependent mechanism. Despite a lack of experimental evidence, the mechanism is predicted to involve HCMV episome tethering to host mitotic chromosomes via nucleosome binding by IE1x4 resulting in nuclear retention and partitioning of viral genomes across latently infected dividing cells [75]. Although IE2 appears to have relatively little affinity for histones, both IE proteins have been shown to be present in complexes with nucleosome modifying host cell proteins. For example, IE1 binds to histone deacetylase (HDAC) 3 [124], while IE2 binds to HDAC1-3 [124,126,231], lysine acetyltransferases CREB binding protein (CBP, KAT3A), p300 (KAT3B) and p300/CBP-associated factor (KAT2B) [232,233], and lysine methyltransferases G9A (euchromatic histone lysine methyltransferase 2, EHMT2) and suppressor of variegation 3-9 homolog 1 (SUV39H1) [126]. Accordingly, transcriptional activation of viral IE and early genes by IE1 correlates with histone acetylation, while transcriptional repression of the MIEP by IE2 involves histone deacetylation and methylation [130,131,234].
