Discovery of a Novel Antiviral Effect of the Restriction Factor SPOC1 against Human Cytomegalovirus.

The chromatin-remodeler SPOC1 (PHF13) is a transcriptional co-regulator and has been identified as a restriction factor against various viruses, including human cytomegalovirus (HCMV). For HCMV, SPOC1 was shown to block the onset of immediate-early (IE) gene expression under low multiplicities of infection (MOI). Here, we demonstrate that SPOC1-mediated restriction of IE expression is neutralized by increasing viral titers. Interestingly, our study reveals that SPOC1 exerts an additional antiviral function beyond the IE phase of HCMV replication. Expression of SPOC1 under conditions of high MOI resulted in severely impaired viral DNA replication and viral particle release, which may be attributed to inefficient viral transcription. With the use of click chemistry, the localization of viral DNA was investigated at late time points after infection. Intriguingly, we detected a co-localization of SPOC1, RNA polymerase II S5P and polycomb repressor complex 2 (PRC2) components in close proximity to viral DNA in areas that are hypothesized to harbor viral transcription sites. We further identified the N-terminal domain of SPOC1 to be responsible for interaction with EZH2, a subunit of the PRC2 complex. With this study, we report a novel and potent antiviral function of SPOC1 against HCMV that is efficient even with unrestricted IE gene expression.

IE1 of Human Cytomegalovirus Inhibits Necroptotic Cell Death via Direct and Indirect Modulation of the Necrosome Complex.

Programmed necrosis is an integral part of intrinsic immunity, serving to combat invading pathogens and restricting viral dissemination. The orchestration of necroptosis relies on a precise interplay within the necrosome complex, which consists of RIPK1, RIPK3 and MLKL. Human cytomegalovirus (HCMV) has been found to counteract the execution of necroptosis during infection. In this study, we identify the immediate-early 1 (IE1) protein as a key antagonist of necroptosis during HCMV infection. Infection data obtained in a necroptosis-sensitive cell culture system revealed a robust regulation of post-translational modifications (PTMs) of the necrosome complex as well as the importance of IE1 expression for an effective counteraction of necroptosis. Interaction analyses unveiled an association of IE1 and RIPK3, which occurs in an RHIM-domain independent manner. We propose that this interaction manipulates the PTMs of RIPK3 by promoting its ubiquitination. Furthermore, IE1 was found to exert an indirect activity by modulating the levels of MLKL via antagonizing its interferon-mediated upregulation. Overall, we claim that IE1 performs a broad modulation of innate immune signaling to impede the execution of necroptotic cell death, thereby generating a favorable environment for efficient viral replication.

Generation of UL128-shRNA transduced fibroblasts for the release of cell-free virus from clinical human cytomegalovirus isolates.

Working with recent isolates of human cytomegalovirus (HCMV) is complicated by their strictly cell-associated growth with lack of infectivity in the supernatant. Adaptation to cell-free growth is associated with disruption of the viral UL128 gene locus. The authors transduced fibroblasts with a lentiviral vector encoding UL128-specific-shRNA to allow the release of cell-free infectivity without genetic alteration. Transduced cells were cocultured with fibroblasts containing cell-associated isolates, and knockdown of the UL128 protein was validated by immunoblotting. Cell-free infectivity increased 1000-fold in isolate cocultures with UL128-shRNA compared with controls, and virions could be purified by density gradients. Transduced fibroblasts also allowed direct isolation of HCMV from a clinical specimen and cell-free transfer to other cell types. In conclusion, UL128-shRNA-transduced fibroblasts allow applications previously unsuitable for recent isolates.

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H.

The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97-cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97-cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97-cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97-cyclin interaction. High selective pressure on the formation of pUL97-cyclin complexes was identified by the use of clinically relevant mutants.

Promyelocytic Leukemia Protein Potently Restricts Human Cytomegalovirus Infection in Endothelial Cells.

PML nuclear bodies (PML-NBs) are dynamic macromolecular complexes that mediate intrinsic immunity against viruses of different families, including human cytomegalovirus (HCMV). Upon HCMV infection, PML-NBs target viral genomes entering the nucleus and restrict viral immediate-early gene expression by epigenetic silencing. Studies from several groups performed in human fibroblast cells have shown that the major PML-NB components PML, Daxx, Sp100 and ATRX contribute to this repression in a cooperative manner. Their role for HCMV restriction in endothelial cells, however, has not yet been characterized although infected endothelium is thought to play a crucial role for HCMV dissemination and development of vascular disease in vivo. Here, we use conditionally immortalized umbilical vein endothelial cells (HEC-LTT) as a cell culture model to elucidate the impact of PML-NB proteins on lytic HCMV infection. Depletion of individual PML-NB proteins by lentiviral transduction showed a particularly strong antiviral effect of PML in HEC-LTT, compared to human fibroblasts. A closer characterization of this antiviral function revealed that PML may not only effectively inhibit HCMV immediate-early gene expression but also act at later steps of the viral replication cycle. At contrast, we surprisingly noted an antiviral behavior of Daxx in complementary approaches: Depletion of Daxx resulted in decreased viral gene expression, while overexpression of Daxx promoted HCMV infection. In summary, our data demonstrate a cell type-specific effect of PML-NB components on lytic HCMV infection and suggest an important role of PML in the inhibition of HCMV dissemination through infected endothelial cells.

The chromatin remodeling protein ATRX positively regulates IRF3-dependent type I interferon production and interferon-induced gene expression.

The chromatin remodeling protein alpha thalassemia/mental retardation syndrome X-linked (ATRX) is a component of promyelocytic leukemia nuclear bodies (PML-NBs) and thereby mediates intrinsic immunity against several viruses including human cytomegalovirus (HCMV). As a consequence, viruses have evolved different mechanisms to antagonize ATRX, such as displacement from PML-NBs or degradation. Here, we show that depletion of ATRX results in an overall impaired antiviral state by decreasing transcription and subsequent secretion of type I IFNs, which is followed by reduced expression of interferon-stimulated genes (ISGs). ATRX interacts with the transcription factor interferon regulatory factor 3 (IRF3) and associates with the IFN-ß promoter to facilitate transcription. Furthermore, whole transcriptome sequencing revealed that ATRX is required for efficient IFN-induced expression of a distinct set of ISGs. Mechanistically, we found that ATRX positively modulates chromatin accessibility specifically upon IFN signaling, thereby affecting promoter regions with recognition motifs for AP-1 family transcription factors. In summary, our study uncovers a novel co-activating function of the chromatin remodeling factor ATRX in innate immunity that regulates chromatin accessibility and subsequent transcription of interferons and ISGs. Consequently, ATRX antagonization by viral proteins and ATRX mutations in tumors represent important strategies to broadly compromise both intrinsic and innate immune responses.

Cross-Species Analysis of Innate Immune Antagonism by Cytomegalovirus IE1 Protein.

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.

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.

Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers.

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.

Functional regulation of the structure-specific endonuclease FEN1 by the human cytomegalovirus protein IE1 suggests a role for the re-initiation of stalled viral replication forks.

Flap endonuclease 1 (FEN1) is a member of the family of structure-specific endonucleases implicated in regulation of DNA damage response and DNA replication. So far, knowledge on the role of FEN1 during viral infections is limited. Previous publications indicated that poxviruses encode a conserved protein that acts in a manner similar to FEN1 to stimulate homologous recombination, double-strand break (DSB) repair and full-size genome formation. Only recently, cellular FEN1 has been identified as a key component for hepatitis B virus cccDNA formation. Here, we report on a novel functional interaction between Flap endonuclease 1 (FEN1) and the human cytomegalovirus (HCMV) immediate early protein 1 (IE1). Our results provide evidence that IE1 manipulates FEN1 in an unprecedented manner: we observed that direct IE1 binding does not only enhance FEN1 protein stability but also phosphorylation at serine 187. This correlates with nucleolar exclusion of FEN1 stimulating its DSB-generating gap endonuclease activity. Depletion of FEN1 and inhibition of its enzymatic activity during HCMV infection significantly reduced nascent viral DNA synthesis demonstrating a supportive role for efficient HCMV DNA replication. Furthermore, our results indicate that FEN1 is required for the formation of DSBs during HCMV infection suggesting that IE1 acts as viral activator of FEN1 in order to re-initiate stalled replication forks. In summary, we propose a novel mechanism of viral FEN1 activation to overcome replication fork barriers at difficult-to-replicate sites in viral genomes.

Intrinsic Immune Mechanisms Restricting Human Cytomegalovirus Replication.

Cellular restriction factors (RFs) act as important constitutive innate immune barriers against viruses. In 2006, the promyelocytic leukemia protein was described as the first RF against human cytomegalovirus (HCMV) infection which is antagonized by the viral immediate early protein IE1. Since then, at least 15 additional RFs against HCMV have been identified, including the chromatin regulatory protein SPOC1, the cytidine deaminase APOBEC3A and the dNTP triphosphohydrolase SAMHD1. These RFs affect distinct steps of the viral replication cycle such as viral entry, gene expression, the synthesis of progeny DNA or egress. This review summarizes our current knowledge on intrinsic immune mechanisms restricting HCMV replication as well as on the viral strategies to counteract the inhibitory effects of RFs. Detailed knowledge on the interplay between host RFs and antagonizing viral factors will be fundamental to develop new approaches to combat HCMV infection.

Stable and Inducible Gene Knockdown in Primary Human Fibroblasts: A Versatile Tool to Study the Role of Human Cytomegalovirus Host Cell Factors.

Human fibroblasts represent the most extensively used cell type for the investigation of lytic human cytomegalovirus (HCMV) replication. However, analyzing the function of specific proteins during infection can be challenging since primary cells are difficult to transfect. An alternative approach is the use of lentiviral transduction with vectors for stable or inducible shRNA expression. This approach provides a versatile tool to study the role of host cell factors during HCMV infection. The essential steps to achieve an efficient target protein knockdown are shRNA design, cloning, generation of transgenic lentiviral particles, and, finally, transduction of the cells. However, these steps are highly dependent on the selected vector system. Here we focus on two different vector systems and describe how to successfully generate stable and inducible knockdown fibroblasts. Additionally, we demonstrate different methods to validate the knockdown of the target protein.

Vpu modulates DNA repair to suppress innate sensing and hyper-integration of HIV-1.

To avoid innate sensing and immune control, human immunodeficiency virus type 1 (HIV-1) has to prevent the accumulation of viral complementary DNA species. Here, we show that the late HIV-1 accessory protein Vpu hijacks DNA repair mechanisms to promote degradation of nuclear viral cDNA in cells that are already productively infected. Vpu achieves this by interacting with RanBP2-RanGAP1*SUMO1-Ubc9 SUMO E3-ligase complexes at the nuclear pore to reprogramme promyelocytic leukaemia protein nuclear bodies and reduce SUMOylation of Bloom syndrome protein, unleashing end degradation of viral cDNA. Concomitantly, Vpu inhibits RAD52-mediated homologous repair of viral cDNA, preventing the generation of dead-end circular forms of single copies of the long terminal repeat and permitting sustained nucleolytic attack. Our results identify Vpu as a key modulator of the DNA repair machinery. We show that Bloom syndrome protein eliminates nuclear HIV-1 cDNA and thereby suppresses immune sensing and proviral hyper-integration. Therapeutic targeting of DNA repair may facilitate the induction of antiviral immunity and suppress proviral integration replenishing latent HIV reservoirs.

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.

Centrosomal protein TRIM43 restricts herpesvirus infection by regulating nuclear lamina integrity.

Tripartite motif (TRIM) proteins mediate antiviral host defences by either directly targeting viral components or modulating innate immune responses. Here we identify a mechanism of antiviral restriction in which a TRIM E3 ligase controls viral replication by regulating the structure of host cell centrosomes and thereby nuclear lamina integrity. Through RNAi screening we identified several TRIM proteins, including TRIM43, that control the reactivation of Kaposi's sarcoma-associated herpesvirus. TRIM43 was distinguished by its ability to restrict a broad range of herpesviruses and its profound upregulation during herpesvirus infection as part of a germline-specific transcriptional program mediated by the transcription factor DUX4. TRIM43 ubiquitinates the centrosomal protein pericentrin, thereby targeting it for proteasomal degradation, which subsequently leads to alterations of the nuclear lamina that repress active viral chromatin states. Our study identifies a role of the TRIM43-pericentrin-lamin axis in intrinsic immunity, which may be targeted for therapeutic intervention against herpesviral infections.

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.

SUMOylation of IE2p86 is required for efficient autorepression of the human cytomegalovirus major immediate-early promoter.

The human cytomegalovirus (HCMV) IE2p86 protein is pivotal for coordinated regulation of viral gene expression. Besides functioning as a promiscuous transactivator, IE2p86 is also known to negatively regulate its own transcription. This occurs via direct binding of IE2p86 to a 14-bp palindromic DNA element located between the TATA box and the transcription start site of the major immediate-early promoter (MIEP), which is referred to as the cis repression signal (CRS). However, the exact mechanism of IE2p86-based autorepression is still unclear. By testing a series of IE2p86 mutants in transient expression assays, we found that not only did a DNA binding-deficient mutant of IE2p86 fail to repress the MIEP, but SUMOylation-negative mutants also failed to repress it. This finding was further supported by infection studies with primary fibroblasts harbouring a MIEP-driven transgene as a reporter. Here, we observed that a recombinant HCMV expressing SUMOylation-negative IE2p86 was defective in transgene downregulation, in contrast to wild-type HCMV. Interestingly, however, a double-mutant virus in which both the SUMO acceptor sites and the SUMO interaction motif (SIM) of IE2p86 were inactivated regained the ability to silence the MIEP. This correlated with increased expression levels of the IE2 isoforms IE2p40 and IE2p60, suggesting that these late proteins may contribute to MIEP suppression, thus compensating for the loss of IE2p86 SUMOylation. In summary, our results show that autorepression of the MIEP is not only regulated by late isoforms of IE2, but also depends on posttranslational SUMO modification, revealing a novel mechanism to fine-tune the expression of this important viral gene region.

Human Cytomegalovirus Particles Treated with Specific Antibodies Induce Intrinsic and Adaptive but Not Innate Immune Responses.

Human cytomegalovirus (HCMV) persistently infects 40% to 100% of the human population worldwide. Experimental and clinical evidence indicates that humoral immunity to HCMV plays an important role in restricting virus dissemination and protecting the infected host from disease. Specific immunoglobulin preparations from pooled plasma of adults selected for high titers of HCMV antibodies have been used for the prevention of CMV disease in transplant recipients and pregnant women. Even though incubation of HCMV particles with these preparations leads to the neutralization of viral infectivity, it is still unclear whether the antibody-treated HCMV particles (referred to here as HCMV-Ab) enter the cells and modulate antiviral immune responses. Here we demonstrate that HCMV-Ab did enter macrophages. HCMV-Ab did not initiate the expression of immediate early antigens (IEAs) in macrophages, but they induced an antiviral state and rendered the cells less susceptible to HCMV infection upon challenge. Resistance to HCMV infection seemed to be due to the activation of intrinsic restriction factors and was independent of interferons. In contrast to actively infected cells, autologous NK cells did not degranulate against HCMV-Ab-treated macrophages, suggesting that these cells may not be eliminated by innate effector cells. Interestingly, HCMV-Ab-treated macrophages stimulated the proliferation of autologous adaptive CD4+ and CD8+ T cells. Our findings not only expand the current knowledge on virus-antibody immunity but may also be relevant for future vaccination strategies.IMPORTANCE Human cytomegalovirus (HCMV), a common herpesvirus, establishes benign but persistent infections in immunocompetent hosts. However, in subjects with an immature or dysfunctional immune system, HCMV is a major cause of morbidity and mortality. Passive immunization has been used in different clinical settings with variable clinical results. Intravenous hyperimmune globulin preparations (IVIg) are obtained from pooled adult human plasma selected for high anti-CMV antibody titers. While HCMV neutralization can be shown in vitro using different systems, data are lacking regarding the cross-influence of IVIg administration on the cellular immune responses. The aim of this study was to evaluate the effects of IVIg on distinct components of the immune response against HCMV, including antigen presentation by macrophages, degranulation of innate natural killer cells, and proliferation of adaptive CD4+ and CD8+ T cells.

The Human CMV IE1 Protein: An Offender of PML Nuclear Bodies.

PML nuclear bodies (PML-NBs) are SUMOylation-dependent, highly complex protein assemblies that accumulate in the interchromosomal territories of the cell nucleus. Research of the last two decades revealed that many viruses have evolved effector proteins that modify PML-NBs. This correlates with antagonization of individual PML-NB components which act as host cell restriction factors. The multifunctional immediate-early protein IE1 of human cytomegalovirus directly interacts with the PML protein resulting in a disruption of the dot-like structure of PML-NBs. This review summarizes recent advances on the functional consequences of PML-NB modification by IE1. In particular, we describe that PML exerts a novel co-regulatory role during the interferon response which is abrogated by IE1. Via binding to PML, IE1 is able to compromise both intrinsic antiviral defense mechanisms and classical innate immune responses. These interactions of IE1 with innate host defenses are crucial for the onset of lytic replication and, consequently, may represent promising targets for antiviral strategies.

The ND10 Component Promyelocytic Leukemia Protein Acts as an E3 Ligase for SUMOylation of the Major Immediate Early Protein IE1 of Human Cytomegalovirus.

Previous studies identified the nuclear domain 10 (ND10) components promyelocytic leukemia protein (PML), hDaxx, and Sp100 as factors of an intrinsic immune response against human cytomegalovirus (HCMV). This antiviral function of ND10, however, is antagonized by viral effector proteins like IE1p72, which induces dispersal of ND10. Furthermore, we have shown that both major immediate early proteins of HCMV, IE1p72 and IE2p86, transiently colocalize with ND10 subnuclear structures and undergo modification by the covalent attachment of SUMO. Since recent reports indicate that PML acts as a SUMO E3 ligase, we asked whether the SUMOylation of IE1p72 and IE2p86 is regulated by PML. To address this, PML-depleted fibroblasts, as well as cells overexpressing individual PML isoforms, were infected with HCMV. Western blot experiments revealed a clear correlation between the degree of IE1p72 SUMO conjugation and the abundance of PML. On the other hand, the SUMOylation of IE2p86 was not affected by PML. By performing in vitro SUMOylation assays, we were able to provide direct evidence that IE1p72 is a substrate for PML-mediated SUMOylation. Interestingly, disruption of the RING finger domain of PML, which is proposed to confer SUMO E3 ligase activity, abolished PML-induced SUMOylation of IE1p72. In contrast, IE1p72 was still efficiently SUMO modified by a SUMOylation-defective PML mutant, indicating that intact ND10 bodies are not necessary for this effect. Thus, this is the first report that the E3 ligase PML is capable of stimulating the SUMOylation of a viral protein which is supposed to serve as a cellular mechanism to compromise specific functions of IE1p72.IMPORTANCE The major immediate early proteins of human cytomegalovirus, termed IE1p72 and IE2p86, have previously been shown to undergo posttranslational modification by covalent coupling to SUMO moieties at specific lysine residues. However, the enzymatic activities that are responsible for this modification have not been identified. Here, we demonstrate that the PML protein, which mediates an intrinsic immune response against HCMV, specifically serves as an E3 ligase for SUMO modification of IE1p72. Since SUMO modification of IE1p72 has previously been shown to interfere with STAT factor binding, thus compromising the interferon-antagonistic function of this viral effector protein, our finding highlights an additional mechanism through which PML is able to restrict viral infections.