*3.4. Role in Inhibition of Innate Immunity*

Post-attachment events associated with HCMV entry and the recognition of virion components by pattern recognition receptors including foreign DNA sensors trigger the induction of cytokine and chemokine genes [262–264]. Many of these cytokines and chemokines are important components of our innate immune system, especially type I, II and III IFNs. The synthesis and secretion of these IFNs activates signalling pathways that involve the phosphorylation of STAT family members including STAT1 and STAT2. Activated STAT proteins form homodimers or heteromeric complexes that subsequently bind to and stimulate transcription from promoters of ISGs many of which encode proteins that interfere with viral replication at various points.

IE1 confers increased type I IFN resistance to HCMV [225]. This phenotype was largely attributed to nuclear complex formation between IE1 and STAT2 depending on amino acids 410–420 in the presumably disordered "acidic domain" of the viral protein downstream from the central core domain and upstream of the CTD [209,222,225,226]. The IE1-STAT2 interaction causes reduced sequence-specific DNA binding by ISGF3 and diminished activation of type I ISGs (e.g., CXCL10, IFIT2, ISG15, MX1) [209,225,226,265,266]. The C-terminal part of IE1 has also been reported to disrupt type II ISG activation by STAT1 homodimers, although IE1 is not believed to bind to STAT1 directly (only indirectly via STAT heterodimers) [222,225,226,267]. The ability of IE1 to inhibit type I ISG induction via STAT2 interaction facilitates HCMV replication and appears to be conserved among IE1 homologs of other betaherpesviruses [209,226,268]. Besides STAT2 interaction, complex formation between PML and IE1 (see Section 3.3) may also contribute to the inhibition of ISG induction during HCMV infection [216,269].

IE2 is not known to interact with STAT family members, but this protein limits HCMV-induced expression of antiviral cytokine and proinflammatory chemokine genes (e.g., IFNB1, CCL3, CCL5, CCL8, CXCL8, CXCL9) [270,271]. A very recent report has also shown that IE2 targets interleukin 1 beta (IL1B) at both the transcript and protein level [272]. The underlying mechanism appears to involve inhibition of virus- or tumor necrosis factor alpha-induced binding of NF-κB to the IFN-β promoter, resulting in attenuated target gene expression [273]. Another recent report has demonstrated that IE2 inhibits IFN-β promoter activation induced by STING, a critical sensor of intracellular DNA and adaptor for type I IFN signalling. IE2 facilitated the proteasome-dependent degradation of STING and inhibited cGAMP-mediated induction of IFNB1 and CXCL10 [274]. Taken together, these studies suggest that IE2 targets STING (and likely other proteins) post-translationally resulting in inhibition of

NF-κB-dependent induction of cytokine and chemokine genes relevant to the innate immune response to HCMV infection.

#### *3.5. Role in Inflammation and Adaptive Immunity*

HCMV reactivation and replication are typically linked to a strong inflammatory host response that involves numerous cytokines and chemokines, which often contributes to pathogenesis [27,28,275]. Despite their roles as intrinsic and innate immune antagonists (see Sections 3.3 and 3.4), the major IE proteins may also facilitate inflammation, most obviously by driving HCMV replication. However, IE1 and IE2 may promote inflammation even in the absence of viral replication [223,276–281]. Consistent with this idea, the IE1-specific host cell transcriptome is largely characterized by downregulation of genes responsive to IL6-type cytokines and upregulation of ISGs normally induced by IFN-γ (see Section 3.1) [222,223]. IE1-dependent gene activation proved to be independent of IFN-γ and other IFNs, yet required phosphorylated STAT1. Accordingly, IE1 induced phosphorylation, nuclear accumulation and binding of STAT1 to type II ISG promoters. Moreover, the repression of STAT3- and the activation of STAT1-responsive genes by IE1 turned out to be coupled. By targeting STAT3, IE1 rewires upstream STAT3 to downstream STAT1 signalling. Consequently, genes normally induced by IL6 are repressed while genes normally induced by IFN-γ become responsive to IL6 in the presence of IE1. Thus, IE1 merges two central cellular signalling pathways diverting cytokine responses relevant to inflammation and (neuro)pathogenesis [222,282].

Adaptive antibody as well T cell responses are thought to be important for long-term control of HCMV. Studies on T cell immunity in HCMV have traditionally focused on pUL83/pp65 and IE1. However, it has become clear that both IE1 and IE2 are highly immunogenic CD4+ and CD8+ T cell antigens adding to their complex roles in the immune response to HCMV infection [283–285]. Based on the stimulatory effect IE1 exerts on cellular adaptive immunity, the protein has been utilized in the development of both diagnostic assays as well as vaccine candidates [286–288].
