Binding STAT2 by the Acidic Domain of Human Cytomegalovirus IE1 Promotes Viral Growth and Is Negatively Regulated by SUMO

Abstract

The human cytomegalovirus (HCMV) 72-kDa immediate-early 1 (IE1) protein is thought to modulate cellular antiviral functions impacting on promyelocytic leukemia (PML) nuclear bodies and signal transducer and activator of transcription (STAT) signaling. IE1 consists of four distinct regions: an amino-terminal region required for nuclear localization, a large central hydrophobic region responsible for PML targeting and transactivation activity, an acidic domain, and a carboxyl-terminal chromatin tethering domain. We found that the acidic domain of IE1 is required for binding to STAT2. A mutant HCMV encoding IE1(⌬421-475) with the acidic domain deleted was generated. In mutant virus-infected cells, IE1(⌬421-475) failed to bind to STAT2. The growth of mutant virus was only slightly delayed at a high multiplicity of infection (MOI) but was severely impaired at a low MOI with low-level accumulation of viral proteins. When cells were pretreated with beta interferon, the mutant virus showed an additional 1,000-fold reduction in viral growth, even at a high MOI, compared to the wild type. The inhibition of STAT2 loading on the target promoter upon infection was markedly reduced with mutant virus. Furthermore, sumoylation of IE1 at this acidic domain was found to abolish the activity of IE1 to bind to STAT2 and repress the interferon-stimulated genes. Our results provide genetic evidence that IE1 binding to STAT2 requires the 55-amino-acid acidic domain and promotes viral growth by interfering with interferon signaling and demonstrate that this viral activity is negatively regulated by a cellular sumoylation pathway.

Involvement of the N-Terminal Deubiquitinating Protease Domain of Human Cytomegalovirus UL48 Tegument Protein in Autoubiquitination, Virion Stability, and Virus Entry

“…The HCMV Towne virus stocks used in this study were prepared as previously described (30). Viral titers were determined on HF cells using infectious center assays employing an anti-IE1 antibody (31).…”

Upregulation of Nrf2 expression by human cytomegalovirus infection protects host cells from oxidative stress

“…Viral stocks were aliquoted and stored at 270 uC. The titres of viral stocks were determined as an infectious centre unit (ICU) after the measurement of the IE1/IE2-positive cells by infectious centre assay (Huh et al, 2008). Clinical strain JHC (passage 3), originally isolated from patients undergoing bone marrow transplantation, was provided by Dr Chan Hee Lee (Chungbuk National University, Cheongju, Korea), and passaged one more time in HFFs (Jung et al, 2011).…”

Characterization of Recombinant Human Cytomegaloviruses Encoding IE1 Mutants L174P and 1-382 Reveals that Viral Targeting of PML Bodies Perturbs both Intrinsic and Innate Immune Responses

“…It has been shown that IE1 interacts with both STAT1 and STAT2 and inhibits DNA binding of these proteins, thereby affecting the level of ISG transcripts. As a result, IE1-deleted HCMV displays increased sensitivity to IFN-␣ treatment compared to wild-type HCMV (41,42). In contrast to its influence on type I IFN signaling, expression of IE1 triggers a transcriptional response characterized by an upregulation of immune stimulatory and proinflammatory genes that are normally induced by IFN-␥, the only representative of type II IFNs (43).…”

“…Mutations, e.g., the L174P mutation, or deletions within IE1 CORE were shown to abolish the interaction with PML and, consequently, the effects on PML SUMOylation and ND10 integrity in transfection assays (34,(37)(38)(39)(40). These mutations, however, do not affect the ability of IE1 to interact with STAT proteins through binding sites located in the disordered C-terminal region (5,41). It has been shown that IE1 interacts with both STAT1 and STAT2 and inhibits DNA binding of these proteins, thereby affecting the level of ISG transcripts.…”