Herpes simplex virus 1 (HSV-1) utilizes cellular RNA polymerase II (Pol) to transcribe its genes in one of two phases. In the latent phase, viral transcription is highly restricted, but during the productive lytic phase, more than 80 genes are expressed in a temporally coordinated cascade. In this study, we used Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to characterize early viral transcriptional events using HSV-1 immediate early (IE) gene mutants, corresponding genetically repaired viruses, and wild-type virus. Unexpectedly, in the absence of the IE genes ICP4, ICP22, and ICP0 at 1.5 hours postinfection (hpi), we observed high levels of aberrant transcriptional activity across the mutant viral genomes but substantially less on either wild-type or the congenic repaired virus genomes. This feature was particularly prominent in the absence of ICP4 expression. Cycloheximide treatment during infection with both the ICP4 and ICP22 mutants and their respective genetic repairs did not alter the relative distribution of Pol activity, but it increased overall activity across both viral genomes, indicating that both virion components and at least some de novo protein synthesis were required for full repression. Overall, these data reveal that prior to their role in transcriptional activation, IE gene products and virion components first repress transcription and that the HSV-1 lytic transcriptional cascade is mediated through subsequent derepression steps. IMPORTANCE HSV-1 transcription during productive replication is believed to comprise a series of activation steps leading to a specific sequence of gene expression. Here, we show that virion components and IE gene products ICP0, ICP4, and ICP22 first repress viral gene transcription to various degrees before subsequently activating specific gene subsets. It follows that the entire HSV transcriptional program involves a series of steps to sequentially reverse this repression. This previously uncharacterized repressive activity of IE genes very early in infection may represent an important checkpoint allowing HSV-1 to orchestrate either the robust lytic transcriptional cascade or the more restricted transcriptional program during latency.

Initiation of and progression through chondrogenesis is driven by changes in the cellular microenvironment. At the onset of chondrogenesis, resting mesenchymal stem cells are mobilized in vivo and a complex, step-wise chondrogenic differentiation program is initiated. Differentiation requires coordinated transcriptomic reprogramming and increased progenitor proliferation; both processes require chromatin remodeling. The nature of early molecular responses that relay differentiation signals to chromatin is poorly understood. We here show that immediate early genes are rapidly and transiently induced in response to differentiation stimuli in vitro. Functional ablation of the immediate early factor EGR1 severely deregulates expression of key chondrogenic control genes at the onset of differentiation. In addition, differentiating cells accumulate DNA damage, activate a DNA damage response and undergo a cell cycle arrest and prevent differentiation associated hyper-proliferation. Failed differentiation in the absence of EGR1 affects global acetylation and terminates in overall histone hypermethylation. We report novel molecular connections between EGR1 and Polycomb Group function: Polycomb associated histone H3 lysine27 trimethylation (H3K27me3) blocks chromatin access of EGR1. In addition, EGR1 ablation results in abnormal Ezh2 and Bmi1 expression. Consistent with this functional interaction, we identify a number of co-regulated targets genes in a chondrogenic gene network. We here describe an important role for EGR1 in early chondrogenic epigenetic programming to accommodate early gene-environment interactions in chondrogenesis.

Restriction factors are potent antiviral proteins that constitute a first line of intracellular defense by blocking viral replication and spread. During co-evolution, however, viruses have developed antagonistic proteins to modulate or degrade the restriction factors of their host. To ensure the success of lytic replication, the herpesvirus human cytomegalovirus (HCMV) expresses the immediate-early protein IE1, which acts as an antagonist of antiviral, subnuclear structures termed PML nuclear bodies (PML-NBs). IE1 interacts directly with PML, the key protein of PML-NBs, through its core domain and disrupts the dot-like multiprotein complexes thereby abrogating the antiviral effects. Here we present the crystal structures of the human and rat cytomegalovirus core domain (IE1CORE). We found that IE1CORE domains, also including the previously characterized IE1CORE of rhesus CMV, form a distinct class of proteins that are characterized by a highly similar and unique tertiary fold and quaternary assembly. This contrasts to a marked amino acid sequence diversity suggesting that strong positive selection evolved a conserved fold, while immune selection pressure may have fostered sequence divergence of IE1. At the same time, we detected specific differences in the helix arrangements of primate versus rodent IE1CORE structures. Functional characterization revealed a conserved mechanism of PML-NB disruption, however, primate and rodent IE1 proteins were only effective in cells of the natural host species but not during cross-species infection. Remarkably, we observed that expression of HCMV IE1 allows rat cytomegalovirus replication in human cells. We conclude that cytomegaloviruses have evolved a distinct protein tertiary structure of IE1 to effectively bind and inactivate an important cellular restriction factor. Furthermore, our data show that the IE1 fold has been adapted to maximize the efficacy of PML targeting in a species-specific manner and support the concept that the PML-NBs-based intrinsic defense constitutes a barrier to cross-species transmission of HCMV.

Human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is the major transactivator for viral gene expression and is required for lytic replication. In addition to transcriptional activation, IE2 is known to mediate transcriptional repression of promoters, including the major immediate-early (MIE) promoter and a bidirectional promoter within the lytic origin of replication (oriLyt). The activity of IE2 is modulated by another viral protein, UL84. UL84 is multifunctional and is proposed to act as the origin-binding protein (OBP) during lytic replication. UL84 specifically interacts with IE2 to relieve IE2-mediated repression at the MIE and oriLyt promoters. Originally, UL84 was thought to be indispensable for viral replication, but recent work demonstrated that some strains of HCMV (TB40E and TR) can replicate independently of UL84. This peculiarity is due to a single amino acid change of IE2 (UL122 H388D). Here, we identified that a UL84-dependent (AD169) Δ84 viral mutant had distinct IE2 localization and was unable to synthesize DNA. We also demonstrated that a TB40E Δ84 IE2 D388H mutant containing the reversed IE2 amino acid switch adopted the phenotype of AD169 Δ84. Further functional experiments, including chromatin-immunoprecipitation sequencing (ChIP-seq), suggest distinct protein interactions and transactivation function at oriLyt between strains. Together, these data further highlight the complexity of initiation of HCMV viral DNA replication. IMPORTANCE Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in immunocompromised individuals and is also the leading viral cause of congenital birth defects. After initial infection, HCMV establishes a lifelong latent infection with periodic reactivation and lytic replication. During lytic DNA synthesis, IE2 and UL84 have been regarded as essential factors required for initiation of viral DNA replication. However, previous reports identified that some isolates of HCMV can replicate in a UL84-independent manner due to a single amino acid change in IE2 (H388D). These UL84-independent strains are an important consideration, as they may have implications for HCMV disease and research. This has prompted renewed interest into the functional roles of IE2 and UL84. The work presented here focuses on the described functions of UL84 and ascertains if those required functions are fulfilled by IE2 in UL84-independent strains.

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and can trigger devastating disease in immune-suppressed patients. Cytotoxic lymphocytes (CD8+ T cells and NK cells) control HCMV infection by releasing interferon-γ and five granzymes (GrA, GrB, GrH, GrK, GrM), which are believed to kill infected host cells through cleavage of intracellular death substrates. However, it has recently been demonstrated that the in vivo killing capacity of cytotoxic T cells is limited and multiple T cell hits are required to kill a single virus-infected cell. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we demonstrate that (primary) cytotoxic lymphocytes can block HCMV dissemination independent of host cell death, and interferon-α/β/γ. Prior to killing, cytotoxic lymphocytes induce the degradation of viral immediate-early (IE) proteins IE1 and IE2 in HCMV-infected cells. Intriguingly, both IE1 and/or IE2 are directly proteolyzed by all human granzymes, with GrB and GrM being most efficient. GrB and GrM cleave IE1 after Asp398 and Leu414, respectively, likely resulting in IE1 aberrant cellular localization, IE1 instability, and functional impairment of IE1 to interfere with the JAK-STAT signaling pathway. Furthermore, GrB and GrM cleave IE2 after Asp184 and Leu173, respectively, resulting in IE2 aberrant cellular localization and functional abolishment of IE2 to transactivate the HCMV UL112 early promoter. Taken together, our data indicate that cytotoxic lymphocytes can also employ noncytotoxic ways to control HCMV infection, which may be explained by granzyme-mediated targeting of indispensable viral proteins during lytic infection.

Interferons (IFNs) are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad) replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC) and TERT-immortalized normal human diploid fibroblasts (HDF-TERT). IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib), a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection.

Murine cytomegalovirus (MCMV) immediate-early protein 3 (IE3) is essential for successful viral infection. This study developed MCMVs with an EGFP-fused IE3 gene in order to study IE3 gene expression, subnuclear distribution and biological function, as well as to examine the interaction of IE3 with cellular and viral proteins. The generated viruses included MCMVIE3gfp, in which IE1 was completely removed by the in-frame fusion of exons 3 and 5 and the C terminus of IE3 was tagged with EGFP, and MCMVIE1/3gfp, in which IE1 was kept intact and EGFP was also fused to the C terminus of IE3. Unlike human CMV (HCMV), whose growth was significantly reduced when IE2 (the HCMV homologue of IE3 in MCMV) was tagged with EGFP, MCMVs with IE3-EGFP presented an unchanged replication profile. Using these new constructs, the distribution of IE3 was revealed as well as its interaction with viral and cellular proteins, especially proteins pertaining to DNA replication (M44 and E1) and cellular intrinsic defence [promyelocytic leukemia protein and histone deacetylases (HDACs)]. It was also shown that IE3 domains co-localize with DNA replication domains, and IE3 attracted other required proteins into IE3 domains via protein-protein interactions. In addition, IE3 was shown to interact with HDAC2 and to eliminate the inhibitory effect of HDAC2 on early viral gene production. Together, these results suggest that IE3 acts as a key protein for viral DNA replication by establishing pre-replication domains via recruitment of the required viral and cellular proteins, and by reducing host defences.

The ability to execute different responses in an expedient temporal order is central for efficient goal-directed actions and often referred to as multi-component behaviour. However, the underlying neural mechanisms on a cellular level remain unclear. Here we establish a link between neural activity at the cellular level within functional neuroanatomical structures to this form of goal-directed behaviour by analyzing immediate early gene (IEG) expression in an animal model, the pigeon (Columba livia). We focus on the group of zif268 IEGs and ZENK in particular. We show that when birds have to cascade separate task goals, ZENK expression is increased in the avian equivalent of the mammalian prefrontal cortex, i.e. the nidopallium caudolaterale (NCL) as well as in the homologous striatum. Moreover, in the NCL as well as in the medial striatum (MSt), the degree of ZENK expression was highly correlated with the efficiency of multi-component behaviour. The results provide the first link between cellular IEG expression and behavioural outcome in multitasking situations. Moreover, the data suggest that the function of the fronto-striatal circuitry is comparable across species indicating that there is limited flexibility in the implementation of complex cognition such as multi-component behaviour within functional neuroanatomical structures.

The development of an induced transcript environment was investigated at the supramolecular level through comparative localization of the human cytomegalovirus immediate early (IE) transcripts and specific nuclear domains shortly after infection. Compact aggregates of IE transcripts form only adjacent to nuclear domain 10 (ND10), and the viral protein IE86 accumulates exclusively juxtaposed to the subpopulation of ND10 with transcripts. The stream of transcripts is funneled from ND10 into the spliceosome assembly factor SC35 domain through the accumulation of IE86 protein, which recruits some components of the basal transcription machinery. Concomitantly the IE72 protein binds to ND10 and later disperses them. The domain containing the zinc finger region of IE72 is essential for this dispersal. Positional analysis of proteins IE86 and IE72, IE transcripts, ND10, the spliceosome assembly factor SC35, and basal transcription factors defines spatially and temporally an immediate transcript environment, the basic components of which exist in the cell before viral infection, providing the structural environment for the virus to usurp.

Long-term recognition memory requires protein synthesis, but little is known about the coordinate regulation of specific genes. Here, we examined expression of the plasticity-associated immediate early genes (Arc, Zif268, and Narp) in the dentate gyrus following long-term object-place recognition learning in rats. RT-PCR analysis from dentate gyrus tissue collected shortly after training did not reveal learning-specific changes in Arc mRNA expression. In situ hybridization and immunohistochemistry were therefore used to assess possible sparse effects on gene expression. Learning about objects increased the density of granule cells expressing Arc, and to a lesser extent Narp, specifically in the dorsal blade of the dentate gyrus, while Zif268 expression was elevated across both blades. Thus, object-place recognition triggers rapid, blade-specific upregulation of plasticity-associated immediate early genes. Furthermore, Western blot analysis of dentate gyrus homogenates demonstrated concomitant upregulation of three postsynaptic density proteins (Arc, PSD-95, and alpha-CaMKII) with key roles in long-term synaptic plasticity and long-term memory.

Post-translational modification of proteins by members of the small ubiquitin-like modifier (SUMO) is involved in diverse cellular functions. Many viral proteins are SUMO targets and also interact with the cellular SUMOylation system. During human cytomegalovirus (HCMV) infection, the immediate-early (IE) proteins IE1 and IE2 are covalently modified by SUMO. IE2 SUMOylation promotes its transactivation activity, whereas the role of IE1 SUMOylation is not clear. We performed in silico, genome-wide analysis to identify possible SUMOylation sites in HCMV-encoded proteins and evaluated their modification using the E. coli SUMOylation system and in vitro assays. We found that only IE1 and IE2 are substantially modified by SUMO in E. coli, although US34A was also identified as a possible SUMO target in vitro. We also found that SUMOylation of IE1 and IE2 is temporally regulated during viral infection. Levels of SUMO-modified form of IE1 were increased during the early phase of infection, but decreased in the late phase when IE2 and its SUMO-modified forms were expressed at high levels. IE2 expression inhibited IE1 SUMOylation in cotransfection assays. As in IE2 SUMOylation, PIAS1, a SUMO E3 ligase, interacted with IE1 and enhanced IE1 SUMOylation. In in vitro assays, an IE2 fragment that lacked covalent and non-covalent SUMO attachment sites, but was sufficient for PIAS1 binding, effectively inhibited PIAS1-mediated SUMOylation of IE1, indicating that IE2 expression negatively regulates IE1 SUMOylation. We also found that the IE2-mediated downregulation of IE1 SUMOylation correlates with the IE1 activity to repress the promoter containing the interferon stimulated response elements. Taken together, our data demonstrate that IE1 and IE2 are the main viral SUMO targets in HCMV infection and that temporal regulation of their SUMOylation may be important in the progression of this infection.

Cyprinid herpesvirus 2 (CyHV-2), which infects goldfish and crucian carp causing high mortality, is an emerging viral pathogen worldwide. The genome of CyHV-2 is large and comprises double-stranded DNA, including several genes similar to cyprinid herpesvirus 1, ictalurid herpesvirus-1, cyprinid herpesvirus 3, and ranid herpesvirus-1. Genes of DNA viruses are expressed in three temporal phases: immediate-early (IE), early (E), and late (L) genes. Viral IE genes initiate transcription as soon as the virus enters the host, without viral DNA replication. IE gene products enable the efficient expression of E and L genes or regulate the host to initiate virus replication. In the present study, five IE genes of CyHV-2 were identified, including open reading frame (ORF)54, ORF121, ORF141, ORF147, and ORF155. Time course analysis and reverse transcription polymerase chain reaction confirmed five IE genes, thirty-four E genes, and thirty-nine L genes. In addition, all 150 ORFs identified in the CyHV-2 genome are transcribed, and are expressed in chronological order, similar to other herpesviruses. This study is the first to identify the IE genes of CyHV-2, which will provide more information for viral molecular characterization.

Lysine demethylase KDM7A removes histone modifications H3K9me1/2 and H3K27me1/2. KDM7A plays critical roles in gene expression and contribute to biological processes including tumorigenesis, metabolism, and embryonic development. However, the functions of KDM7A in mammalian nervous system are still poorly explored. In this study, functional roles of KDM7A are comprehensively investigated in neuronal cells by applying CUT&Tag-seq, RNA-seq and mice models. Knockdown of Kdm7a in N2A cells result in the alteration of histone modifications near transcription start sites (TSSs) and the expression changes of a large number of genes. In particular, the expression of immediate early genes (IEGs), a series of genes maintaining the function of the nervous system and associating with neurological disorders, are significantly decreased upon Kdm7a knockdown. Furthermore, in vivo knockdown of Kdm7a in dentate gyrus (DG) neuron of mice hippocampus, via Adeno-associated virus (AAV)-based stereotaxic microinjection, led to a significant decrease of the expression of c-Fos, a marker of neuron activity. Behavior assays in mice further revealed that Kdm7a knockdown in hippocampus repress neuron activity, which leading to impairment of emotion and memory. Collectively, the study reveals that KDM7A affects neuron functions by regulating IEGs, which may provide new clues for understanding epigenetic mechanisms in neurological disorders.

During viral infection, viral immediate-early (IE) genes encode regulatory proteins critical for the viral life cycle. Here we screened white spot syndrome virus (WSSV) IE genes with cycloheximide (CHX)-treated primary culture of crayfish hemocyte and a WSSV genome tiling microarray. Sixteen ORFs, including a known WSSV IE gene (ie1/wsv069), were identified and confirmed by RT-PCR and time course studies. The 16 identified IE proteins contain four proteins (wsv051, wsv069, wsv100, wsv079) with transcription activity, one (wsv083) with Ser/Thr kinase domain and one (wsv249) previously described to function as an ubiquitin E3 ligase. Furthermore, most of the identified WSSV IE genes cluster in a 14 kb genomic region (WSSV China isolate: 36,052 to 50,300 bp). This type of arrangement may facilitate the coordinate control and rapid expression of IE genes.

Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (RNA Pol II). While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22) function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16) was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq). The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq), we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production.IMPORTANCE HSV-1 interacts with many cellular proteins throughout productive infection. Here, we demonstrate the interaction of a viral protein, ICP22, with a subset of cellular proteins known to be involved in transcription elongation. We determined that ICP22 is required to recruit the FACT complex and other transcription elongation factors to viral genomes and that in the absence of ICP22 viral transcription is globally reduced late in productive infection, due to an elongation defect. This insight defines a fundamental role of ICP22 in HSV-1 infection and elucidates the involvement of cellular factors in HSV-1 transcription.

Cataracts are treated by lens fiber cell removal followed by intraocular lens (IOL) implantation into the lens capsule. While effective, this procedure leaves behind numerous lens epithelial cells (LECs) which undergo a wound healing response that frequently leads to posterior capsular opacification (PCO). In order to elucidate the acute response of LECs to lens fiber cell removal which models cataract surgery (post cataract surgery, PCS), RNA-seq was conducted on LECs derived from wild type mice at 0 and 6 h PCS. This analysis found that LECs upregulate the expression of numerous proinflammatory cytokines and profibrotic regulators by 6 h PCS suggesting rapid priming of pathways leading to inflammation and fibrosis PCS. LECs also highly upregulate the expression of numerous immediate early transcription factors (IETFs) by 6 h PCS and immunolocalization found elevated levels of these proteins by 3 h PCS, and this was preceded by the phosphorylation of ERK1/2 in injured LECs. Egr1 and FosB were among the highest expressed of these factors and qRT-PCR revealed that they also upregulate in explanted mouse lens epithelia suggesting potential roles in the LEC injury response. Analysis of lenses lacking either Egr1 or FosB revealed that both genes may regulate a portion of the acute LEC injury response, although neither gene was essential for expression of either proinflammatory or fibrotic markers at later times PCS suggesting that IETFs may work in concert to mediate the LEC injury response following cataract surgery.

Visualizing neuronal activation on a brain-wide scale yet with cellular resolution is a fundamental technical challenge for neuroscience. This would enable analyzing how different neuronal circuits are disrupted in pathology and how they could be rescued by pharmacological treatments. Although this goal would have appeared visionary a decade ago, recent technological advances make it eventually feasible. Here, we review the latest developments in the fields of genetics, sample preparation, imaging, and image analysis that could be combined to afford whole-brain cell-resolution activation mapping. We show how the different biochemical and optical methods have been coupled to study neuronal circuits at different spatial and temporal scales, and with cell-type specificity. The inventory of techniques presented here could be useful to find the tools best suited for a specific experiment. We envision that in the next years, mapping of neuronal activation could become routine in many laboratories, allowing dissecting the neuronal counterpart of behavior.

Lentivirus-based vectors derived from human immunodeficiency viruses type 1 and 2 (HIV-1 and 2) are widely used tools in research and may also be utilized in clinical settings. Like their parental virions, they are known to depend on the cellular machinery for successful gene delivery and integration. While most of the studies on cellular proteomic and transcriptomic changes have focused on the late phase of the transduction, studies of those changes in early time-points, especially in the case of HIV-2 based vectors, are widely lacking. Using second generation HIV-1 and 2 vesicular stomatitis virus G protein (VSV-G) pseudotyped lentiviral vectors, we transduced HEK-293T human embryonic kidney cells and carried out transcriptomic profiling at 0 and 2 h time points, with accompanying proteomic analysis at 2 h following transduction. Significant variations were observed in gene expression profile between HIV-1 and HIV-2 transduced samples. Thrombospondin 1 (THBS1), collagens (COL1A2, COL3A1), and eukaryotic translation factors (EIF3CL) in addition to various genes coding for long non-coding RNA (lncRNA) were significantly upregulated 2 h after HIV-2 transduction compared to HIV-1. Label-free quantification mass spectrometry (MS) indicated that seven proteins involved in RNA binding, mRNA transport, and chaperoning were significantly downregulated. The identification of cellular protein targets of lentiviral vectors and their effect on the cellular transcriptome will undoubtedly shed more light on their complex life cycle and may be utilized against infection by their parental lentiviruses. Furthermore, characterizing the early phase of HIV-2 infection may aid in the understanding of its pathomechanism and long incubation period.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that infects most of the population. The complex 236 kbp genome encodes more than 170 open reading frames, whose expression is temporally regulated by both viral transcriptional regulators and cellular factors that control chromatin and transcription. Here, we have used state of the art genomic technologies to investigate the viral transcriptome in conjunction with 2 key transcriptional regulators: Pol II and H3K27Ac. Although it is well known that the major immediate early (IE) proteins activate early gene expression through both direct and indirect interactions, and that histone modifications play an important role in regulating viral gene expression, the role of the IE proteins in modulating viral chromatin is not fully understood. To address this question, we have used a virus engineered for conditional expression of the IE proteins combined with RNA and Chromatin immunoprecipitation (ChIP) analyses to assess the role of these proteins in modulating both viral chromatin and gene expression. Our results show that (i) there is an enhancer-like element in OriLyt that is extraordinarily enriched in H3K27Ac; (ii) in addition to activation of viral gene expression, the IE proteins play a critical role in recruitment of Pol II and H3K27Ac to this element. IMPORTANCE HCMV is an important human pathogen associated with complications in transplant patients and birth defects. The complex program of viral gene expression is regulated by both viral proteins and host factors. Here, we have investigated the role of the immediate early proteins in regulating the viral epigenome. Our results show that the viral immediate early proteins bring about an enormous enrichment of H3K27Ac marks at the OriLyt RNA4.9 promoter, concomitant with an increase in RNA4.9 expression. This epigenetic characteristic adds importantly to the view that OriLyt has structural and functional characteristics of a strong enhancer that, we now discover, is regulated by IE proteins.

Promyelocytic leukemia (PML) bodies are nuclear organelles implicated in intrinsic and innate antiviral defense. The eponymous PML proteins, central to the self-organization of PML bodies, and other restriction factors found in these organelles are common targets of viral antagonism. The 72-kDa immediate-early protein 1 (IE1) is the principal antagonist of PML bodies encoded by the human cytomegalovirus (hCMV). IE1 is believed to disrupt PML bodies by inhibiting PML SUMOylation, while PML was proposed to act as an E3 ligase for IE1 SUMOylation. PML targeting by IE1 is considered to be crucial for hCMV replication at low multiplicities of infection, in part via counteracting antiviral gene induction linked to the cellular interferon (IFN) response. However, current concepts of IE1-PML interaction are largely derived from mutant IE1 proteins known or predicted to be metabolically unstable and globally misfolded. We performed systematic clustered charge-to-alanine scanning mutagenesis and identified a stable IE1 mutant protein (IE1cc172-176) with wild-type characteristics except for neither interacting with PML proteins nor inhibiting PML SUMOylation. Consequently, IE1cc172-176 does not associate with PML bodies and is selectively impaired for disrupting these organelles. Surprisingly, functional analysis of IE1cc172-176 revealed that the protein is hypermodified by mixed SUMO chains and that IE1 SUMOylation depends on nucleosome rather than PML binding. Furthermore, a mutant hCMV expressing IE1cc172-176 was only slightly attenuated compared to an IE1-null virus even at low multiplicities of infection. Finally, hCMV-induced expression of cytokine and IFN-stimulated genes turned out to be reduced rather than increased in the presence of IE1cc172-176 relative to wild-type IE1. Our findings challenge present views on the relationship of IE1 with PML and the role of PML in hCMV replication. This study also provides initial evidence for the idea that disruption of PML bodies upon viral infection is linked to activation rather than inhibition of innate immunity.