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-β 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-β-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.

The interferon-inducible Sp100 proteins are thought to play roles in the chromatin pathway and in transcriptional regulation. Sp100A, the smallest isoform, is one of the major components of PML nuclear bodies (NBs) that exhibit intrinsic antiviral activity against several viruses. Since PML NBs are disrupted by the immediate-early 1 (IE1) protein during human cytomegalovirus (HCMV) infection, the modulation of Sp100 protein expression or activity during infection has been suggested. Here, we show that Sp100 proteins are lost largely in the late stages of HCMV infection. This event required viral gene expression and involved posttranscriptional control. The mutant virus with deletion of the sequence for IE1 (CR208) did not have Sp100 loss. In CR208 infection, PML depletion by RNA interference abrogated the accumulation of SUMO-modified Sp100A and of certain high-molecular-weight Sp100 isoforms but did not significantly affect unmodified Sp100A, suggesting that the IE1-induced disruption of PML NBs is not sufficient for the complete loss of Sp100 proteins. Sp100A loss was found to require proteasome activity. Depletion of all Sp100 proteins by RNA silencing enhanced HCMV replication and major IE (MIE) gene expression. Sp100 knockdown enhanced the acetylation level of histones associated with the MIE promoter, demonstrating that the repressive effect of Sp100 proteins may involve, at least in part, the epigenetic control of the MIE promoter. Sp100A was found to interact directly with IE1 through the N-terminal dimerization domain. These findings indicate that the IE1-dependent loss of Sp100 proteins during HCMV infection may represent an important requirement for efficient viral growth.During the early stages of human cytomegalovirus (HCMV) infection, the 72-kDa immediate-early 1 (IE1 or IE72) protein targets the subnuclear structures referred to as PML nuclear bodies (NBs) (also known as nuclear domain 10 [ND10] or PML oncogenic domains [PODs]), in which input viral genomes are deposited and IE transcription occurs (22). However, the targeting of IE1 to PML NBs is transient, and subsequently, PML NBs are disrupted in an IE1-dependent manner and both IE1 and the components of PML NBs, including PML and Sp100 proteins, are relocalized into the nucleoplasm (4, 25, 27, 53). Several lines of evidence suggest that this early event promotes viral replication. The overexpression of PML conferred resistance to HCMV infection (3), and the analysis of IE1 mutants demonstrated that the ability of IE1 to disrupt PML NBs was correlated with its transactivation activity and efficient viral growth in cells transfected with HCMVbacterial artificial chromosome (BAC) DNA (30). In addition, the depletion of PML by RNA interference has been reported to promote viral replication efficiency (47). These results support the notion that PML NBs are intrinsic defense sites at which the epigenetic silencing of input viral DNA genome may take place and that the components of PML NBs perform antiviral roles against a variety of DNA and RNA vir...

Interferon-stimulated gene 15 (ISG15) encodes an ubiquitin-like protein that covalently conjugates protein. Protein modification by ISG15 (ISGylation) is known to inhibit the replication of many viruses. However, studies on the viral targets and viral strategies to regulate ISGylation-mediated antiviral responses are limited. In this study, we show that human cytomegalovirus (HCMV) replication is inhibited by ISGylation, but the virus has evolved multiple countermeasures. HCMV-induced ISG15 expression was mitigated by IE1, a viral inhibitor of interferon signaling, however, ISGylation was still strongly upregulated during virus infection. RNA interference of UBE1L (E1), UbcH8 (E2), Herc5 (E3), and UBP43 (ISG15 protease) revealed that ISGylation inhibits HCMV growth by downregulating viral gene expression and virion release in a manner that is more prominent at low multiplicity of infection. A viral regulator pUL26 was found to interact with ISG15, UBE1L, and Herc5, and be ISGylated. ISGylation of pUL26 regulated its stability and inhibited its activities to suppress NF-κB signaling and complement the growth of UL26-null mutant virus. Moreover, pUL26 reciprocally suppressed virus-induced ISGylation independent of its own ISGylation. Consistently, ISGylation was more pronounced in infections with the UL26-deleted mutant virus, whose growth was more sensitive to IFNβ treatment than that of the wild-type virus. Therefore, pUL26 is a viral ISG15 target that also counteracts ISGylation. Our results demonstrate that ISGylation inhibits HCMV growth at multiple steps and that HCMV has evolved countermeasures to suppress ISG15 transcription and protein ISGylation, highlighting the importance of the interplay between virus and ISGylation in productive viral infection.

In human cytomegalovirus (HCMV)-infected cells, the 86 kDa immediate-early (IE) 2 protein plays a key role in transactivating downstream viral genes. Recently, IE2 has been shown to interact with histone deacetylase 1 (HDAC1) and HDAC3. HDAC1 recruited by IE2 was required for IE2-mediated autorepression of the major IE (MIE) promoter, whereas IE2-HDAC3 interaction was suggested to relieve the repressive effect of HDAC3 on viral early promoters. However, whether IE2 indeed inhibits HDAC's deacetylation activity on viral promoters and interacts with other HDACs remains unclear. Here, we provide evidence that IE2 functionally interacts with HDAC2 and negates its repressive effect on the viral polymerase promoter. IE2 interacted with HDAC2 in both virus-infected cells and in vitro, and required the conserved C-terminal half for HDAC2 binding. The subcellular localization of HDAC2 was changed in virus-infected cells, showing colocalization with IE2 in viral transcription and replication sites. The overall HDAC2 protein levels and its deacetylation activity slightly increased during the late stages of infection and the IE2-associated deacetylation activity was still sensitive to an HDAC inhibitor, trichostatin A. In transfection assays, however, histone acetylation of the viral polymerase promoter was suppressed by HDAC2, and this was relieved by IE2 binding. Therefore, our data demonstrate that IE2 functionally interacts with HDAC2 and modulates its deacetylation activity on the viral polymerase promoter. Our results also support the idea that interactions of IE2 with several HDACs to modulate the host epigenetic regulation on viral MIE and early promoters are important events in the process of productive infection.

The human cytomegalovirus (HCMV) UL112-113 region encodes four phosphoproteins with common amino termini (p34, p43, p50, and p84) via alternative splicing and is thought to be required for efficient viral DNA replication. We have previously shown that interactions among the four UL112-113 proteins regulate their intranuclear targeting and enable the recruitment of the UL44 DNA polymerase processivity factor to viral prereplication foci. Here, we show that in virus-infected cells, the UL112-113 proteins form a complex with UL44 and other replication proteins, such as UL84 and IE2. In vitro assays showed that all four phosphoproteins interacted with UL44. Interestingly, p84 required both the shared amino-terminal region and the specific near-carboxy-terminal region for UL44 binding. UL44 required both the carboxy-terminal region and the central region, including the dimerization domain for p84 binding. The production of recombinant virus from mutant Towne bacterial artificial chromosome (BAC) DNA, which encodes intact p34, p43, and p50 and a carboxy-terminally truncated p84 defective in UL44 binding, was severely impaired compared to wild-type BAC DNA. A similar defect was observed when mutant BAC DNA encoded a carboxy-terminally truncated UL44 defective in p84 binding. In cotransfection replication assays using six replication core proteins, UL84, IE2, and UL112-113, the efficient replication of an HCMV oriLyt-containing plasmid required the regions of p84 and UL44 necessary for their interaction. Our data suggest that the UL112-113 proteins form a complex with other replication proteins such as UL44, UL84, and IE2 and that the specific interaction of UL112-113 p84 with UL44 is necessary for efficient viral DNA replication.In human cytomegalovirus (HCMV), viral DNA replication requires six replication core proteins, such as DNA polymerase (UL54) and its associated polymerase processivity factor (UL44), single-stranded DNA-binding protein (UL57), and the heterotrimer consisting of DNA helicase (UL105), primase (UL70), and primase-associated factor (UL102) subunits. Their genes are initially predicted by their homology to essential herpes simplex virus type 1 (HSV-1) replication genes (8, 9) and are highly conserved among all herpesviruses. In cotransfection replication assays using oriLyt-containing plasmid DNA, five additional genetic loci (UL36-38 loci, TRS1/IRS1, IE2, UL84, and UL112-113) have also been found to contribute to effective viral DNA replication (24,25,32,33,41,42).The UL112-113 region encodes four nuclear phosphoproteins (p34, p43, p50, and p84) with common amino termini of 252 amino acids via alternative splicing (35,39,40). The UL112-113 proteins were previously shown to have DNAbinding activity (14) and enhance the IE2-mediated transactivation of the viral polymerase (UL54) promoter (13,15,19). Interestingly, the UL112-113 gene was recently found to activate the lytic cycle of Kaposi's sarcoma-associated herpesvirus (38). A more direct role of the UL112-113 proteins in viral DNA replication was ...

DNA-dependent activator of interferon regulatory factor (DAI) acts as a cytosolic B-form DNA sensor that induces type I interferons. However, DAI is not required for DNA sensing in certain cell types due to redundancy of the DNA sensing system. Here, we investigated the effect of DAI on herpes simplex virus 1 (HSV-1) infection in HepG2 hepatocellular carcinoma cells. DAI transcription was induced after gamma interferon (IFN-␥) treatment or HSV-1 infection. HSV-1 replication was enhanced by DAI knockdown, and ectopic DAI expression repressed viral replication in a manner requiring the Z␤ and D3 domains, but not the Z␣ domain. This activity of DAI was more prominent at low multiplicity of infection (MOI) and correlated with the reduced expression of viral immediate-early genes. Consistently, DAI repressed the activation of ICP0 promoter in reporter gene assays. DNA-dependent activator of interferon (IFN) regulatory factor (DAI), which is also referred to as Z-DNA binding protein 1 (ZBP1) or DLM-1, was initially identified as a highly upregulated protein in mouse tumor stromal cells and in macrophages treated by gamma IFN (IFN-␥) or lipopolysaccharide (1). Structural analyses have revealed that DAI/ZBP1/DLM-1 (referred to as DAI hereafter) contains the amino-terminal Z-form DNA-binding domains, Z␣ and Z␤, which are homologous to those of adenosine deaminase that acts on RNA (ADAR1), an RNA editing enzyme (2-5). Since Z-DNA is located near the transcription start sites of certain genes in the genome, a role of DAI in transcriptional regulation has been suggested (6, 7). Induction of DAI was also observed in mouse hepatocytes infected with hepatitis B virus (HBV) (8) and in mouse embryonic fibroblasts (MEFs) stimulated by B-form DNA (9).Recently, DAI was shown to act as a cytosolic B-form DNA sensor that initiates IFN responses via activation of the nuclear factor-B (NF-B) and interferon regulatory transcription factor 3 (IRF3) pathways in mice (10). In addition to the Z-DNA-binding domains, a region termed the D3 domain was demonstrated to primarily contribute to the recognition of B-DNA in vitro (10). However, all of the Z␣, Z␤, and D3 domains were required for efficient B-DNA binding in vivo and DAI was suggested to undergo DNA-mediated multimerization to evoke activation of IFN responses (11). The carboxyl-terminal region of DAI was responsible for recruitment of both IRF3 and TANK-binding kinase 1 (TBK1), an IB kinase that activates IRF3 (10). The mechanism by which DAI activates the NF-B pathway was shown to involve recruitment of receptor-interacting protein kinase 1 (RIP1) and RIP3 through a RIP homotypic interaction motif (RHIM)-dependent interaction with DAI (12, 13). Recently, the binding of DAI with RIP3 was shown to mediate virus-induced programmed necrosis (14).The requirement of DAI in induction of IFN response by cytosolic stimulation of B-DNA is dependent on cell type. DAI played a role in the DNA-mediated IFN production in mouse fibroblast L929 (10, 12, 15) and mouse SVEC4-10 endothelial cells (12)...

Four phosphoproteins, of 34, 43, 50, and 84 kDa, with common amino termini are synthesized via alternative splicing from the UL112-113 region of the human cytomegalovirus genome. Although genetic studies provided evidence that both the UL112 and UL113 loci in the viral genome are required for efficient viral replication, whether the four proteins play specific roles or cooperate in replication is not understood. Here we present evidence, using in vitro and in vivo coimmunoprecipitation assays, that the four UL112-113 proteins both self-interact and interact with each other. A mapping study of the 84-kDa protein showed that the N-terminal region encompassing amino acids 1 to 125, which is shared in all UL112-113 proteins and highly conserved among betaherpesviruses, is required for both self-interaction and nuclear localization as foci. Further localization studies revealed that, unlike the 43-, 50-, and 84-kDa proteins, which were distributed as nuclear punctate forms, the 34-kDa form was located predominantly in the cytoplasm. However, when all four proteins were coexpressed simultaneously, all of the UL112-113 proteins were efficiently localized to the promyelocytic leukemia oncogenic domains. We also found that the ability of the UL112-113 proteins to relocate UL44 (the viral polymerase processivity factor) to prereplication foci relied on self-interaction and reached maximal levels when the four proteins were coexpressed. Therefore, our data suggest that interactions occurring among UL112-113 proteins via their shared N-terminal regions are important to both their intranuclear targeting and the recruitment of UL44 to subnuclear sites for viral replication.

NF-E2 related factor 2 (Nrf2) is a transcription factor that plays a key role(s) in cellular defence against oxidative stress. In this study, we showed that the expression of Nrf2 was upregulated in primary human foreskin fibroblasts (HFFs), following human cytomegalovirus (HCMV/HHV-5) infection. The expression of haem oxygenase-1, a downstream target of Nrf2, was also increased by HCMV infection, and this induction was suppressed in HFFs expressing a small hairpin RNA (shRNA) against Nrf2. The HCMV-mediated increase in Nrf2 expression was abolished when UVirradiated virus was used or when the activity of casein kinase 2 was inhibited. Host cells infected by HCMV had higher survival rates following oxidative stress induced by buthionine sulfoximine compared with uninfected control cells, but this cell-protective effect was abolished by the use of Nrf2 shRNA. Our results suggest that HCMV-mediated activation of Nrf2 might be beneficial to the virus by increasing the host cell's ability to cope with oxidative stress resulting from viral infection and/or inflammation.