Research Group of T. Stamminger (since 01/2018: Institute of Virology, Ulm University)

Virology

Head of Institute:
Prof. Dr. med. Klaus Überla

Human Cytomegalovirus Gene Regulation

Human cytomegalovirus (HCMV) remains an important cause of mortality and morbidity in immuno-compromised patients such as transplant recipients on immunosuppressive therapy. Furthermore, HCMV is the most common cause of intrauterine infection which can lead to sensorineural hearing loss and mental retardation. Although progress in the diagnosis of HCMV infections has led to improved therapeutic strategies, treatment of HCMV induced disease suffers from the toxicity of the currently available antiviral drugs. In addition, in patients where longer therapy is necessary the emergence of drug-resistant viral strains is frequent. Therefore, the development of novel antiviral drugs is urgently required in order to improve the therapy of HCMV infections. Thus, one important aim of our laboratory is the characterization of molecular events that can be used as targets for new antiviral strategies.

PML nuclear bodies: a cellular structure that mediates intrinsic immunity against viruses

Microbial infections are not only controlled by innate and adaptive immune mechanisms but also by cellular restriction factors which give cells the capacity to resist pathogens. Unlike the innate and adaptive part of the immune system, that require pathogen-induced signaling cascades in order to be switched on, these so-called intrinsic immune mechanisms are mediated by cellular proteins that are constitutively expressed and active before a pathogen enters the cell, thus serving as a front-line defense. Our laboratory investigates whether a subnuclear structure, termed PML nuclear bodies (PML-NBs), contributes to the resistance of cells against herpesvirus infections. PML-NBs are dot-like structures of the cell nucleus, that are defined by the distinct accumulation of specific cellular proteins like PML, hDaxx, Sp100 and ATRX (Figure 1). During the last ten years our laboratory could show, that these proteins act as cellular restriction factors by inducing a silencing of viral gene expression. Moreover, our research revealed that specific viral proteins (pp71 and IE1 of HCMV) are able to antagonize this cellular silencing mechanism (Figure 1). This establishes a delicate balance between cellular defense and viral antagonism which determines whether the herpesvirus enters the productive cycle or viral gene expression is silenced.

Figure 1: PML-NBs: a cellular structure that mediates intrinsic immunity against human cytomegalovirus. Viral effector proteins that are known to antagonize PML-NBs are shown in the left part of the figure.

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Molecular mechanism of PML-NB disruption

During the last two years we could delineate the molecular mechanism how IE1 antagonizes PML-NBs. As evident from the crystal structure of IE1 that we solved in collaboration with Prof. Y. Muller and Prof. H. Sticht, this protein directly interacts with the PML coiled-coil domain via its globular core region and disrupts NB foci by inducing a loss of PML SUMOylation. We were able to demonstrate that IE1 acts via abrogating the de novo SUMOylation of PML. In order to overcome reversible SUMOylation dynamics, we made use of a cell-based assay that combines inducible IE1 expression with a SUMO mutant resistant to SUMO proteases. Interestingly, we observed that IE1 expression did not affect preSUMOylated PML, however, it clearly prevented de novo SUMO conjugation. Consistent results were obtained by in vitro SUMOylation assays demonstrating that IE1 alone is sufficient for this effect. Furthermore, IE1 acts in a selective manner since K160 was identified as the main target lysine. This is strengthened by the fact that IE1 also prevents As2O3-mediated hyper-SUMOylation of K160 thereby blocking PML degradation. Since IE1 did not interfere with coiled-coil mediated PML dimerization we propose that IE1 either affects PML autoSUMOylation by directly abrogating PML E3 ligase function or by preventing the access to SUMO sites. Thus, our data suggest a novel mechanism how a viral protein counteracts a cellular restriction factor by selectively preventing the de novo SUMOylation at specific lysine residues without affecting global protein SUMOylation.

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SPOC1 acts as a novel cellular restriction factor against HCMV

SPOC1 (survival time-associated PHD finger protein in ovarian cancer 1) functions as a regulator of chromatin structure and DNA damage response. It is able to bind H3K4me2/3 containing chromatin and promotes DNA condensation by recruiting H3K9 methyltransferases, such as SETDB1. SPOC1 was demonstrated to act as a restriction factor for Adenovirus (AdV) infection by repressing viral gene expression at the transcriptional level. To antagonize this repressive function, AdV targets SPOC1 for degradation immediately upon infection. Our results show that, in contrast to Ad-infected cells, SPOC1 is transiently upregulated during the early phase of HCMV infection. Furthermore, we demonstrate that SPOC1 overexpression severely impairs HCMV replication by repressing the initiation of viral immediate early (IE) gene expression. In accordance, a depletion of SPOC1 leads to an enhancement of IE gene expression. Since nuclear domain 10 (ND10) structures are crucial regulators of the onset of lytic infection, we investigated a possible inter-regulation of SPOC1 and ND10. Therefore, we compared the replication of wild-type HCMV and recombinant viruses lacking ND10 antagonistic proteins in primary human fibroblasts (HFF) with a stable SPOC1 knockout. While multistep growth curve analyses with wild-type HCMV strains displayed only a mild increase in replication efficiency, mutant viruses deprived of either immediate-early protein 1 (IE1) or pp71 replicated with clearly pronounced efficiency in the absence of endogenous SPOC1. We next analyzed the impact of SPOC1 depletion in combination with a knockdown of individual ND10 components on the onset of viral IE gene expression. Interestingly, the restrictive effect of SPOC1 was decreased in ND10 knockdown HFFs, indicating a cross-talk of different cellular factors in the intrinsic defense against HCMV. Since SPOC1 was shown to modulate the chromatin association of specific DNA compaction factors like KAP-1 and HP1-a, this inter-regulation suggests epigenetic synergy concerning the silencing mechanisms of SPOC1 and ND10.

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The autophagy initiating protein kinase Ulk1 exerts a proviral role during HCMV infection

Research conducted during the last few years revealed that the cellular protein kinase Ulk1 exerts critical regulatory functions at the intersection of autophagy, innate immunity and inflammatory disorders. This protein was first described as autophagy-initiating protein kinase, however, recent evidence suggests that Ulk1 is an important component of different protein complexes involved in pathogen recognition. Furthermore, Ulk1 not only controls the onset of macroautophagy and mitophagy but also fine-tunes and negatively regulates inflammatory processes thus preventing immunopathology. We observed that Ulk1 is strongly upregulated after infection of primary human fibroblasts with human cytomegalovirus (HCMV). In addition, we detected an enhanced phosphorylation of Ulk1 at various serine residues which are typically targeted by the cellular AMP-activated protein kinase (AMPK), a metabolic stress response kinase. Inhibition of AMPK activity reversed this HCMV-induced modulation of Ulk1 which correlated with an impairment of viral replication. Evidence for a proviral role of Ulk1 was also obtained by generating primary human fibroblasts with a stable Ulk1-knockdown which revealed a profound growth defect of HCMV in the absence of Ulk1. Furthermore, small molecule inhibition of Ulk1 kinase activity strongly interfered with HCMV replication. Collectively, these data suggest that Ulk1 may serve as a novel target molecule for antiviral therapy. Moreover, viral dysregulation of Ulk1 may contribute to immunopathology frequently observed in connection with HCMV infection.

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The UL69 of human cytomegalovirus: a regulator of viral mRNA export that is controlled by distinct post-translational modifications

Human cytomegalovirus (HCMV) encodes the viral mRNA export factor pUL69, which facilitates the cytoplasmic accumulation of unspliced mRNA via interaction with the cellular RNA-helicases UAP56 or URH49. We reported before that pUL69 is posttranslationally phosphorylated by cellular CDKs and/or their viral orthologue pUL97. Here we set out to identify phosphorylation sites within pUL69 and characterize their in vivo importance for HCMV-replication. First, we performed MassSpec-based phosphosite mapping of pUL69 after immunopurification from AD169-infected HFF cells and identified several phosphorylation sites within the protein at 72hpi. However, in contrast to in silico analyses, which predicted numerous phosphosites within the functionally important N-terminus of pUL69, we failed to find any of those by our MassSpec analyses. We therefore compared the expression profiles of pUL69-mutants that carry individual or combinatorial substitutions of putative phosphosites within the pUL69 N-terminus by Phos-tag-SDS-PAGE, and provide evidence that S46, S49 as well as 2 of the serines 132, 133 or 134 were phosphorylated when pUL97 wildtype but not when its catalytically inactive derivative pUL97-K355M were coexpressed. Since pS46 and pS49 within alpha-helix 2 are both preceding a proline, we speculated that they might be substrate for Pin1-mediated peptidyl-prolyl cis-trans-isomerization and confirmed a complex formation of pUL69 and Pin1 by CoIP experiments. By performing NMR-experiments with (un)phosphorylated pUL69-peptides, we were able to exclude that phosphorylation affects the secondary structure of pUL69. However, our NMR-results strongly suggest that purified Pin1 catalyzes the cis/trans-isomerization of Proline 50 when S49 is phosphorylated. In accordance with our biochemical in vitro studies, multistep growth-curve analyses of recombinant HB15-derived viruses that carry S/T to A-substitutions within the pUL69 N-terminus revealed a severe growth defect. We therefore identified numerous phosphorylation sites within pUL69 and demonstrated that its N-terminal pUL97-phosphosite(s) are crucial for Pin1-mediated cis/trans-isomerization and efficient HCMV replication.

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Signaling of HCMV-encoded G protein-coupled receptors pUS27

Herpesviruses encode multiple G protein-coupled receptor homologues (vGPCRs) that have acquired unique properties to modulate cellular signaling. We identified a novel signaling capability of the vGPCR pUS27 of human cytomegalovirus (HCMV) that leads to strong NF-κB activation thus inducing the expression of chemoattractant cytokines. We demonstrate that the C-terminus of pUS27 directly interacts with TNF receptor-associated factor (TRAF) 6 resulting in canonical NF-κB activation. Intriguingly, signaling by pUS27 does not depend on G protein-coupling and correlates with internalization to endosomes. Interestingly, disruption of a PDZ domain-binding motif at the C-terminus of pUS27 strongly enhances signaling indicating that the activity of this vGPCR is restrained by cellular PDZ proteins. A mass spectrometry-based analysis was performed to identify the factors that interact with the US27 C-terminus. Most prominent among the identified proteins were components of signaling complexes which are involved in the establishment of apico-basolateral polarity in epithelial cells. In order to further investigate the consequences of pUS27-dependent NF-κB activation, we performed a cDNA array to identify cellular genes regulated by US27. Using a 293 cell line with doxycycline-inducible expression of US27-FLAG we show that pUS27 strongly induces the mRNA levels of specific chemoattractant cytokines when it is uncoupled from negative regulation by PDZ proteins. Infection experiments of epithelial cells with US27 mutant cytomegaloviruses suggest that pUS27 also contributes to the upregulation of specific cytokines during viral infection. Since inflammatory bowel disease (IBD) is associated with a downregulation of polarity complex proteins this may unleash pUS27 signaling thus contributing to enhanced viral dissemination via the secretion of chemoattractant cytokines. In summary, our data reveal a unique and switchable TRAF6-dependent signaling activity of this vGPCR which may foster viral dissemination upon inflammation-mediated downregulation of constraining epithelial factors.

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