Biology subjects

Stamminger, T.

Publications and source records attributed to Stamminger, T..

9 recordsLinked to original sources

bioRxiv2026

Human cytomegalovirus antagonizes SMC5/6 driven genome silencing via UL35 instituted proteasomal degradation of SLF2

Recent studies demonstrated that the structural maintenance of chromosomes complex 5/6 (SMC5/6) acts as an important antiviral restriction factor that silences episomal DNA. Consequently, viruses have evolved strategies to antagonize this defence system. Interestingly, it was shown that SMC5/6-mediated silencing of extrachromosomal DNA depends on its recruitment to PML nuclear bodies which is mediated by a protein termed SMC5/6 localization factor 2 (SLF2). Since human cytomegalovirus (HCMV) dissociates PML nuclear bodies (PML-NBs) during the first hours of infection, we asked for the fate of SMC5/6 components during the HCMV replicative cycle. We first investigated the expression levels of SMC5/6 core components at the onset of HCMV infection and found that they were not downregulated by HCMV. Instead, we observed a distinct decrease of SLF2 protein levels which correlated with a complete dispersal of the SMC5/6 complex from PML-NBs. This was also observed after infection with UV inactivated HCMV and could not be blocked by cycloheximide treatment suggesting the involvement of an imported structural component of the virion. While we could exclude a role of the HCMV tegument protein pp71 (UL82), a targeted screen identified the tegument protein UL35 as being required and sufficient for proteasomal degradation of SLF2. In line with these results, SLF2 levels remained stable after infection with a recombinant HCMV harbouring a stop codon within the UL35 gene region and this correlated with a defect in the onset of HCMV immediate early gene expression. Furthermore, we observed an enhanced recruitment of SMC5/6 to PML-NBs and an overall increase in the association of parental viral genomes with SMC5/6 containing PML-NBs in the absence of UL35. Congruently, siRNA mediated depletion of SLF2 resulted in a reversal of this phenotype. This strongly suggests that UL35 serves as a novel viral SLF2 antagonist to prevent the initial silencing of incoming viral genomes by SMC5/6. Author summaryHuman cytomegalovirus (HCMV) is an important human pathogen which establishes a life-long persistent infection. Previous research revealed that host intrinsic defense mechanisms can silence HCMV gene expression and thereby foster latency. However, during lytic infection, viral effector proteins like the structural protein pp71 (UL82) and the viral immediate-early protein IE1 act as antagonists of this defense. Here, we report on the identification of a novel antagonist of host-mediated viral gene silencing which targets the SMC5/6 complex. SMC5/6 belongs to a highly conserved family of protein complexes that act as loop extrusion machines to facilitate the folding of chromatin. While SMC5/6 core factors were not attacked by HCMV, we found that the SMC5/6 associated protein SLF2 was degraded by the viral tegument protein UL35, that is imported into infected cells. This abrogates the accumulation of SMC5/6 at PML nuclear bodies which compromises the entrapment of viral genomes in these subnuclear structures. Since SLF2 is also important for genome stability and pathogenic variants of SLF2 cause the neurodevelopmental disease Atelis syndrome, we speculate that depletion of this factor via a viral tegument protein may also contribute to DNA damage induced during congenital HCMV infection.

microbiology↗

bioRxiv2026

Herpes simplex virus infection promotes ALS pathology through ICP0-mediated PML body disruption

Transactive response DNA binding protein 43 kDa (TDP-43) pathology, is a central molecular hallmark of amyotrophic lateral sclerosis (ALS). However, the underlying triggers are incompletely understood. Here, we show that infection with herpes simplex virus (HSV) induces molecular hallmarks of ALS in various in vitro and in vivo models and is associated with an increased risk of ALS in human population data. German healthcare provider data (n = 238,440) and herpesvirus serology of an ALS patient and control cohort (n = 1,100) showed that HSV infection elevated the ALS risk by 210% and odds by [~]65%, respectively. On a molecular level, HSV infection promoted TDP-43 pathology in neuronal cell models, human iPSC-derived motoneurons and cerebral organoids, mice, and human tissue sections. This effect was triggered by HSV-1 or 2, but not by several other related herpesviruses. Mechanistically, the infected cell protein 0 (ICP0) of HSV-1/2 drives TDP-43 pathology by disturbance of promyelocytic leukemia nuclear bodies (PML-NBs), thereby abrogating TDP-43 SUMO2/3ylation. Taken together, we reveal a previously unrecognized association between HSV infection and ALS and clarify the underlying molecular mechanism that drives TDP-43 pathology. Our data may guide future studies into therapeutic and prophylactic interventions against ALS.

neuroscience↗

bioRxiv2025

Virus-against-virus dominant-negative interference strategy targeting a viral CC chemokine prevents cytomegalovirus-related neurodevelopmental pathogenesis

BackgroundCongenital cytomegalovirus (CMV) infections are one leading cause of human neurodevelopmental disorders. Increasing evidence for the pathogenic involvement of brain immune alterations was obtained in the recent years. Host and virus-encoded chemokines might play important roles in CMV-related neuropathogenesis by regulating leukocyte trafficking and microglia recruitment in the CMV-infected brains, and by interfering with key neurodevelopmental steps. In a rat model of CMV infection of the fetal brain in utero that leads to detrimental neurologic and other severe phenotypes postnatally, we reported on the early alteration of microglia and on the infiltration of the infected brains by lymphoid and myeloid cells. Particularly, expression of the r129 gene encoding the viral chemokine RCK3 was detected as early as 24h post-infection; together with the previously reported chemotaxis properties exerted by RCK3 on lymphocytes and on macrophages in vitro, this suggested that RCK3 might be involved in the brain immune cell alterations seen in the CMV-infected developing brains, and in the related neuropathogenesis. MethodsInfection of the rat fetal brain was done by intracerebroventricular injections in utero of either rat CMV encoding wild-type (wt) RCK3 (RCMV-wt), or a mutant CMV counterpart encoding RCK3 with a deletion in its chemokine domain (RCMV-r129{Delta}NT). As RCMV-r129{Delta}NT had shown dominant-negative effects on the chemotaxis properties of RCK3 in vitro, simultaneous and successive co-infection rescue assays were also performed. The detrimental postnatal phenotypes in vivo and the epileptiform activity ex vivo usually detected after infection of the rat developing brain with RCMV-wt were monitored in the RCMV-r129{Delta}NT condition and in co-infection assays. ResultsIn sharp contrast with RCMV-wt whose infection of the fetal brain led to decreased postnatal survival, impaired sensorimotor development, hindlimb hyperextension and epileptic seizures in neonatal pups, RCMV-r129{Delta}NT infection was not associated with any severe postnatal phenotype in vivo. Consistently, the epileptiform activity recorded in most neocortical slices from RCMV-wt-infected pups was not detected in any slice from RCMV-r129{Delta}NT-infected pups. Simultaneous co-infection assays led to dramatic prevention against the postnatal phenotypes in vivo and the altered network activity ex vivo, revealing a dose-dependent rescuing effect exerted by RCMV-r129{Delta}NT on RCMV-wt. Importantly, successful rescue was also obtained when the mutant RCMV-r129{Delta}NT was inoculated in the fetal brains either before or after infection with RCMV-wt. SignificanceOur data demonstrate the crucial neuropathogenic role of RCK3 CMV chemokine in vivo. The dramatic success and apparent safety of the dominant-negative RCK3 rescue assays in vivo provide a proof-of-principle for the beneficial use of a virus-against-virus approach against CMV-related pathogenesis.

neuroscience↗

bioRxiv2025

Intrinsic immunity against HAdV is achieved by a novel epigenetic silencing complex

The DNA double-stranded genome of human adenoviruses (HAdV) is preferentially targeted by host factors involved in chromatin remodeling, such as SPOC1 and KAP1 to inhibit efficient viral gene expression. HAdV genomes undergo alterations through association with host histones and epigenetic modifications; however, the precise underlying mechanism remains elusive. A recently discovered silencing mechanism for retrotransposons and retroviruses involves the Human Silencing Hub (HUSH) complex. This complex of MPP8, TASOR, and PPHLN1 safeguards the human genome by utilizing histone H3 Lys9 trimethylation (H3K9me3) to block transcription. Through the recruitment of SETDB1 and MORC2, the HUSH complex silences host genes and condenses target genomes to combat infections such as HIV, MLV, and AAV. Here, we present evidence that the HUSH complex effectively restricts HAdV infection. To counteract the repressive function of this epigenetic silencing complex, HUSH factors are inhibited through binding of HAdV proteins and subsequent relocalization. We observe that MPP8 is targeted by the adenoviral E3 ubiquitin ligase, thus recruited by the viral early proteins E1B-55K and E4orf6 for proteasomal degradation. In summary, we provide evidence that the HUSH complex is a previously unrecognized host factor that restricts HAdV gene expression and replication. Based on these novel findings, we propose that HUSH represents a promising therapeutic target to combat HAdV infection.

cell biology↗

bioRxiv2025

Exonuclease ISG20 inhibits human cytomegalovirus replication by inducing an innate immune defense signature

ISG20 is an interferon-regulated protein that exhibits RNase activity thereby inhibiting the replication of a broad spectrum of RNA viruses. By single cell RNA sequencing, we identified ISG20 as an antiviral factor for human cytomegalovirus (HCMV) as it was upregulated in a population of HCMV-resistant cells. In accordance with an antiviral role on herpesviruses, overexpression of ISG20 in primary human fibroblasts led to reduced HCMV and HSV-1 replication, while knockdown of ISG20 enhanced virus growth. In Western blot kinetics, we observed that inhibition of HCMV replication by ISG20 occurs at the early stage of infection which correlated with reduced amounts of viral early and late transcripts. However, neither the half-life of viral and cellular RNAs nor of viral DNA was decreased in ISG20-expressing cells, indicating that ISG20 does not exert its antiviral effect via a degradation of RNAs or DNA. Instead, RNA-seq analysis revealed an innate immune defense signature upon ISG20 expression that comprised the upregulation of a distinct set of interferon stimulated genes (ISGs), zinc finger proteins (ZNFs) and of transposable elements (TEs). Our data indicate that this gene signature augments both IFN production and response of the host cell. Consistently, the JAK-STAT inhibitor ruxolitinib rescued HCMV gene expression in ISG20-expressing cells. We conclude that ISG20 induces a so far unprecedented immune defense signature that serves to amplify the IFN-mediated host cell defense thus explaining its broad antiviral activity.

microbiology↗

bioRxiv2024

DNA virus infections shape transposable elements activity in vitro and in vivo

Transposable elements (TEs) are implicated in a variety of processes including placental and preimplantation development and a variety of human diseases. TEs are known to be activated in the context of some viral infections, but the mechanisms and consequences are not understood. We show strong activation of TEs upon DNA virus infection, in particular the MLT- and THE1-class of LTR-containing retrotransposons as well as a subset of LINE-1-, Alu-elements and HERVs. Mechanistically, two key pathways induce TEs upregulation: inhibition of the KAP1/TRIM28 repressive complex by phosphorylation, and expression of the pioneer transcription factor double-homeobox 4 (DUX4), which is known to be involved in TE-induction during zygotic genome activation in embryonic development. DUX4 is induced by DNA viruses, it binds to TEs upon infection and analysis of genes adjacent to TEs shows pathways that are important for DNA virus infections. Analysis of knockdown, knockout and overexpression data reveal that almost all TEs expressed upon herpesviral infection are regulated by KAP1/TRIM28 and DUX4. Interestingly, analysis of single cell sequencing data from patients with DNA virus-associated cancers showed that in vivo TEs expression strongly correlates with virus infection, indicating a possible role in viral oncogenesis.

genomics↗

bioRxiv2021

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.

microbiology↗

bioRxiv2020

An enzyme-based immunodetection assay to quantify SARS-CoV-2 infection

SARS-CoV-2 is a novel pandemic coronavirus that caused a global health and economic crisis. The development of efficient drugs and vaccines against COVID-19 requires detailed knowledge about SARS-CoV-2 biology. Several techniques to detect SARS-CoV-2 infection have been established, mainly based on counting infected cells by staining plaques or foci, or by quantifying the viral genome by PCR. These methods are laborious, time-consuming and expensive and therefore not suitable for a high sample throughput or rapid diagnostics. We here report a novel enzyme-based immunodetection assay that directly quantifies the amount of de novo synthesized viral spike protein within fixed and permeabilized cells. This in-cell ELISA enables a rapid and quantitative detection of SARS-CoV-2 infection in microtiter format, regardless of the virus isolate or target cell culture. It follows the established method of performing ELISA assays and does not require expensive instrumentation. Utilization of the in-cell ELISA allows to e.g. determine TCID50 of virus stocks, antiviral efficiencies (IC50 values) of drugs or neutralizing activity of sera. Thus, the in-cell spike ELISA represents a promising alternative to study SARS-CoV-2 infection and inhibition and may facilitate future research. HighlightsO_LIDetermination of SARS-CoV-2 infection by enzymatically quantifying the expression of viral spike protein in bulk cell cultures C_LIO_LITargeting a highly conserved region in the S2 subunit of the S protein allows broad detection of several SARS-CoV-2 isolates in different cell lines C_LIO_LIScreening of antivirals in microtiter format and determining the antiviral activity as inhibitory concentrations 50 (IC50) C_LI

microbiology↗

bioRxiv2019

The thrombopoietin receptor agonist eltrombopag inhibits human cytomegalovirus replication via iron chelation

The thrombopoietin receptor agonist eltrombopag was successfully used against human cytomegalovirus (HCMV)-associated thrombocytopenia refractory to immunomodulatory and antiviral drugs. These effects were ascribed to effects of eltrombopag on megakaryocytes. Here, we tested whether eltrombopag may also exert direct antiviral effects. Therapeutic eltrombopag concentrations inhibited HCMV replication in human fibroblasts and adult mesenchymal stem cells infected with six different virus strains and drug-resistant clinical isolates. Eltrombopag also synergistically increased the anti-HCMV activity of the mainstay drug ganciclovir. Time-of-addition experiments suggested that eltrombopag interferes with HCMV replication after virus entry. Eltrombopag was effective in thrombopoietin receptor-negative cells, and addition of Fe3+ prevented the anti-HCMV effects, indicating that it inhibits HCMV replication via iron chelation. This may be of particular interest for the treatment of cytopenias after haematopoietic stem cell transplantation, as HCMV reactivation is a major reason for transplantation failure. Since therapeutic eltrombopag concentrations are effective against drug-resistant viruses and synergistically increase the effects of ganciclovir, eltrombopag is also a drug repurposing candidate for the treatment of therapy-refractory HCMV disease.

pharmacology and toxicology↗