• cancer
• chromatin
• genome maintenance
• herpesviruses
• mitosis

• Humans
• Cytomegalovirus/genetics
• Cytomegalovirus/metabolism
• Cytomegalovirus/physiology
• Nucleosomes/metabolism
• Nucleosomes/genetics
• S Phase
• Lysine/metabolism
• Lysine/genetics
• Cytomegalovirus Infections/virology
• Cytomegalovirus Infections/metabolism
• Viral Proteins/metabolism
• Viral Proteins/genetics
• Protein Binding
• Immediate-Early Proteins/metabolism
• Immediate-Early Proteins/genetics
• Amino Acid Motifs

• R01 AI130089 NIAID NIH HHS

The human cytomegalovirus (CMV) immediate early 1 (IE1) protein has evolved as a multifunctional antagonist of intrinsic and innate immune mechanisms. In addition, this protein serves as a transactivator and potential genome maintenance protein. Recently, the crystal structures of the human and rat CMV IE1 (hIE1, rIE1) core domain were solved. Despite low sequence identity, the respective structures display a highly similar, all alpha-helical fold with distinct variations. To elucidate which activities of IE1 are either species-specific or conserved, this study aimed at a comparative analysis of hIE1 and rIE1 functions. To facilitate the quantitative evaluation of interactions between IE1 and cellular proteins, a sensitive NanoBRET assay was established. This confirmed the species-specific interaction of IE1 with the cellular restriction factor promyelocytic leukemia protein (PML) and with the DNA replication factor flap endonuclease 1 (FEN1). To characterize the respective binding surfaces, helix exchange mutants were generated by swapping hIE1 helices with the corresponding rIE1 helices. Interestingly, while all mutants were defective for PML binding, loss of FEN1 interaction was confined to the exchange of helices 1 and 2, suggesting that FEN1 binds to the stalk region of IE1. Furthermore, our data reveal that both hIE1 and rIE1 antagonize human STAT2; however, distinct regions of the respective viral proteins mediated the interaction. Finally, while PML, FEN1, and STAT2 binding were conserved between primate and rodent proteins, we detected that rIE1 lacks a chromatin tethering function suggesting that this activity is dispensable for rat CMV. In conclusion, our study revealed conserved and distinct functions of primate and rodent IE1 proteins, further supporting the concept that IE1 proteins underwent a narrow co-evolution with their respective hosts to maximize their efficacy in antagonizing innate immune mechanisms and supporting viral replication.

• FEN1
• HCMV
• IE1
• PML
• RCMV
• STAT2
• cytomegalovirus
• immediate early
• innate immunity

• Animals
• Cytomegalovirus/genetics
• Cytomegalovirus Infections
• Humans
• Immediate-Early Proteins/genetics
• Immediate-Early Proteins/metabolism
• Immunity, Innate
• Promyelocytic Leukemia Protein/genetics

• chromatin fiber
• epigenetic regulation
• linker histone
• structural dynamics
• tetranucleosome
• transcription

Tethering of viral genomes to host chromosomes has been recognized in a variety of DNA and RNA viruses. It can occur during both the productive cycle and latent infection and may impact viral genomes in manifold ways including their protection, localization, transcription, replication, integration, and segregation. Tethering is typically accomplished by dedicated viral proteins that simultaneously associate with both the viral genome and cellular chromatin via nucleic acid, histone and/or non-histone protein interactions. Some of the most prominent tethering proteins have been identified in DNA viruses establishing sustained latent infections, including members of the papillomaviruses and herpesviruses. Herpesvirus particles have linear genomes that circularize in infected cell nuclei and usually persist as extrachromosomal episomes. In several γ-herpesviruses, tethering facilitates the nuclear retention and faithful segregation of viral episomes during cell division, thus contributing to persistence of these viruses in the absence of infectious particle production. However, it has not been studied whether the genomes of human Cytomegalovirus (hCMV), the prototypical β-herpesvirus, are tethered to host chromosomes. Here we provide evidence by fluorescence in situ hybridization that hCMV genomes associate with the surface of human mitotic chromosomes following infection of both non-permissive myeloid and permissive fibroblast cells. This chromosome association occurs at lower frequency in the absence of the immediate-early 1 (IE1) proteins, which bind to histones and have been implicated in the maintenance of hCMV episomes. Our findings point to a mechanism of hCMV genome maintenance through mitosis and suggest a supporting but non-essential role of IE1 in this process.

• chromatin tethering
• fluorescence in situ hybridization
• genome maintenance
• human cytomegalovirus
• human herpesvirus
• immediate-early 1
• mitotic chromosome
• viral episome

• cancer
• chromatin
• herpes
• latency
• mitosis
• transcription

• Cell Line
• Cells, Cultured
• Chromatin/genetics
• Chromatin/metabolism
• Cytomegalovirus/genetics
• Cytomegalovirus/physiology
• Fibroblasts/virology
• Genome, Viral
• HEK293 Cells
• Host-Pathogen Interactions
• Humans
• Immediate-Early Proteins/genetics
• Mitosis/genetics

• R01 AI130089 NIAID NIH HHS
• R01 AI139180 NIAID NIH HHS
• T32 CA009135 NCI NIH HHS
• HHS | National Institutes of Health (NIH)
• HHS | NIH | NIH Office of the Director (OD)

Human cytomegalovirus (HCMV) is a prevalent viral pathogen, which can cause severe clinical consequences in neonates, immunocompromised individuals, patients with AIDS, and organ and stem cell transplant recipients. HCMV inhibits the host cell cycle progress while the immediate-early protein 1 (IE1) tethers to condensed chromatin in mitotic cells. The present study investigated the effect of HCMV on the cell cycle in human glioblastoma cells, as well as the role of RhoA GTPase during mitosis in the same context. Live cell microscopy showed that despite the apparent cell cycle arrest at late stages of mitosis in normal fibroblasts, HCMV-infected U373MG cells successfully went through all stages of cell division. HCMV IE1 protein exhibited a remarkably tight association with mitotic chromosomes from early mitosis to late cytokinesis. Depletion of RhoA significantly impaired the proliferation rate of HCMV-infected U373MG cells; consistent with this observation, the number of cells entering mitosis was also decreased. These results demonstrated the differential behavior of HCMV during mitosis in a normal and a cancer background. Furthermore, RhoA may be a critical component for the efficient cell division of HCMV-infected glioblastoma cells, which subsequently ensures the maintenance of viral genomes.

• Cell Line
• Cytomegalovirus/physiology
• Cytomegalovirus Infections/genetics
• Cytomegalovirus Infections/immunology
• Cytomegalovirus Infections/pathology
• Gene Expression Regulation, Viral/immunology
• Humans
• Immediate-Early Proteins/genetics
• Immediate-Early Proteins/immunology
• Immunity, Innate
• Mutation
• Promyelocytic Leukemia Protein/genetics
• Promyelocytic Leukemia Protein/immunology
• Sumoylation/immunology
• Virus Replication/genetics
• Virus Replication/immunology

• Wellcome Trust
• MR/P022146/1 Medical Research Council
• Tenovus
• Erasmus+

Understanding how the innate immune system keeps human cytomegalovirus (HCMV) in check has recently become a critical issue in light of the global clinical burden of HCMV infection in newborns and immunodeficient patients. Innate immunity constitutes the first line of host defense against HCMV as it involves a complex array of cooperating effectors – e.g., inflammatory cytokines, type I interferon (IFN-I), natural killer (NK) cells, professional antigen-presenting cells (APCs) and phagocytes – all capable of disrupting HCMV replication. These factors are known to trigger a highly efficient adaptive immune response, where cellular restriction factors (RFs) play a major gatekeeping role. Unlike other innate immunity components, RFs are constitutively expressed in many cell types, ready to act before pathogen exposure. Nonetheless, the existence of a positive regulatory feedback loop between RFs and IFNs is clear evidence of an intimate cooperation between intrinsic and innate immunity. In the course of virus-host coevolution, HCMV has, however, learned how to manipulate the functions of multiple cellular players of the host innate immune response to achieve latency and persistence. Thus, HCMV acts like an orchestra conductor able to piece together and rearrange parts of a musical score (i.e., innate immunity) to obtain the best live performance (i.e., viral fitness). It is therefore unquestionable that innovative therapeutic solutions able to prevent HCMV immune evasion in congenitally infected infants and immunocompromised individuals are urgently needed. Here, we provide an up-to-date review of the mechanisms regulating the interplay between HCMV and innate immunity, focusing on the various strategies of immune escape evolved by this virus to gain a fitness advantage.

• NK cells
• antigen presenting cell (APC)
• apoptosis
• human cytomegalovirus
• innate immunity
• interferon system
• restriction factors

Before a pathogen even enters a cell, intrinsic immune defenses are active. This first-line defense is mediated by a variety of constitutively expressed cell proteins collectively termed “restriction factors” (RFs), and they form a vital element of the immune response to virus infections. Over time, however, viruses have evolved in a variety ways so that they are able to overcome these RF defenses via mechanisms that are specific for each virus. This review provides a summary of the universal characteristics of RFs, and goes on to focus on the strategies employed by some of the most important RFs in their attempt to control human cytomegalovirus (HCMV) infection. This is followed by a discussion of the counter-restriction mechanisms evolved by viruses to circumvent the host cell’s intrinsic immune defenses. RFs include nuclear proteins IFN-γ inducible protein 16 (IFI16) (a Pyrin/HIN domain protein), Sp100, promyelocytic leukemia, and hDaxx; the latter three being the keys elements of nuclear domain 10 (ND10). IFI16 inhibits the synthesis of virus DNA by down-regulating UL54 transcription - a gene encoding a CMV DNA polymerase; in response, the virus antagonizes IFI16 via a process involving viral proteins UL97 and pp65 (pUL83), which results in the mislocalizing of IFI16 into the cytoplasm. In contrast, viral regulatory proteins, including pp71 and IE1, seek to modify or disrupt the ND10 proteins and thus block or reverse their inhibitory effects upon virus replication. All in all, detailed knowledge of these HCMV counter-restriction mechanisms will be fundamental for the future development of new strategies for combating HCMV infection and for identifying novel therapeutic agents.

• Human cytomegalovirus
• Intrinsic immunity
• Restriction factors
• Viral escape mechanisms
• DNA sensors

Human cytomegalovirus (hCMV) immediate early 1 (IE1) protein associates with condensed chromatin of the host cell during mitosis. We have determined the structure of the chromatin-tethering domain (CTD) of IE1 bound to the nucleosome core particle, and discovered that the specific interaction between IE1-CTD and the H2A-H2B acidic patch impairs the compaction of higher-order chromatin structure. Our results suggest that IE1 loosens up the folding of host chromatin during hCMV infections.

DOI:http://dx.doi.org/10.7554/eLife.11911.001

Most of the DNA in a cell is tightly wrapped around groups of proteins called histones, which gives the impression of beads on a string. These bead-like structures are called nucleosomes, and interactions between histones in different nucleosomes can link one nucleosome to another, to package the DNA into a very condensed form.

Viruses sometimes interact with this condensed DNA; for example, a virus called human cytomegalovirus is known to attach to condensed DNA when cells are preparing to divide. But the consequences of these interactions are not always clear.

Now, Fang, Chen et al. have worked out the three-dimensional structure of a protein from the cytomegalovirus while it is attached to a nucleosome. This structure revealed that the viral protein connects to same part of the histones that otherwise helps pull the nucleosomes together.

Further experiments then compared how the cytomegalovirus protein attaches to nucleosomes with the interaction between nucleosomes and a similar protein from a different virus. Both viral proteins were seen to interact with the same part of the histone protein, but in different ways.

Next, Fang, Chen et al. showed that the DNA is more loosely packed when the cytomegalovirus protein is attached to the nucleosomes. This was not the case for the similar protein from the other virus. The experiments show that small differences in the ways viral proteins interact with condensed DNA can change their effects on DNA packaging. Additionally, these findings may help scientists to better understand how the binding of the cytomegalovirus protein to the nucleosomes might affect this virus’s ability to infect or cause illness in humans.

DOI:http://dx.doi.org/10.7554/eLife.11911.002

• biochemistry
• biophysics
• chromatin
• crystal structure
• cytomegalovirus
• human
• nucleosome
• structural biology
• virus

• Cytomegalovirus/physiology
• DNA Topoisomerases, Type II/metabolism
• DNA, Viral/metabolism
• DNA-Binding Proteins/metabolism
• Gene Expression
• Gene Expression Profiling
• Host-Pathogen Interactions
• Humans
• Immediate-Early Proteins/genetics
• Immediate-Early Proteins/metabolism
• Protein Binding
• Sp1 Transcription Factor/metabolism
• Virus Latency
• Virus Replication

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.

For productive viral infection, virus needs to overcome successive host defenses including intrinsic defense and innate and acquired immunity. Promyelocytic leukemia protein (PML) has been shown to play an important role in intrinsic defense by acting as a nuclear restriction factor that suppresses incoming viral genomes. In this study, we demonstrate that PML also positively regulates type I interferon response by promoting transcription of interferon-stimulated genes (ISGs). Therefore, PML is a key player in both intrinsic and 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 interferon-stimulated 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.

• Cytomegalovirus/genetics
• Cytomegalovirus/metabolism
• HEK293 Cells
• Histone Deacetylase 1/genetics
• Histone Deacetylase 1/metabolism
• Histone Deacetylase 2/genetics
• Histone Deacetylase 2/metabolism
• Humans
• Immediate-Early Proteins/genetics
• Immediate-Early Proteins/metabolism
• Interferon Type I/genetics
• Interferon Type I/metabolism
• Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics
• Interferon-Stimulated Gene Factor 3, gamma Subunit/metabolism
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism
• Promyelocytic Leukemia Protein
• STAT1 Transcription Factor/genetics
• STAT1 Transcription Factor/metabolism
• STAT2 Transcription Factor/genetics
• STAT2 Transcription Factor/metabolism
• Transcription Factors/genetics
• Transcription Factors/metabolism
• Tumor Suppressor Proteins/genetics
• Tumor Suppressor Proteins/metabolism

• Cell Line
• Cytomegalovirus/metabolism
• Cytomegalovirus Infections/metabolism
• Escherichia coli/metabolism
• Humans
• Immediate-Early Proteins/metabolism
• Protein Processing, Post-Translational
• Sumoylation
• Trans-Activators/metabolism

The parameters involved in human cytomegalovirus (HCMV) latent infection in CD14 (+) and CD34 (+) cells remain poorly identified. Using next generation sequencing we deduced the transcriptome of HCMV latently infected CD14 (+) and CD34 (+) cells in experimental as well as natural latency settings. The gene expression profile from natural infection in HCMV seropositive donors closely matched experimental latency models, and included two long non-coding RNAs (lncRNAs), RNA4.9 and RNA2.7 as well as the mRNAs encoding replication factors UL84 and UL44. Chromatin immunoprecipitation assays on experimentally infected CD14 (+) monocytes followed by next generation sequencing (ChIP-Seq) were employed to demonstrate both UL84 and UL44 proteins interacted with the latent viral genome and overlapped at 5 of the 8 loci identified. RNA4.9 interacts with components of the polycomb repression complex (PRC) as well as with the MIE promoter region where the enrichment of the repressive H3K27me3 mark suggests that this lncRNA represses transcription. Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE), which identifies nucleosome-depleted viral DNA, was used to confirm that latent mRNAs were associated with actively transcribed, FAIRE analysis also showed that the terminal repeat (TR) region of the latent viral genome is depleted of nucleosomes suggesting that this region may contain an element mediating viral genome maintenance. ChIP assays show that the viral TR region interacts with factors associated with the pre replication complex and a plasmid subclone containing the HCMV TR element persisted in latently infected CD14 (+) monocytes, strongly suggesting that the TR region mediates viral chromosome maintenance.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus where infection is usually subclinical. HCMV initial infection is followed by the establishment of latency in CD34 (+) myeloid cells and CD14 (+) monocytes. Primary infection or reactivation from latency can be associated with significant morbidity and mortality can occur in immune compromised patients. Latency is marked by the persistence of the viral genome, lack of production of infectious virus and the expression of only a few previously recognized latency associated transcripts. Despite the significant interest in HCMV latent infection, little is known regarding the mechanism involved in establishment or maintenance of the viral chromosome. We have now identified the transacting factors present in latently infected CD14 (+) monocytes and CD34 (+) progenitor cells as well as identification of a region of the HCMV genome, the terminal repeat locus that mediates viral DNA maintenance. This is a major step toward understanding the mechanism of HCMV latent infection.

• Animals
• Cell Line
• Cytomegalovirus/genetics
• Genetic Complementation Test
• Herpesvirus 1, Human/genetics
• Herpesvirus 1, Human/physiology
• Humans
• Immediate-Early Proteins/deficiency
• Immediate-Early Proteins/genetics
• Immediate-Early Proteins/metabolism
• Ubiquitin-Protein Ligases/deficiency
• Viral Plaque Assay
• Viral Proteins/genetics
• Viral Proteins/metabolism
• Virus Replication

• MC_U130169966 Medical Research Council