During the first wave of the pandemic, we compared the occurrence of subjectively experienced COVID‐19‐like symptoms and true severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) ...seroconversion rates among medical personnel in general practices. This cross‐sectional study determined the SARS‐CoV‐2‐specific immunoglobulin G (IgG) antibody status of medical staff from 100 outpatient practices in Germany. Study cohort characteristics and COVID‐19‐like symptoms were obtained by questionnaires. The initial screening for SARS‐CoV‐2‐recognizing antibodies was performed using a commercial chemiluminescence microparticle immunoassay. Positive results were controlled with another approved test. Samples with discrepant results were subjected to a third IgG‐binding assay and a neutralization test. A total of 861 participants were included, 1.7% (n = 15) of whom tested positive for SARS‐CoV‐specific IgG in the initial screening test. In 46.6% (n = 7) of positive cases, test results were confirmed by an independent test. In the eight samples with discrepant results, neither spike‐specific antibodies nor in vitro neutralizing capacity were detectable, resulting in a genuine seroprevalence rate of 0.8%. 794 participants completed the questionnaire. Intriguingly, a total of 53.7% (n = 426) of them stated episodes of COVID‐19‐like symptoms. Except for smell and taste dysfunction, there were no significant differences between the groups with and without laboratory‐confirmed SARS‐CoV‐2 seroconversion. Our results demonstrated that only 0.8% of participants acquired SARS‐CoV‐2 even though 53.7% of participants reportedly experienced COVID‐19‐like symptoms. Thus, even among medical staff, self‐diagnosis based on subjectively experienced symptoms does not have a relevant predictive value.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Primary and recurrent cytomegalovirus (CMV) infections frequently cause CMV colitis in immunocompromised as well as inflammatory bowel disease (IBD) patients. Additionally, colitis occasionally ...occurs upon primary CMV infection in patients who are apparently immunocompetent. In both cases, the underlying pathophysiologic mechanisms are largely elusive ‐ in part due to the lack of adequate access to specimens. We employed the mouse cytomegalovirus (MCMV) model to assess the association between CMV and colitis. During acute primary MCMV infection of immunocompetent mice, the gut microbial composition was affected as manifested by an altered ratio of the Firmicutes to Bacteroidetes phyla. Interestingly, these microbial changes coincided with high‐titer MCMV replication in the colon, crypt hyperplasia, increased colonic pro‐inflammatory cytokine levels, and a transient increase in the expression of the antimicrobial protein Regenerating islet‐derived protein 3 gamma (Reg3γ). Further analyses revealed that murine and human intestinal epithelial cell lines, as well as primary intestinal crypt cells and organoids represent direct targets of CMV infection causing increased cell death. Accordingly, in vivo MCMV infection disrupted the intestinal epithelial barrier and increased apoptosis of intestinal epithelial cells. In summary, our data show that CMV transiently induces colitis in immunocompetent hosts by altering the intestinal homeostasis.
Acute cytomegalovirus (CMV) infection of immunocompetent mice alters the gut microbiota, involves high‐titer CMV replication in the colon, andepithelial barrier disruption. In vitro studies employing murine and human primary intestinal organoids reveal epithelial cells as a direct target of CMV with high rates of cell mortality upon CMV infection.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Human cytomegalovirus (CMV) is a ubiquitously distributed pathogen whose rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted global proteome profiling ...of rat CMV‐infected cells and uncovered a pronounced loss of the transcription factor STAT2, which is crucial for antiviral interferon signalling. Via deletion mutagenesis, we found that the viral protein E27 is required for CMV‐induced STAT2 depletion. Cellular and in vitro analyses showed that E27 exploits host‐cell Cullin4‐RING ubiquitin ligase (CRL4) complexes to induce poly‐ubiquitylation and proteasomal degradation of STAT2. Cryo‐electron microscopy revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors called DCAFs (DDB1‐ and Cullin4‐associated factors), thereby displacing them from the catalytic core of CRL4. Moreover, structural analyses showed that E27 recruits STAT2 through a bipartite binding interface, which partially overlaps with the IRF9 binding site. Structure‐based mutations in M27, the murine CMV homologue of E27, impair the interferon‐suppressing capacity and virus replication in mouse models, supporting the conserved importance of DCAF mimicry for CMV immune evasion.
Synopsis
Viruses can hijack the cellular ubiquitin–proteasome system to induce the degradation of antiviral host factors. Here, a combination of proteomic, biochemical, structural and virological analyses explains how cytomegaloviruses hijack Cullin4‐RING ubiquitin ligases for the removal of STAT2, a key signalling hub of the antiviral interferon response.
The rat cytomegalovirus‐encoded protein E27 induces proteasomal STAT2 degradation
E27 binds and exploits DDB1‐containing Cullin4‐RING ubiquitin ligases (CRL4) by mimicking endogenous CRL4 substrate receptors, DCAFs
Cryo‐EM shows that E27 recruits STAT2 via a bipartite interface, positioning STAT2 for CRL4‐catalysed ubiquitylation
The homologous mouse cytomegalovirus factor M27 establishes interferon resistance in mouse models via CRL4‐mediated STAT2 degradation
Cytomegaloviruses exploit host ubiquitin ligases to induce ubiquitylation and proteasomal degradation of STAT2, a key signalling hub of the antiviral interferon response.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
•Cytomegaloviruses exploit the ubiquitin proteasome system (UPS) to regulate virus-encoded proteins and to manipulate the host proteome.•Cytomegaloviruses repurpose the UPS to prevent programmed cell ...death.•Cytomegaloviruses repurpose the UPS to regulate the cell cycle.•Cytomegaloviruses repurpose the UPS to manipulate DNA-damage responses.•Cytomegaloviruses repurpose the UPS to antagonize intrinsic, innate, and adaptive immunity.
Human cytomegalovirus (HCMV) is a ubiquitous member of the Betaherpesvirinae subfamily, causing life-threatening diseases in individuals with impaired, immature, or senescent immunity. Accordingly, HIV-infected AIDS patients, transplant recipients, and congenitally infected neonates frequently suffer from symptomatic episodes of HCMV replication.
Like all viruses, HCMV has a split relationship with the host proteome. Efficient virus replication can only be achieved if proteins involved in intrinsic, innate, and adaptive immune responses are sufficiently antagonized. Simultaneously, the abundance and function of proteins involved in the synthesis of chemical building blocks required for virus production, such as nucleotides, amino acids, and fatty acids, must be preserved or even enriched. The ubiquitin (Ub) proteasome system (UPS) constitutes one of the most relevant protein decay systems of eukaryotic cells. In addition to the regulation of the turn-over and abundance of thousands of proteins, the UPS also generates the majority of peptides presented by major histocompatibility complex (MHC) molecules to allow surveillance by T lymphocytes. Cytomegaloviruses exploit the UPS to regulate the abundance of viral proteins and to manipulate the host proteome in favour of viral replication and immune evasion.
After summarizing the current knowledge of CMV-mediated misuse of the UPS, we discuss the evolution of viral proteins utilizing the UPS for the degradation of defined target proteins. We propose two alternative routes of adapter protein development and their mechanistic consequences.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Human cytomegalovirus (HCMV) causes diseases in individuals with immature or compromised immunity. To evade immune control, HCMV evolved numerous antagonists targeting the interferon system at ...multiple levels. By comparative analysis of naturally arising variants of the most widely studied HCMV strain, AD169, and a panel of targeted mutants, we uncover the UL145 gene as indispensable for STAT2 downregulation. Ribosome profiling confirms the translation of the canonical pUL145 protein (pUL145-Long) and newly identifies a shorter isoform (pUL145-Short). Both isoforms recruit DDB1-containing ubiquitin ligases to induce proteasomal degradation of STAT2. An alanine-scanning mutagenesis discloses the DDB1 interaction motif of pUL145 that resembles the DDB1-binding interface of cellular substrate receptors of DDB1-containing ubiquitin ligases. Thus, pUL145 constitutes a viral DDB1-cullin-associated factor (vDCAF), which mimics cellular DCAFs to exploit the ubiquitin-proteasome system to impede antiviral immunity. Notably, the viral exploitation of the cullins can be targeted to restore the efficacy of the host immune response.
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•HCMV strains differ in the ULb′ genome region and the capacity to degrade STAT2•The ULb′ gene UL145 is associated with a posttranscriptional loss of STAT2•pUL145s mimic cellular DCAFs and exploit DDB1-cullin RING ubiquitin ligases (CRLs)•Inhibition of CRLs elicits antiviral activity, which is potentiated by interferon
Le-Trilling et al. utilize intra-strain differences occurring in the HCMV strain AD169 to probe into viral IFN antagonism. They uncover UL145 as a key factor for HCMV-encoded STAT2 degradation. The pUL145 proteins constitute druggable viral DCAFs (vDCAFs) that mimic the DDB1-recognizing interface of cellular DCAFs to impede antiviral immunity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that frequently causes morbidity and mortality in individuals with insufficient immunity, such as transplant recipients, AIDS patients, ...and congenitally infected newborns. Several antiviral drugs are approved to treat HCMV infections. However, resistant HCMV mutants can arise in patients receiving long-term therapy. Additionally, side effects and the risk to cause birth defects limit the use of currently approved antivirals against HCMV. Therefore, the identification of new drug targets is of clinical relevance. Recent work identified DNA-damage binding protein 1 (DDB1) and the family of the cellular cullin (Cul) RING ubiquitin (Ub) ligases (CRLs) as host-derived factors that are relevant for the replication of human and mouse cytomegaloviruses. The first-in-class CRL inhibitory compound Pevonedistat (also called MLN4924) is currently under investigation as an anti-tumor drug in several clinical trials. Cytomegaloviruses exploit CRLs to regulate the abundance of viral proteins, and to induce the proteasomal degradation of host restriction factors involved in innate and intrinsic immunity. Accordingly, pharmacological blockade of CRL activity diminishes viral replication in cell culture. In this review, we summarize the current knowledge concerning the relevance of DDB1 and CRLs during cytomegalovirus replication and discuss chances and drawbacks of CRL inhibitory drugs as potential antiviral treatment against HCMV.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Viruses are by definition fully dependent on the cellular translation machinery, and develop diverse mechanisms to co-opt this machinery for their own benefit. Unlike many viruses, human ...cytomegalovirus (HCMV) does suppress the host translation machinery, and the extent to which translation machinery contributes to the overall pattern of viral replication and pathogenesis remains elusive. Here, we combine RNA sequencing and ribosomal profiling analyses to systematically address this question. By simultaneously examining the changes in transcription and translation along HCMV infection, we uncover extensive transcriptional control that dominates the response to infection, but also diverse and dynamic translational regulation for subsets of host genes. We were also able to show that, at late time points in infection, translation of viral mRNAs is higher than that of cellular mRNAs. Lastly, integration of our translation measurements with recent measurements of protein abundance enabled comprehensive identification of dozens of host proteins that are targeted for degradation during HCMV infection. Since targeted degradation indicates a strong biological importance, this approach should be applicable for discovering central host functions during viral infection. Our work provides a framework for studying the contribution of transcription, translation and degradation during infection with any virus.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
During viral infections, type I interferons (IFN) are induced and play a key role in counteracting initial viral spread. Twelve different human IFNα subtypes exist that bind the same receptor; ...however, they elicit unique host responses and display distinct potencies of antiviral activities. Our previous studies on human immunodeficiency virus (HIV) and hepatitis B virus (HBV) demonstrated that the clinically used IFNα2 is not the most effective one among the IFNα subtypes. By sequence modeling, we identified a region in helix B with mainly conserved residues at the outside facing IFNAR1, but variable residues at the inside facing the core of IFNα, potentially representing a putative tunable anchor to tune pleiotropic IFN responses. Using site-directed mutagenesis, various mutations were introduced into the IFNα2b backbone targeting sites which are important for binding to IFNAR1 and IFNAR2, the putative tunable anchor, or outside these three regions. Selected mutations were based on sequence differences to high antiviral subtypes IFNα6 and IFNα14. Treatment assays against HBV and HIV identified several critical residues for the antiviral activity of IFNα mainly in the IFNAR1 binding region. Combined mutations of the IFNα2 IFNAR1/2 binding sites or the IFNAR1 binding region plus the putative tunable anchor by those of IFNα14 further augmented activation of different downstream signaling cascades providing a molecular correlate for the enhanced antiviral activity. We describe here important functional residues within IFNα subtype molecules, which enabled us to design novel and innovative drugs that may have the potential to be used in clinical trials against a variety of different viral infections.IMPORTANCEThe potency of interferon (IFN)α to restrict viruses was already discovered in 1957. However, until today, only IFNα2 out of the 12 distinct human IFNα subtypes has been therapeutically used against chronic viral infections. There is convincing evidence that other IFNα subtypes are far more efficient than IFNα2 against many viruses. In order to identify critical antiviral residues within the IFNα subtype sequence, we designed hybrid molecules based on the IFNα2 backbone with individual sequence motifs from the more potent subtypes IFNα6 and IFNα14. In different antiviral assays with HIV or HBV, residues binding to IFNAR1 as well as combinations of residues in the IFNAR1 binding region, the putative tunable anchor, and residues outside these regions were identified to be crucial for the antiviral activity of IFNα. Thus, we designed artificial IFNα molecules, based on the clinically approved IFNα2 backbone, but with highly improved antiviral activity against several viruses.
Mammalian cell culture is indispensable for most aspects of current biomedical research. Immortalized cell lines are very convenient, but transforming principles (e.g. oncogenic viruses or their ...oncogenes) can heavily influence the experimental outcome. Primary cells do not share this apparent disadvantage but are more laborious to generate. Certain viruses (e.g. mouse cytomegalovirus) do not replicate efficiently in most transformed cell lines. In the past, such viruses have been routinely propagated on primary mouse embryonic fibroblasts (MEF) established around day 17 (d17) of gestation. According to new regulations of the European Union, experiments using gravid mammals and/or their embryos in the last trimester (>d14 in the case of mice) of gestation do require explicit permission of the local authorities responsible for animal care and use. Applying for such permission is time-consuming and often inflexible. Embryonic fibroblasts could also be produced at earlier time points of pregnancy from younger and smaller embryos. Obviously, this approach consumes more pregnant mice and embryos. Newborn mice are larger thus yielding more cells per sacrificed animal and the new Directive (2010/63/EU) excludes the killing of animals solely for the use of their organs or tissues. We established a convenient protocol to generate adherent mouse newborn cells (MNC). A direct comparison of MNC with MEF revealed that MNC fully recapitulate all tested aspects of a broad panel of virological parameters (plaque size, final titers, viral replication kinetics, viral gene expression, drug and interferon susceptibility as well as species specificity). The herein described approach allows researchers the legal use of primary cells and contributes to the 3R (replace, reduce, refine) guiding principles-especially the 'reduce' aspect-for the use of animals in scientific research. Additionally, it offers the option to directly compare in vitro and in vivo experiments when MNC are generated from littermates of animals included in the in vivo experiments.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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