AMPK protects endothelial cells against HSV-1 replication via inhibition of mTORC1 and ACC1.

Herpes simplex virus type 1 (HSV-1) is a widespread contagious pathogen, mostly causing mild symptoms on the mucosal entry side. However, systemic distribution, in particular upon reactivation of the virus in immunocompromised patients, may trigger an innate immune response and induce damage of organs. In these conditions, HSV-1 may infect vascular endothelial cells, but little is known about the regulation of HSV-1 replication and possible defense mechanisms in these cells. The current study addresses the question of whether the host cell protein AMP-activated protein kinase (AMPK), an important metabolic sensor, can control HSV-1 replication in endothelial cells. We show that downregulation of the catalytic subunits AMPKα1 and/or AMPKα2 increased HSV-1 replication as monitored by TCID50 titrations, while a potent AMPK agonist, MK-8722, strongly inhibited it. MK-8722 induced a persistent phosphorylation of the AMPK downstream targets acetyl-CoA carboxylase (ACC) and the rapamycin-sensitive adaptor protein of mTOR (Raptor) and, related to this, impairment of ACC1-mediated lipid synthesis and the mechanistic target of the rapamycin complex-1 (mTORC1) pathway. Since blockade of mTOR by Torin-2 as well as downregulation of ACC1 by siRNA also decreased HSV-1 replication, MK-8722 is likely to exert its anti-viral effect via mTORC1 and ACC1 inhibition. Importantly, MK-8722 was able to reduce virus replication even when added after HSV-1. Together, our data highlight the importance of endothelial cells as host cells for HSV-1 replication upon systemic infection and identify AMPK, a metabolic host cell protein, as a potential target for antiviral strategies against HSV-1 infection and its severe consequences. IMPORTANCE Herpes simplex virus type 1 (HSV-1) is a common pathogen that causes blisters or cold sores in humans. It remains latent in infected individuals and can be reactivated multiple times. In adverse conditions, for instance, in immunocompromised patients, HSV-1 can lead to serious complications such as encephalitis, meningitis, or blindness. In these situations, infection of endothelial cells lining the surface of blood vessels may contribute to the manifestation of disease. Here, we describe the role of AMP-activated protein kinase (AMPK), a potent regulator of cellular energy metabolism, in HSV-1 replication in endothelial cells. While downregulation of AMPK potentiates HSV-1 replication, pharmacological AMPK activation inhibits it by limiting the availability of required host cell macromolecules such as proteins or fatty acids. These data highlight the role of metabolic host cell proteins as antiviral targets and reveal activation of endothelial AMPK as a potential strategy to protect from severe consequences of HSV-1 infection.

STAT1 N-terminal domain discriminatively controls type I and type II IFN signaling.

The seven signal transducers of transcription (STATs) are cytokine-inducible modular transcription factors. They transmit the stimulation of cells with type I interferons (IFN-α/IFN-ß) and type II interferon (IFN-É£) into altered gene expression patterns. The N-terminal domain (NTD) of STAT1 is a surface for STAT1/STAT1 homodimer and STAT1/STAT2 heterodimer formation and allows the cooperative DNA binding of STAT1. We investigated whether the STAT1 NTD-mediated dimerization affected the IFN-induced tyrosine phosphorylation of STAT1, its nuclear translocation, STAT1-dependent gene expression, and IFN-dependent antiviral defense. We reconstituted human STAT1-negative and STAT2-negative fibrosarcoma cells with STAT1, NTD-mutated STAT1 (STAT1AA), STAT1 with a mutated DNA-binding domain (DBD), or STAT2. We treated these cells with IFN-α and IFN-É£ to assess differences between IFN-α-induced STAT1 homo- and heterodimers and IFN-É£-induced STAT1 homodimers. Our data demonstrate that IFNs induce the phosphorylation of STAT1 and STAT1AA at Y701 and their nuclear accumulation. We further reveal that STAT1AA can be phosphorylated in response to IFN-α in the absence of STAT2 and that IFN-É£-induced STAT1AA can activate gene expression directly. However, STAT1AA largely fails to bind STAT2 and to activate IFN-α-induced expression of endogenous antiviral STAT1/STAT2 target proteins. Congruent herewith, both an intact STAT1 NTD and STAT2 are indispensable to establish an antiviral state with IFN-α. These data provide new insights into the biological importance of the STAT1 NTD.

Performance of the RT-LAMP-based eazyplex® SARS-CoV-2 as a novel rapid diagnostic test.

BACKGROUND: Diagnostic assays for severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) that are easy to perform and produce fast results are essential for timely decision making regarding the isolation of contagious individuals. OBJECTIVE: We evaluated the CE-approved eazyplex® SARS-CoV-2, a ready-to-use real time RT-LAMP assay for identification of the SARS-CoV-2 N and ORF8 genes from swabs in less than 30 min without RNA extraction. STUDY DESIGN: Oropharyngeal and nasal swabs from 100 positive and 50 negative patients were inoculated into 0.9 % saline and tested by NeuMoDx™ RT-PCR. An aliquot was diluted fivefold in Copan sputum liquefying (SL) solution and directly analyzed by eazyplex® SARS-CoV-2. In addition, 130 patient swabs were prospectively tested with both methods in parallel. Analytical sensitivity of the assay was determined using virus stock dilutions. RESULTS: Positive percent agreement (PPA) between the eazyplex® SARS-CoV-2 and RT-PCR was 74 % for samples with Ct values < 35. When using a Ct cut-off ≤ 28 the PPA increased to 97.4 %. In the prospective part of the study overall PPA of the eazyplex® kit was 66.7 % but increased to 100 % when only Ct values ≤ 28 were considered. There were no false positive results. The median time to positivity was 12.5 min for the N gene and 16.75 min for ORF8. Analytical sensitivity was 3.75 TCID50/mL. 105 virus copies/mL were reproducibly detected. CONCLUSION: The eazyplex® SARS-CoV-2 is a rapid assay that accurately identifies samples with high viral loads. It may be useful for near-patient testing outside of a molecular diagnostic laboratory.

Early postmortem mapping of SARS-CoV-2 RNA in patients with COVID-19 and the correlation with tissue damage.

Clinical observations indicate that COVID-19 is a systemic disease. An investigation of the viral distribution within the human body and its correlation with tissue damage can aid in understanding the pathophysiology of SARS-CoV-2 infection. We present a detailed mapping of the viral RNA in 61 tissues and organs of 11 deceased patients with COVID-19. The autopsies were performed within the early postmortem interval (between 1.5 and 15 hr, mean: 5.6 hr) to minimize the bias due to viral RNA and tissue degradation. Very high viral loads (>104copies/ml) were detected in most patients' lungs, and the presence of intact viral particles in the lung tissue could be verified by transmission electron microscopy. Interestingly, viral RNA was detected throughout various extrapulmonary tissues and organs without visible tissue damage. The dissemination of SARS-CoV-2-RNA throughout the body supports the hypothesis that there is a maladaptive host response with viremia and multiorgan dysfunction.

Since the discovery of the new coronavirus that causes COVID-19, scientists have been scrambling to understand the different features of the virus. While a lot more is now known about SARS-CoV-2, several key questions have proved more difficult to answer. For example, it remained unclear where the virus travels to in the body and causes the most harm. To help answer this question, Deinhardt-Emmer, Wittschieber et al. performed postmortem examinations on 11 patients who had recently died of COVID-19. After sampling 61 different organs and tissues from each patient, several tests were used to detect traces of SARS-CoV-2. The experiments showed that the largest pool of SARS-CoV-2 was present in the lungs, where it had caused severe damage to the alveolae, the delicate air sacs at the end of the lungs' main air tubes. Small amounts of the virus were also detected in other organs and tissues, but no severe tissue damage was seen. In addition, Deinhardt-Emmer, Wittschieber et al. found that each patient had increased levels of some of the proteins involved in inflammation and blood clotting circulating their bloodstream. This suggests that the inflammation caused by SARS-CoV-2 leads to an excessive immune reaction throughout the entire body. This research provides important new insights into which areas of the body are most impacted by SARS-CoV-2. These findings may help to design more effective drug treatments that target the places SARS-CoV-2 is most likely to accumulate and help patients fight off the infection at these regions.

SARS-CoV-2 causes severe epithelial inflammation and barrier dysfunction.

Infections with SARS-CoV-2 can be asymptomatic, but they can also be accompanied by a variety of symptoms that result in mild to severe coronavirus disease-19 (COVID-19) and are sometimes associated with systemic symptoms. Although the viral infection originates in the respiratory system, it is unclear how the virus can overcome the alveolar barrier, which is observed in severe COVID-19 disease courses. To elucidate the viral effects on the barrier integrity and immune reactions, we used mono-cell culture systems and a complex human chip model composed of epithelial, endothelial, and mononuclear cells. Our data show that SARS-CoV-2 efficiently infected epithelial cells with high viral loads and inflammatory response, including interferon expression. By contrast, the adjacent endothelial layer was neither infected nor did it show productive virus replication or interferon release. With prolonged infection, both cell types were damaged, and the barrier function was deteriorated, allowing the viral particles to overbear. In our study, we demonstrate that although SARS-CoV-2 is dependent on the epithelium for efficient replication, the neighboring endothelial cells are affected, e.g., by the epithelial cytokines or components induced during infection, which further results in the damage of the epithelial/endothelial barrier function and viral dissemination.IMPORTANCESARS-CoV-2 challenges healthcare systems and societies worldwide in unprecedented ways. Although numerous new studies have been conducted, research to better understand the molecular pathogen-host interactions are urgently needed. For this, experimental models have to be developed and adapted. In the present study we used mono cell-culture systems and we established a complex chip model, where epithelial and endothelial cells are cultured in close proximity. We demonstrate that epithelial cells can be infected with SARS-CoV-2, while the endothelium did not show any infection signs. Since SARS-CoV-2 is able to establish viremia, the link to thromboembolic events in severe COVID-19 courses is evident. However, whether the endothelial layer is damaged by the viral pathogens or whether other endothelial-independent homeostatic factors are induced by the virus is essential for understanding the disease development. Therefore, our study is important as it demonstrates that the endothelial layer could not be infected by SARS-CoV-2 in our in vitro experiments, but we were able to show the destruction of the epithelial-endothelial barrier in our chip model. From our experiments we can assume that virus-induced host factors disturbed the epithelial-endothelial barrier function and thereby promote viral spread.

The PP2A subunit PR130 is a key regulator of cell development and oncogenic transformation.

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase. This enzyme is involved in a plethora of cellular processes, including apoptosis, autophagy, cell proliferation, and DNA repair. Remarkably, PP2A can act as a context-dependent tumor suppressor or promoter. Active PP2A complexes consist of structural (PP2A-A), regulatory (PP2A-B), and catalytic (PP2A-C) subunits. The regulatory subunits define the substrate specificity and the subcellular localization of the holoenzyme. Here we condense the increasing evidence that the PP2A B-type subunit PR130 is a critical regulator of cell identity and oncogenic transformation. We summarize knowledge on the biological functions of PR130 in normal and transformed cells, targets of the PP2A-PR130 complex, and how diverse extra- and intracellular stimuli control the expression and activity of PR130. We additionally review the impact of PP2A-PR130 on cardiac functions, neuronal processes, and anti-viral defense and how this might affect cancer development and therapy.

Use of the variplex™ SARS-CoV-2 RT-LAMP as a rapid molecular assay to complement RT-PCR for COVID-19 diagnosis.

BACKGROUND: Molecular assays based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) may be useful for rapid diagnosis of the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) because of the easy performance and the option to bypass RNA extraction. OBJECTIVES: This study was designed to evaluate the clinical performance of the CE-labeled variplexTM real time SARS-CoV-2 RT-LAMP assay in comparison to commercial RT-PCRs. STUDY DESIGN: RNA extracted from pharyngeal swabs was tested by variplex™ RT-LAMP and Corman's LightMix™ E gene RT-PCR as reference. Samples of respiratory secretions from Coronavirus infection disease (COVID-19) and negative control patients were analyzed by variplex™ without RNA extraction and tested in parallel with the Allplex™ and VIASURE BD MAX RT-PCRs. RESULTS: Using isolated RNA variplex™ RT-LAMP showed a sensitivity of 75 % compared to LightMix E gene RT-PCR but contrary to the latter it produced no false-positive results. For the evaluation of samples from respiratory secretions concordance analysis showed only a moderate agreement between the variplex™ RT-LAMP conducted on unprocessed samples and Allplex™ and VIASURE RT-PCRs (Cohen's κ ranging from 0.52-0.56). Using the approach to define a sample as true-positive when at least two assays gave a positive result the clinical sensitivities were as follows: 76.3 % for variplex™, 84.2 % for Allplex™ and 68.4 % for VIASURE. However, when results of RT-PCR and RT-LAMP were combined diagnostic sensitivity was increased to 92-100 %. CONCLUSION: The variplex RT-LAMP may serve as a rapid test to be combined with a RT-PCR assay to increase the diagnostic accuracy in patients with suspected COVID-19 infection.

The role of sphingosine-1-phosphate signaling in HSV-1-infected human umbilical vein endothelial cells.

Infections with the herpes simplex virus type 1 (HSV-1) are common and widespread. Most infections remain undetected but severe forms may develop in newborns and in immunocompromised patients. Moreover, HSV-1 might be involved in the pathogenesis of atherosclerosis, which may include viral infection of the endothelium. Antiviral therapy is efficient to treat symptomatic patients. However, an increasing accumulation of resistance-associated mutations has been observed in the viral genome. Thus, new antiviral strategies are focused on host factors. Among others, signaling of bioactive sphingolipids seems to be important in mediating HSV-1 replication. With the present study, regulation and function of sphingosine-1-phosphate (S1P)-based signaling were analyzed in HSV-1-infected human umbilical vein endothelial cells (HUVEC). Our data indicate that viral replication in endothelial cells relies on sphingosine kinase (SK) activity and S1P receptor (S1PR)1,3-5 signaling, which involves the activation of phosphatidylinositol-3-kinase (PI3K) and the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac-1). Inhibitor- or siRNA-meditated reduction of Rac-1 activity decreased HSV-1 replication. In general, targeting S1P-related signaling may be a successful strategy to establish new anti-HSV-1 therapies.

Influence of sphingosine-1-phosphate signaling on HCMV replication in human embryonal lung fibroblasts.

The human cytomegalovirus (HCMV) is a common pathogen, which causes severe or even deadly diseases in immunocompromised patients. In addition, congenital HCMV infection represents a major health concern affecting especially the lung tissue of the susceptible individuals. Antivirals are a useful strategy to treat HCMV-caused diseases. However, all approved drugs target viral proteins but significant toxicity and an increasing resistance against these compounds have been observed. In infected cells, numerous host molecules have been identified to play important roles during HCMV replication. Among others, HCMV infection depends on the presence of bioactive sphingolipids. In this study, the role of sphingosine-1-phosphate (S1P) signaling in HCMV-infected human embryonal lung fibroblasts (HELF) was analyzed. Viral replication depended on the functional activity of sphingosine kinases (SK). During SK inhibition, addition of extracellular S1P restored HCMV replication. Moreover, neutralization of extracellular S1P by anti-S1P antibodies decreased HCMV replication as well. While the application of FTY720 as an functional antagonist of S1P receptor (S1PR)1,3-5 signaling did not reduce HCMV replication significantly, JTE-013, an inhibitor of S1PR2, decreased viral replication. Furthermore, inhibition of Rac-1 activity reduced HCMV replication, whereas inhibition of the Rac-1 effector protein Rac-1-activated kinase 1 (PAK1) had no influence. In general, targeting S1P-induced pathways, which are essential for a successful HCMV replication, may represent a valuable strategy to develop new antiviral drugs.

A severe pediatric infection with a novel enterovirus A71 strain, Thuringia, Germany.

Infection by Enterovirus A71 (EV-A71) is an important cause of hand, foot, and mouth disease (HFMD). Outbreaks including severe cases with neurological and cardiopulmonary complications have been reported particularly from Southeast Asia. In Europe, the epidemiology of EV-A71 is not well understood. In summer 2015, a two-year-old girl from Thuringia, Germany, presented with rhombencephalitis/brainstem encephalitis associated with severe neurological and cardiopulmonary complications. EV-A71 was detected in stool and almost the entire viral genome was amplified and sequenced. While the capsid protein VP1-encoding region belongs to the EV-A71 subgenogroup C1, the 3D polymerase encoding region represents a unique lineage. Thus, the data suggest that the Thuringian EV-A71 sequence likely represents a recombinant. The case underlines the importance of intensified EV-A71 surveillance in Germany and Europe including analysis of full-genome data.

Drug resistance of clinical varicella-zoster virus strains confirmed by recombinant thymidine kinase expression and by targeted resistance mutagenesis of a cloned wild-type isolate.

In this study, approaches were developed to examine the phenotypes of nonviable clinical varicella-zoster virus (VZV) strains with amino acid substitutions in the thymidine kinase (TK) (open reading frame 36 [ORF36]) and/or DNA polymerase (Pol) (ORF28) suspected to cause resistance to antivirals. Initially, recombinant TK proteins containing amino acid substitutions described as known or suspected causes of antiviral resistance were analyzed by measuring the TK activity by applying a modified commercial enzyme immunoassay. To examine the effects of these TK and Pol substitutions on the replication of recombinant virus strains, the method of en passant mutagenesis was used. Targeted mutations within ORF36 and/or ORF28 and an autonomously expressed gene of the monomeric red fluorescent protein for plaque identification were introduced into the European wild-type VZV strain HJO. Plaque reduction assays revealed that the amino acid substitutions with unknown functions in TK, Q303stop, N334stop, A163stop, and the deletion of amino acids 7 to 74 aa (Δaa 7 to 74), were associated with resistance against acyclovir (ACV), penciclovir, or brivudine, whereas the L73I substitution and the Pol substitutions T237K and A955T revealed sensitive viral phenotypes. The results were confirmed by quantitative PCR by measuring the viral load under increasing ACV concentrations. In conclusion, analyzing the enzymatic activities of recombinant TK proteins represent a useful tool for evaluating the significance of amino acid substitutions in the antiviral resistance of clinical VZV strains. However, direct testing of replication-competent viruses by the introduction of nonsynonymous mutations in a VZV bacterial artificial chromosome using en passant mutagenesis led to reliable phenotypic characterization results.

The anti-obesity drug orlistat reveals anti-viral activity.

The administration of drugs to inhibit metabolic pathways not only reduces the risk of obesity-induced diseases in humans but may also hamper the replication of different viral pathogens. In order to investigate the value of the US Food and Drug Administration-approved anti-obesity drug orlistat in view of its anti-viral activity against different human-pathogenic viruses, several anti-viral studies, electron microscopy analyses as well as fatty acid uptake experiments were performed. The results indicate that administrations of non-cytotoxic concentrations of orlistat reduced the replication of coxsackievirus B3 (CVB3) in different cell types significantly. Moreover, orlistat revealed cell protective effects and modified the formation of multi-layered structures in CVB3-infected cells, which are necessary for viral replication. Lowering fatty acid uptake from the extracellular environment by phloretin administrations had only marginal impact on CVB3 replication. Finally, orlistat reduced also the replication of varicella-zoster virus moderately but had no significant influence on the replication of influenza A viruses. The data support further experiments into the value of orlistat as an inhibitor of the fatty acid synthase to develop new anti-viral compounds, which are based on the modulation of cellular metabolic pathways.

Evaluation of commercial herpes simplex virus IgG and IgM enzyme immunoassays.

Serological methods are used widely for the determination of herpes simplex virus (HSV) IgG and IgM antibodies in virological laboratories. The present study evaluates the automated performance of the Virion\Serion (Würzburg, Germany) and Orgentec (Mainz, Germany) enzyme-linked immunosorbent assays (ELISA) for the determination of HSV type-common and type-specific IgG and IgM antibodies. Two hundred sixty-three sera from HSV-negative children, healthy blood donors as well as patients without and with acute HSV infections were included. The Serion ELISAs classic HSV 1+2, HSV 1 and HSV 2 IgG showed sensitivities between 89.1% and 98.0% and specificities from 82.8% to 100%. Sensitivities of the Orgentec ELISAs Anti-HSV-1 and Anti-HSV-2 IgG were calculated as 91.0-96.0% and 88.5-95.4% accompanied by specificities between 93.1% and 100%. The HSV type-common Serion IgM ELISA revealed also a high sensitivity and specificity. However, the single-type HSV-1 and HSV-2 IgM ELISAs from both companies did not detect reliably HSV-1- and HSV-2-specific IgM antibodies. In conclusion, the automated performance of Serion ELISAs classic HSV 1+2, HSV 1 and HSV 2 IgG as well Orgentec ELISAs Anti-HSV-1 and Anti-HSV-2 IgG provide highly dependable results for identifying HSV-1 and HSV-2 IgG-positive or -negative individuals. While HSV type-common IgM ELISAs can be useful to confirm acute newly acquired HSV infections, the use of single-type IgM ELISAs on the basis of whole-virus antigen is dispensable.

Interferon alpha-armed nanoparticles trigger rapid and sustained STAT1-dependent anti-viral cellular responses.

Interferon-α (IFNα) has enormous potential for anti-proliferative and anti-viral treatments. However, clinical success is still hampered due to its limited bioavailability and thus, lack of sustained modulation of disease-relevant protective programs. Consequently, we here examined whether IFNα immobilized on nanoscale ferromagnetic R-Chitosan carriers is capable of inducing rapid and sustained activation of STAT1 signaling. We report the spontaneous formation of a stable nanoparticle-IFNα protein corona, which was exploited to generate IFNα-loaded spheres, obviating the need to specifically couple the cytokine to the nanoparticles (NPs). Notably, comprehensive experimental approaches ensure that formation of the IFNα NP-corona does not affect the biological activity of the cytokine, as STAT1 signaling was efficiently activated. Employing human prostate cancer and melanoma cell models, we found that the intensity and duration of STAT1 phosphorylation as well as the downstream activation of pathobiologically relevant genes were dose and particle dependent. In comparison with free IFNα, IFNα-loaded spheres resulted in a more sustained biologically relevant STAT1 activation, demonstrated also by conferring innate cellular immunity against vesicular stomatitis virus (VSV) infection. For one, our study demonstrates the advantages of biodegradable IFNα-coated R-Chitosan NPs for controlled cytokine release, and thereby improved therapy. Second, we reveal that the permanent presence of IFNα and not just the initial STAT1 phosphorylation ensures sustained IFNα-dependent signaling.

Identification of gene expression profiles in HeLa cells and HepG2 cells infected with Coxsackievirus B3.

Viral infections of host cells cause multiple changes of cellular metabolism including immediate defense mechanisms as well as processes to support viral replication. Coxsackievirus B3 (CVB3) is a member of the Picornavirus family and is responsible for a wide variety of mild or severe infections including acute and chronic inflammations. Thereby, intracellular signaling can be changed very comprehensively. In order to compare the influence of CVB3 replication on gene expression pattern of two different cell lines, DNA microarray systems were used to study a set of 780 genes related to inflammation. Expression analysis of HeLa cells and HepG2 cells infected with CVB3 identified 34 genes whose mRNA levels were altered significantly upon infection. The expression of additional 16 genes in HepG2 cells and 31 genes in HeLa cells were found to be influenced during CVB3 replication as well. All genes expressed differentially were sorted with regard to their functions and interpreted in view of known contributors to the infection process. The activation of the tumor necrosis factor pathways by CVB3 represents one peculiar observation, including apoptosis, stress, and inflammation responses.

Induction of a broad spectrum of inflammation-related genes by Coxsackievirus B3 requires Interleukin-1 signaling.

UNLABELLED: Coxsackievirus B3 (CVB3) is a major cause of acute and chronic forms of myocarditis. Previously, direct viral injury and post-infectious autoimmune response were suspected as main pathogenetic mechanisms. However, induction of pro-inflammatory cytokines may be crucial for pathogenesis in spite of host protein shut off caused by CVB3 replication. We investigated the global expression profile of pro-inflammatory genes induced by acute and persistent (carrier state) CVB3 infection in human fibroblast cell cultures with DNA microarrays, quantitative RT-PCR and ELISA. Rapid induction of a typical spectrum of about 30 inflammation-related genes (e.g., PTGS2, CCL2, IL-1ß, IL-6, IL-8, CSF2, MMP-1, MMP-3, and MMP-15) suggested an essential, autocrine role of IL-1. This hypothesis was confirmed by over-expression of IL-1RI, which resulted in a cytokine response upon CVB3 infection in HEK 293 cells otherwise refractory to CVB3-caused gene expression. Blocking IL-1 receptor type I (IL-1RI)-signaling during CVB3 infection with an IL-1 receptor antagonist (IL-1ra) as well as knockdown of IL-1RI using siRNA abrogated cytokine response in human fibroblasts. Both IL-1α and IL-1ß are relevant for the induction of inflammation-related genes during CVB3 infection as shown by neutralization experiments. Paracrine effects of IL-1 on the subset of non-infected cells in carrier state infected fibroblast cultures enhanced induction of inflammation-related genes. CONCLUSIONS: A broad spectrum of inflammatory cytokines was induced by CVB3 replication via a pathway that requires IL-1 signaling. Our results suggest that IL-1ra may be used as a therapeutic agent to limit inflammation and tissue destruction in myocarditis.

Significance of amino acid substitutions in the thymidine kinase gene of herpes simplex virus type 1 for resistance.

The analysis of the viral thymidine kinase (TK) genotype is of rising significance for testing resistance of herpes simplex virus (HSV) to antivirals especially acyclovir. However, numerous of the described amino acid (aa) substitutions are diagnostically less conclusive because of the pronounced natural polymorphism of this gene. In this study, several aa substitutions in the TK sequence of HSV-1 with unclear significance for resistance were analyzed by expression of recombinant TK proteins and determination of enzymatic activity on the basis of an enzyme linked immunosorbent assay using bromodeoxyuridine (BrdU) as TK substrate. The recombinant TK wild-type protein resulted in high TK activity and TK mutant with stop of translation showed negative results. The recombinant TK proteins containing the aa substitutions R41H or V348I had high phosphorylation activities suggesting most likely natural gene polymorphisms. By contrast, the aa changes Y53H, L139V, R163H, L298A and L315S were accompanied by negative or weakly positive TK activities indicating resistance association. In conclusion, the combination of methods described here represents a useful tool to evaluate the significance of aa substitutions for resistance of clinical HSV-1 strains.

Histone deacetylase inhibitors block IFNγ-induced STAT1 phosphorylation.

Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation.

Inhibition of nuclear factor kappa B activation reduces Coxsackievirus B3 replication in lymphoid cells.

Interactions between viral replication machineries and host cell metabolism display interesting information how certain viruses capitalize cellular pathways to support progeny production. Among those pathogens, Coxsackievirus B3 (CVB3) has been identified to manipulate intracellular signaling very comprehensively. Next to others, this human pathogenic virus causes acute and chronic forms of myocarditis, pancreatitis, and meningitis. Here, activation of nuclear factor kappa B (NFκB) signaling appears to be involved in successful infection. Viral replication is not restricted to solid organs but involves susceptible immune cells as well. In the present study, p65 phosphorylation as one aspect of NFκB activation and inhibition via BAY 11-7085 administration was analyzed in the context of CVB3 replication in lymphoid cells. During CVB3 infection, an up-regulation of p65 translation is detectable, which is accompanied by noticeable phosphorylation. Inhibition of NFκB signaling reduces viral replication in a dose- and time-dependent manner. Taken together, these results indicate that during CVB3 replication in human and murine lymphoid cells, NFκB signaling is activated and facilitates viral replication. Therefore, antiviral strategies to target such central cellular signaling pathways may represent potential possibilities for the development of new virostatica.

Inhibition of fatty acid synthase by amentoflavone reduces coxsackievirus B3 replication.

Coxsackievirus B3 (CVB3) is a human pathogen that causes acute and chronic infections, but an antiviral drug to treat these diseases has not yet been developed for clinical use. Several intracellular pathways are altered to assist viral transcription, RNA replication, and progeny release. Among these, fatty acid synthase (FAS) expression is increased. In order to test the potential of FAS inhibition as an anti-CVB3 strategy, several experiments were performed, including studies on the correlation of CVB3 replication and FAS expression in human Raji cells and an analysis of the time and dose dependence of the antiviral effect of FAS inhibition due to treatment with amentoflavone. The results demonstrate that CVB3 infection induces an up-regulation of FAS expression already at 1 h postinfection (p.i.). Incubation with increasing concentrations of amentoflavone inhibited CVB3 replication significantly up to 8 h p.i. In addition, suppression of p38 MAP kinase activity by treatment with SB239063 decreased FAS expression as well as viral replication. These data provide evidence that FAS inhibition via amentoflavone administration might present a target for anti-CVB3 therapy.