Dual signaling via interferon and DNA damage response elicits entrapment by giant PML nuclear bodies.

PML nuclear bodies (PML-NBs) are dynamic interchromosomal macromolecular complexes implicated in epigenetic regulation as well as antiviral defense. During herpesvirus infection, PML-NBs induce epigenetic silencing of viral genomes, however, this defense is antagonized by viral regulatory proteins such as IE1 of human cytomegalovirus (HCMV). Here, we show that PML-NBs undergo a drastic rearrangement into highly enlarged PML cages upon infection with IE1-deficient HCMV. Importantly, our results demonstrate that dual signaling by interferon and DNA damage response is required to elicit giant PML-NBs. DNA labeling revealed that invading HCMV genomes are entrapped inside PML-NBs and remain stably associated with PML cages in a transcriptionally repressed state. Intriguingly, by correlative light and transmission electron microscopy (EM), we observed that PML cages also entrap newly assembled viral capsids demonstrating a second defense layer in cells with incomplete first-line response. Further characterization by 3D EM showed that hundreds of viral capsids are tightly packed into several layers of fibrous PML. Overall, our data indicate that giant PML-NBs arise via combined interferon and DNA damage signaling which triggers entrapment of both nucleic acids and proteinaceous components. This represents a multilayered defense strategy to act in a cytoprotective manner and to combat viral infections.

A Noncanonical Function of Polycomb Repressive Complexes Promotes Human Cytomegalovirus Lytic DNA Replication and Serves as a Novel Cellular Target for Antiviral Intervention.

Chromatin-based modifications of herpesviral genomes play a crucial role in dictating the outcome of infection. Consistent with this, host cell multiprotein complexes, such as polycomb repressive complexes (PRCs), were proposed to act as epigenetic regulators of herpesviral latency. In particular, PRC2 has recently been shown to contribute to the silencing of human cytomegalovirus (HCMV) genomes. Here, we identify a novel proviral role of PRC1 and PRC2, the two main polycomb repressive complexes, during productive HCMV infection. Western blot analyses revealed strong HCMV-mediated upregulation of RING finger protein 1B (RING1B) and B lymphoma Moloney murine leukemia virus insertion region 1 homolog (BMI1) as well as of enhancer of zeste homolog 2 (EZH2), suppressor of zeste 12 (SUZ12), and embryonic ectoderm development (EED), which constitute the core components of PRC1 and PRC2, respectively. Furthermore, we observed a relocalization of PRC components to viral replication compartments, whereas histone modifications conferred by the respective PRCs were specifically excluded from these sites. Depletion of individual PRC1/PRC2 proteins by RNA interference resulted in a significant reduction of newly synthesized viral genomes and, in consequence, a decreased release of viral particles. Furthermore, accelerated native isolation of protein on nascent DNA (aniPOND) revealed a physical association of EZH2 and BMI1 with nascent HCMV DNA, suggesting a direct contribution of PRC proteins to viral DNA replication. Strikingly, substances solely inhibiting the enzymatic activity of PRC1/2 did not exert antiviral effects, while drugs affecting the abundance of PRC core components strongly compromised HCMV genome synthesis and particle release. Taken together, our data reveal an enzymatically independent, noncanonical function of both PRC1 and PRC2 during HCMV DNA replication, which may serve as a novel cellular target for antiviral therapy.IMPORTANCE Polycomb group (PcG) proteins are primarily known as transcriptional repressors that modify chromatin and contribute to the establishment and maintenance of cell fates. Furthermore, emerging evidence indicates that overexpression of PcG proteins in various types of cancers contributes to the dysregulation of cellular proliferation. Consequently, several inhibitors targeting PcG proteins are presently undergoing preclinical and clinical evaluation. Here, we show that infection with human cytomegalovirus also induces a strong upregulation of several PcG proteins. Our data suggest that viral DNA replication depends on a noncanonical function of polycomb repressor complexes which is independent of the so-far-described enzymatic activities of individual PcG factors. Importantly, we observe that a subclass of inhibitory drugs that affect the abundance of PcG proteins strongly interferes with viral replication. This principle may serve as a novel promising target for antiviral treatment.

Seasonal Variations in the Metabolome and Bioactivity Profile of Fucus vesiculosus Extracted by an Optimised, Pressurised Liquid Extraction Protocol.

The metabolism of seaweeds depends on environmental parameters, the availability of nutrients, and biotic/abiotic stresses; therefore, their chemical composition fluctuates throughout the year. This study investigated seasonal variations in the metabolome of the Baltic Sea brown alga Fucus vesiculosus and its potential relation to the bioactivity profile. By using a definitive screening design (DSD) combined with pressurised liquid extraction (PLE), an optimised protocol was developed to extract algal biomass monthly for a full calendar year. An untargeted metabolomics approach using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MSn)-based molecular networking and manual dereplication was employed. The extracts were simultaneously screened for their in vitro antimicrobial, anticancer/apoptotic, and free radical scavenging activities. 44 compounds were putatively dereplicated in the metabolome. Many compounds were found to vary with the sampling month; phlorotannin total ion count (TIC) was highest in summer, whilst chlorophylls, lipids, and carotenoids peaked in winter and spring. The greatest radical scavenging and apoptotic activities against pancreas cancer cells observed in the summer months were attributed to high phlorotannin TIC. Methicillin-resistant Staphylococcus aureus (MRSA) inhibitory activity was produced year-round without a clear seasonal trend. This is the first study applying DSD-based optimised PLE extraction combined with a metabolome analysis of F. vesiculosus for the identification of seasonal variations in both metabolome and bioactivity.

Chromatin-Remodeling Factor SPOC1 Acts as a Cellular Restriction Factor against Human Cytomegalovirus by Repressing the Major Immediate Early Promoter.

The cellular protein SPOC1 (survival time-associated PHD [plant homeodomain] finger protein in ovarian cancer 1) acts as a regulator of chromatin structure and the DNA damage response. It binds H3K4me2/3-containing chromatin and promotes DNA condensation by recruiting corepressors such as KAP-1 and H3K9 methyltransferases. Previous studies identified SPOC1 as a restriction factor against human adenovirus (HAdV) infection that is antagonized by E1B-55K/E4-orf6-dependent proteasomal degradation. Here, we demonstrate that, in contrast to HAdV-infected cells, SPOC1 is transiently upregulated during the early phase of human cytomegalovirus (HCMV) replication. We show that the expression of immediate early protein 1 (IE1) is sufficient and necessary to induce SPOC1. Additionally, we discovered that during later stages of infection, SPOC1 is downregulated in a glycogen synthase kinase 3ß (GSK-3ß)-dependent manner. We provide evidence that SPOC1 overexpression severely impairs HCMV replication by repressing the initiation of viral immediate early (IE) gene expression. Consistently, we observed that SPOC1-depleted primary human fibroblasts displayed an augmented initiation of viral IE gene expression. This occurs in a multiplicity of infection (MOI)-dependent manner, a defining hallmark of intrinsic immunity. Interestingly, repression requires the presence of high SPOC1 levels at the start of infection, while later upregulation had no negative impact, suggesting distinct temporal roles of SPOC1 during the HCMV replicative cycle. Mechanistically, we observed a highly specific association of SPOC1 with the major immediate early promoter (MIEP), strongly suggesting that SPOC1 inhibits HCMV replication by MIEP binding and the subsequent recruitment of heterochromatin-building factors. Thus, our data add SPOC1 as a novel factor to the endowment of a host cell to restrict cytomegalovirus infections.IMPORTANCE Accumulating evidence indicates that during millennia of coevolution, host cells have developed a sophisticated compilation of cellular factors to restrict cytomegalovirus infections. Defining this equipment is important to understand cellular barriers against viral infection and to develop strategies to utilize these factors for antiviral approaches. So far, constituents of PML nuclear bodies and interferon gamma-inducible protein 16 (IFI16) were known to mediate intrinsic immunity against HCMV. In this study, we identify the chromatin modulator SPOC1 as a novel restriction factor against HCMV. We show that preexisting high SPOC1 protein levels mediate a silencing of HCMV gene expression via a specific association with an important viral cis-regulatory element, the major immediate early promoter. Since SPOC1 expression varies between cell types, this factor may play an important role in tissue-specific defense against HCMV.

Attenuation of chemokine receptor function and surface expression as an immunomodulatory strategy employed by human cytomegalovirus is linked to vGPCR US28.

BACKGROUND: Some herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled receptors that cause reprogramming of cell signaling to facilitate dissemination of the virus, prevent immune surveillance and establish life-long latency. Human GPCRs are known to function in complex signaling networks involving direct physical interactions as well as indirect crosstalk of orthogonal signaling networks. The human chemokine receptor CXCR4 is expressed on hematopoietic stem cells, leukocytes, endothelial and epithelial cells, which are infected by HCMV or display reservoirs of latency. RESULTS: We investigated the potential heteromerization of US28 with CXCR4 as well as the influence of US28 on CXCR4 signaling. Using Bioluminescence Resonance Energy Transfer and luciferase-complementation based methods we show that US28 expression exhibits negative effects on CXCR4 signaling and constitutive surface expression in HEK293T cells. Furthermore, we demonstrate that this effect is not mediated by receptor heteromerization but via signaling crosstalk. Additionally, we show that in HCMV, strain TB40E, infected HUVEC the surface expression of CXCR4 is strongly downregulated, whereas in TB40E-delUS28 infected cells, CXCR4 surface expression is not altered in particular at late time points of infection. CONCLUSIONS: We show that the vGPCR US28 is leading to severely disturbed signaling and surface expression of the chemokine receptor CXCR4 thereby representing an effective mechanism used by vGPCRs to reprogram host cell signaling. In contrast to other studies, we demonstrate that these effects are not mediated via heteromerization.

Contribution of the Major ND10 Proteins PML, hDaxx and Sp100 to the Regulation of Human Cytomegalovirus Latency and Lytic Replication in the Monocytic Cell Line THP-1.

Promyelocytic leukemia nuclear bodies, also termed nuclear domain 10 (ND10), have emerged as nuclear protein accumulations mediating an intrinsic cellular defense against viral infections via chromatin-based mechanisms, however, their contribution to the control of herpesviral latency is still controversial. In this study, we utilized the monocytic cell line THP-1 as an in vitro latency model for human cytomegalovirus infection (HCMV). Characterization of THP-1 cells by immunofluorescence andWestern blot analysis confirmed the expression of all major ND10 components. THP-1 cells with a stable, individual knockdown of PML, hDaxx or Sp100 were generated. Importantly, depletion of the major ND10 proteins did not prevent the terminal cellular differentiation of THP-1 monocytes. After construction of a recombinant, endotheliotropic human cytomegalovirus expressing IE2-EYFP, we investigated whether the depletion of ND10 proteins affects the onset of viral IE gene expression. While after infection of differentiated, THP-1-derived macrophages as well as during differentiation-induced reactivation from latency an increase in the number of IE-expressing cells was readily detectable in the absence of the major ND10 proteins, no effect was observed in non-differentiated monocytes. We conclude that PML, hDaxx and Sp100 primarily act as cellular restriction factors during lytic HCMV replication and during the dynamic process of reactivation but do not serve as key determinants for the establishment of HCMV latency.