Positive Role of Promyelocytic Leukemia Protein in Type I Interferon Response and Its Regulation by Human Cytomegalovirus

March Positive Role of Promyelocytic Leukemia Protein in Type I Interferon Response and Its Regulation by Human Cytomegalovirus Young-Eui Kim 0 1 2 Jin-Hyun Ahn 0 1 2 0 Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine , Suwon , Republic of Korea 1 Funding: This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIP) (2012R1A2A2A01002551). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Editor: Roger D Everett, University of Glasgow , UNITED KINGDOM Promyelocytic leukemia protein (PML), a major component of PML nuclear bodies (also known as nuclear domain 10), is involved in diverse cellular processes such as cell proliferation, apoptosis, gene regulation, and DNA damage response. PML also acts as a restriction factor that suppresses incoming viral genomes, therefore playing an important role in intrinsic defense. Here, we show that PML positively regulates type I interferon response by promoting transcription of interferon-stimulated genes (ISGs) and that this regulation by PML is counteracted by human cytomegalovirus (HCMV) IE1 protein. Small hairpin RNA-mediated PML knockdown in human fibroblasts reduced ISG induction by treatment of interferon- or infection with UV-inactivated HCMV. PML was required for accumulation of activated STAT1 and STAT2, interacted with them and HDAC1 and HDAC2, and was associated with ISG promoters after HCMV infection. During HCMV infection, viral IE1 protein interacted with PML, STAT1, STAT2, and HDACs. Analysis of IE1 mutant viruses revealed that, in addition to the STAT2-binding domain, the PML-binding domain of IE1 was necessary for suppression of interferon--mediated ISG transcription, and that IE1 inhibited ISG transcription by sequestering interferon-stimulated gene factor 3 (ISGF3) in a manner requiring its binding of PML and STAT2, but not of HDACs. In conclusion, our results demonstrate that PML participates in type I interferon-induced ISG expression by regulating ISGF3, and that this regulation by PML is counteracted by HCMV IE1, highlighting a widely shared viral strategy targeting PML to evade intrinsic and innate defense mechanisms. - Competing Interests: The authors have declared that no competing interests exist. innate host defenses. We further show that this regulation by PML in type I interferon response is inhibited by human cytomegalovirus (HCMV) IE1 protein, which forms a complex with PML, STAT1, STAT2, and HDACs in virus-infected cells. By analyzing mutant viruses, we demonstrate that IE1 inhibits ISG transcription by sequestering interferonstimulated gene factor 3 (ISGF3) in a manner requiring its binding of PML and STAT2, but not of HDACs. Our findings reveal that PML is a regulator of ISGF3 in type I interferon response and that this PML activity is counteracted by HCMV IE1. Our study explains why PML targeting activity is widely conserved among many viruses. Type I interferons (IFNs) are multifunctional cytokines that act as key components of innate immune response to viral infection. Virus infections rapidly trigger induction of IFN and/or IFN through activating nuclear factor-kappa B (NF-B) and interferon regulatory factor 3 (IRF3) transcription factors. The binding of newly synthesized IFN and/or IFN to their receptors leads to tyrosine phosphorylation of cytoplasmic signal transducers and activators of transcription (STAT1 and STAT2) via Janus kinase 1 (Jak1). Phosphorylated STAT1 and STAT2 heterodimerize and rapidly translocate to the nucleus, where they assemble with IFN regulatory factor 9 (IRF9) to form a transcription complex known as IFN-stimulated gene factor 3 (ISGF3), which sequence-specifically binds to an IFN-stimulated response element (ISRE) present in type I IFN-stimulated genes (ISGs), many of which exhibit antiviral activity [1]. ISGF3 specifically interacts with several coactivators including histone acetyltransferases (HATs) [2, 3], histone deacetylases (HDACs) [47], and nucleosome remodeling factors [8]. Promyelocytic leukemia protein (PML), also named TRIM19, belongs to the tripartite motif family (TRIM) of proteins that contain a RING finger, two B-boxes, and an -helical coiled-coil (RBCC) domain [9, 10]. As a major component of PML nuclear bodies (NBs) (also known as nuclear domain 10) [11], PML is involved in diverse cellular processes, including proliferation, apoptosis, gene transcription, and DNA damage response [1214]. PML expression is increased by IFNs [15, 16]. Various PML isoforms are expressed via alternative splicing by sharing the same amino terminus [10, 17]. PML and other major components of PML NBs, such as Sp100, Daxx, and ATRX, exhibit antiviral activities as nuclear intrinsic restriction factors that suppress incoming viral genomes [1821]. Many viruses encode proteins that interfere with the antiviral activity of PML and most research has focused on the viral countermeasures against the antiviral activity of PML as an intrinsic restriction factor that recognizes incoming viral genomes and suppresses the initiation of viral gene expression [18, 21]. The most widely studied example is ICP0 protein of herpes simplex virus type-1 (HSV-1). ICP0 acts as a ubiquitin E3 ligase that preferentially targets the SUMO-modified PML isoforms, leading to their degradation [22]. In human cytomegalovirus (HCMV) infections, immediate-early (IE) 1 protein interacts with PML and disrupts PML NBs [2326]. This activity of IE1 correlates with the functional activities of IE1 during infection and the antiviral role of PML in HCMV replication is well established using PML-overexpressing and knockdown cells [2730]. We previously demonstrated that the central hydrophobic region of IE1 is required for the activities of IE1 to bind PML and induce PML deSUMOylation leading to PML NB disruption and to transactivate several viral and cellular promoters [28, 31]. IE1 also interacts with STAT2 and to a lesser extent with STAT1, and promotes efficient viral growth by down-regulating type I IFN signaling [3234]. The IE1-STAT2 interaction requires the near Cterminal region of IE1 including its acidic domain [3335]. Recently, the crystal structure of the central hydrophobic region of IE1, named IE1core, has been solved and the data show that IE1core shares secondary structure features with the coiled-coil domain of TRIM proteins [36]. IE1 has been shown to interact with HDAC3 [37]. Recently, a new role of PML in IFN-induced gene expression has been demonstrated. In PML-/- mouse embryonic fibroblasts and PML-depleted cells, INF-induced phosphorylation of STAT1 and its binding to the promoters of ISGs were diminished [38]. PML was also shown to enhance IFN-induced MHC class II gene expression by targeting class II transactivator (CIITA) and preventing its degradation [39]. In addition, a specif (...truncated)