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Coxsackievirus B3 (CVB3) encodes proteinases that are essential for processing of the translated viral polyprotein. Viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. While some host protein substrates of the CVB3 3C and 2A cysteine proteinases have been identified, the full repertoire of targets is not known. Here, we utilize an unbiased quantitative proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) to conduct a global analysis of CVB3 protease-generated N-terminal peptides in both human HeLa and mouse cardiomyocyte (HL-1) cell lines infected with CVB3. We identified >800 proteins that are cleaved in CVB3-infected HeLa and HL-1 cells including the viral polyprotein, known substrates of viral 3C proteinase such as PABP, DDX58, and HNRNPs M, K, and D and novel cellular proteins. Network and GO-term analysis showed an enrichment in biological processes including immune response and activation, RNA processing, and lipid metabolism. We validated a subset of candidate substrates that are cleaved under CVB3 infection and some are direct targets of 3C proteinase in vitro . Moreover, depletion of a subset of TAILS-identified target proteins decreased viral yield. Characterization of two target proteins showed that expression of 3C pro -targeted cleaved fragments of emerin and aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 modulated autophagy and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, respectively. The comprehensive identification of host proteins targeted during virus infection provides insights into the cellular pathways manipulated to facilitate infection., Importance: RNA viruses encode proteases that are responsible for processing viral proteins into their mature form. Viral proteases also target and cleave host cellular proteins; however, the full catalog of these target proteins is incomplete. We use a technique called terminal amine isotopic labeling of substrates (TAILS), an N-terminomics to identify host proteins that are cleaved under virus infection. We identify hundreds of cellular proteins that are cleaved under infection, some of which are targeted directly by viral protease. Revealing these target proteins provides insights into the host cellular pathways and antiviral signaling factors that are modulated to promote virus infection and potentially leading to virus-induced pathogenesis., Competing Interests: The authors declare no conflict of interest.

Aims: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15 kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown., Methods and Results: Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system., Conclusion: Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction., Competing Interests: Conflict of interest: JK conducts paid consultancy for Centogene GmbH. All other authors declare no conflict of interest. The patent application EP21174633 with the title “Computer assisted method for the evaluation of cardiac metabolism” was filed by Charité – Universitätsmedizin Berlin as the employer of NB and Titus Kuehne, with both holding inventorship for this patent application., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Dysregulated cell death machinery and an excessive inflammatory response in Coxsackievirus B3(CVB3)-infected myocarditis are hallmarks of an abnormal host response. Complement C4 and C3 are considered the central components of the classical activation pathway and often participate in the response process in the early stages of virus infection., Methods: In our study, we constructed a mouse model of CVB3-related viral myocarditis via intraperitoneal injection of Fer-1 and detected myocarditis and ferroptosis markers in the mouse myocardium. Then, we performed co-IP and protein mass spectrometry analyses to explore which components interact with the ferroptosis gene transferrin receptor (TFRC). Finally, functional experiments were conducted to verify the role of complement components in regulating ferroptosis in CVB3 infection., Results: It showed that the ferroptosis inhibitor Fer-1 could alleviate the inflammation in viral myocarditis as well as ferroptosis. Mechanistically, during CVB3 infection, the key factor TFRC was activated and inhibited by Fer-1. Fer-1 effectively prevented the consumption of complement C3 and overload of the complement product C4b. Interestingly, we found that TFRC directly interacts with complement C4, leading to an increase in the product of C4b and a decrease in the downstream complement C3. Functional experiments have also confirmed that regulating the complement C4/C3 pathway can effectively rescue cell ferroptosis caused by CVB3 infection., Conclusions: In this study, we found that ferroptosis occurs through crosstalk with complement C4 in viral myocarditis through interaction with TFRC and that regulating the complement C4/C3 pathway may rescue ferroptosis in CVB3-infected cardiomyocytes., Competing Interests: Declaration of Competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Evidence is emerging on the roles of long noncoding RNAs (lncRNAs) as regulatory factors in a variety of viral infection processes, but the mechanisms underlying their functions in coxsackievirus group B type3 (CVB3)-induced acute viral myocarditis have not been explicitly delineated. We previously demonstrated that CVB3 infection decreases miRNA-21 expression; however, lncRNAs that regulate the miRNA-21-dependent CVB3 disease process have yet to be identified. To evaluate lncRNAs upstream of miRNA-21, differentially expressed lncRNAs in CVB3-infected mouse hearts were identified by microarray analysis and lncRNA/miRNA-21 interactions were predicted bioinformatically. MEG3 was identified as a candidate miRNA-21-interacting lncRNA upregulated in CVB3-infected mouse hearts. MEG3 expression was verified to be upregulated in HeLa cells 48 h post CVB3 infection and to act as a competitive endogenous RNA of miRNA-21. MEG3 knockdown resulted in the upregulation of miRNA-21, which inhibited CVB3 replication by attenuating P38-MAPK signaling in vitro and in vivo. Knockdown of MEG3 expression before CVB3 infection inhibited viral replication in mouse hearts and alleviated cardiac injury, which improved survival. Furthermore, the knockdown of CREB5, which was predicted bioinformatically to function upstream of MEG3, was demonstrated to decrease MEG3 expression and CVB3 viral replication. This study identifies the function of the lncRNA MEG3/miRNA-21/P38 MAPK axis in the process of CVB3 replication, for which CREB5 could serve as an upstream modulator., Competing Interests: Declaration of Competing Interest All authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Exploring the immune mechanism of coxsackievirus B3 (CVB3)-induced myocarditis may provide a promising therapeutic strategy. Here, we investigated the regulatory role of macrophage CAPN4 in the phenotypic transformation of macrophages and NOD-like receptor protein 3 (NLRP3) inflammasome activation. We found that CAPN4 was the most upregulated subtype of the calpain family in CVB3-infected bone marrow-derived macrophages (BMDMs) and Raw 264.7 cells after CVB3 infection and was upregulated in cardiac macrophages from CVB3-infected mice. Conditional knockout of CAPN4 (CAPN4 flox/flox ; LYZ2-Cre, CAPN4-cKO mice) ameliorated inflammation and myocardial injury and improved cardiac function and survival after CVB3 infection. Enrichment analysis revealed that macrophage differentiation and the interleukin signaling pathway were the most predominant biological processes in macrophages after CVB3 infection. We further found that CVB3 infection and the overexpression of CAPN4 promoted macrophage M1 polarization and NLRP3 inflammasome activation, while CAPN4 knockdown reversed these changes. Correspondingly, CAPN4-cKO alleviated CVB3-induced M1 macrophage transformation and NLRP3 expression and moderately increased M2 transformation in vivo. The culture supernatant of CAPN4-overexpressing or CVB3-infected macrophages impaired cardiac fibroblast function and viability. Moreover, macrophage CAPN4 could upregulate C/EBP-homologous protein (chop) expression, which increased proinflammatory cytokine release by activating the phosphorylation of transducer of activator of transcription 1 (STAT1) and 3 (STAT3). Overall, these results suggest that CAPN4 increases M1-type and inhibits M2-type macrophage polarization through the chop-STAT1/STAT3 signaling pathway to mediate CVB3-induced myocardial inflammation and injury. CAPN4 may be a novel target for viral myocarditis treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lactiplantibacillus plantarum is a lactic acid bacterium widely used in food production. Coxsackievirus B3 (CVB3) is an important human pathogen associated with acute pancreatitis development, and no antiviral therapeutics or vaccines are approved to treat or prevent its infection. However, whether L. plantarum could inhibit CVB3 infection remains unclear. Here, L. plantarum FLPL05 showed antiviral activity against CVB3 infection in vivo and in vitro . Pretreatment with L. plantarum FLPL05 reduced serum amylase levels, CVB3 viral load in the pancreas, serum pro-inflammatory cytokine levels, and macrophage infiltration in CVB3-infected mice. In mice, L. plantarum FLPL05 inhibited CVB3-induced pancreas apoptosis via the B cell leukemia/lymphoma 2 (BCL2)/BCL2-associated X protein (BAX)/caspase-3 (CASP3) signaling pathway. Furthermore, L. plantarum FLPL05 reduced CVB3 replication, protected cells from the cytopathic effect of CVB3 infection, and inhibited cell apoptosis. Moreover, L. plantarum FLPL05's exopolysaccharide (EPS) had activity against CVB3 in vitro , reducing the CVB3 titer and improving cell activity. Therefore, L. plantarum FLPL05 pretreatment improved CVB3-induced pancreatitis by partially reversing pancreatitis, which might be associated with EPS. Consequently, L. plantarum FLPL05 could be a potential probiotic with antiviral activity against CVB3.

Among enteroviruses, echovirus can cause severe illnesses in neonates or infants, with high morbidity and mortality. Autophagy, a central component of host defense mechanisms, can function against diverse infections. In the present study, we investigated the interplay between echovirus and autophagy. We demonstrated that echovirus infection increases LC3-II expression dose-dependently, accompanied by an increased intracellular LC3 puncta level. In addition, echovirus infection induces the formation of autophagosome. These results suggest that echovirus infection induces autophagy machinery. Furthermore, phosphorylated mTOR and ULK1 were both decreased upon echovirus infection. In contrast, both levels of the vacuolar protein sorting 34 (VPS34) and Beclin-1, the downstream molecules which play essential roles in promoting the formation of autophagic vesicles, increased upon virus infection. These results imply that the signaling pathways involved in autophagosome formation were activated by echovirus infection. Moreover, induction of autophagy promotes echovirus replication and viral protein VP1 expression, while inhibition of autophagy impairs VP1 expression. Our findings suggest that autophagy can be induced by echovirus infection via regulating mTOR/ULK1 signaling pathway and exhibits a proviral function, revealing the potential role of autophagy in echovirus infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer YX declared a shared parent affiliation with the author SL at the time of review., (Copyright © 2023 Wu, Zeng, Yi, Miao, Liu, Wang, Liu, Peng, Zheng and Xia.)

The coxsackievirus and adenovirus receptor (CAR) is very well known as an epithelial tight junction and cardiac intercalated disc protein; it mediates attachment and infection via the coxsackievirus B3 (CVB3) and type 5 adenovirus. Macrophages play important roles in early immunity during viral infections. However, the role of CAR in macrophages is not well studied in relation to CVB3 infection. In this study, the function of CAR was observed in the Raw264.7 mouse macrophage cell line. CAR expression was stimulated by treatment with lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α). In thioglycollate-induced peritonitis, the peritoneal macrophage was activated and CAR expression was increased. The macrophage-specific CAR conditional knockout mice (KO) were generated from lysozyme Cre mice. The expression of inflammatory cytokine (IL-1β and TNF-α) was attenuated in the KO mice's peritoneal macrophage after LPS treatment. In addition, the virus was not replicated in CAR-deleted macrophages. The organ virus replication was not significantly different in both wild-type (WT) and KO mice at days three and seven post-infection (p.i). However, the inflammatory M1 polarity genes (IL-1β, IL-6, TNF-α and MCP-1) were significantly increased, with increased rates of myocarditis in the heart of KO mice compared to those of WT mice. In contrast, type1 interferon (IFN-α and β) was significantly decreased in the heart of KO mice. Serum chemokine CXCL-11 was increased in the KO mice at day three p.i. compared to the WT mice. The attenuation of IFN-α and β in macrophage CAR deletion induced higher levels of CXCL-11 and more increased CD4 and CD8 T cells in KO mice hearts compared to those of WT mice at day seven p.i. These results demonstrate that macrophage-specific CAR deletion increased the macrophage M1 polarity and myocarditis in CVB3 infection. In addition, chemokine CXCL-11 expression was increased, and stimulated CD4 and CD8 T cell activity. Macrophage CAR may be important for the regulation of innate-immunity-induced local inflammation in CVB3 infection.

Group B enteroviruses, including coxsackievirus B3 (CVB3), can persistently infect cardiac tissue and cause dilated cardiomyopathy. Persistence is linked to 5' terminal deletions of viral genomic RNAs that have been detected together with minor populations of full-length genomes in human infections. In this study, we explored the functions and interactions of the different viral RNA forms found in persistently infected patients and their putative role(s) in pathogenesis. Since enterovirus cardiac pathogenesis is linked to the viral proteinase 2A, we investigated the effect of different terminal genomic RNA deletions on 2A activity. We discovered that 5' terminal deletions in CVB3 genomic RNAs decreased the levels of 2A proteinase activity but could not abrogate it. Using newly generated viral reporters encoding nano-luciferase, we found that 5' terminal deletions resulted in decreased levels of viral protein and RNA synthesis in singly transfected cardiomyocyte cultures. Unexpectedly, when full-length and terminally deleted forms were cotransfected into cardiomyocytes, a cooperative interaction was observed, leading to increased viral RNA and protein production. However, when viral infections were carried out in cells harboring 5' terminally deleted CVB3 RNAs, a decrease in infectious particle production was observed. Our results provide a possible explanation for the necessity of full-length viral genomes during persistent infection, as they would stimulate efficient viral replication compared to that of the deleted genomes alone. To avoid high levels of viral particle production that would trigger cellular immune activation and host cell death, the terminally deleted RNA forms act to limit the production of viral particles, possibly as trans -dominant inhibitors. IMPORTANCE Enteroviruses like coxsackievirus B3 are able to initiate acute infections of cardiac tissue and, in some cases, to establish a long-term persistent infection that can lead to serious disease sequelae, including dilated cardiomyopathy. Previous studies have demonstrated the presence of 5' terminally deleted forms of enterovirus RNAs in heart tissues derived from patients with dilated cardiomyopathy. These deleted RNAs are found in association with very low levels of full-length enterovirus genomic RNAs, an interaction that may facilitate continued persistence while limiting virus particle production. Even in the absence of detectable infectious virus particle production, these deleted viral RNA forms express viral proteinases at levels capable of causing viral pathology. Our studies provide mechanistic insights into how full-length and deleted forms of enterovirus RNA cooperate to stimulate viral protein and RNA synthesis without stimulating infectious viral particle production. They also highlight the importance of targeting enteroviral proteinases to inhibit viral replication while at the same time limiting the long-term pathologies they trigger.

Background: Myocardial inflammation and apoptosis are key processes in coxsackievirus B3 (CVB3)-induced acute viral myocarditis (AVMC). Accumulating evidence reveals the essential roles of long noncoding RNAs (lncRNAs) in the pathogenesis of AVMC. Here, we aimed to evaluate the biological functions of a novel lncRNA guanylate-binding protein 9 (lncGBP9) in AVMC progression and further explore its underlying mechanisms., Methods: Initially, mouse models of AVMC were constructed by CVB3 infection. The expression and localization of lncGBP9 in heart tissues were analyzed using RT-qPCR and FISH. Adeno-associated virus serotype 9 (AAV9)-mediated lncGBP9 knockdown was then employed to clarify its roles in survival, cardiac function, and myocardial inflammation and apoptosis. Moreover, the mRNA and protein levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) were detected by RT-qPCR and ELISA, and the regulation of lncGBP9 knockdown on the NF-κB signaling pathway was investigated by Western blotting. Using an in vitro model of HL-1 cardiomyocytes exposed to CVB3 infection, the effects of lncGBP9 knockdown on cell viability, inflammation, and apoptosis were further evaluated in vitro., Results: Increased lncGBP9 expression was detected in the heart tissues of AVMC mice and CVB3-infected HL-1 cells, and was mainly located in the cytoplasm. Knockdown of lncGBP9 remarkably alleviated the severity of AVMC in CVB3-infected mice, as verified by improved cardiac function, and reduced myocardial inflammation and apoptosis. Additionally, lncGBP9 knockdown suppressed the NF-κB signaling pathway and consequently reduced productions of pro-inflammatory cytokines in vivo. In vitro functional assays further confirmed that lncGBP9 knockdown promoted cell viability, inhibited cell apoptosis, and reduced pro-inflammatory cytokines release in CVB3-infected HL-1 cells through suppressing NF-κB activation., Conclusions: Collectively, lncGBP9 knockdown exerts anti-inflammatory and anti-apoptotic effects in CVB3-induced AVMC, which may be mediated in part via NF-κB signaling pathway. These findings highlight lncGBP9 as an attractive target for therapeutic interventions., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Echoviruses, for which there are currently no approved vaccines or drugs, are responsible for a range of human diseases, for example echovirus 11 (E11) is a major cause of serious neonatal morbidity and mortality. Decay-accelerating factor (DAF, also known as CD55) is an attachment receptor for E11. Here, we report the structure of the complex of E11 and the full-length ectodomain of DAF (short consensus repeats, SCRs, 1-4) at 3.1 Å determined by cryo-electron microscopy (cryo-EM). SCRs 3 and 4 of DAF interact with E11 at the southern rim of the canyon via the VP2 EF and VP3 BC loops. We also observe an unexpected interaction between the N-linked glycan (residue 95 of DAF) and the VP2 BC loop of E11. DAF is a receptor for at least 20 enteroviruses and we classify its binding patterns from reported DAF/virus complexes into two distinct positions and orientations, named as E6 and E11 poses. Whilst 60 DAF molecules can attach to the virion in the E6 pose, no more than 30 can attach to E11 due to steric restrictions. Analysis of the distinct modes of interaction and structure and sequence-based phylogenies suggests that the two modes evolved independently, with the E6 mode likely found earlier.

Coxsackievirus B (CVB) 3C protease (3C pro ) plays a specific cleavage role on AU-rich binding factor (AUF1, also called hnRNP D), which consequently disputes the regulation of AUF1 on downstream molecules. In our study, the iTRAQ approach was first used to quantify the differentially expressed cellular proteins in AUF1-overexpressing HeLa cells, which provides straightforward insight into the role of AUF1 during viral infection. A total of 1,290 differentially expressed proteins (DEPs), including 882 upregulated and 408 downregulated proteins, were identified. The DEPs are involved in a variety of cellular processes via GO terms, protein-protein interactions, and a series of further bioinformatics analyses. Among the DEPs, some demonstrated important roles in cellular metabolism. In particular, DDX5 was further verified to be negatively regulated by AUF1 and increased in CVB-infected cells, which in turn promoted CVB replication. These findings provide potential novel ideas for exploring new antiviral therapy targets., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Tianying Wang et al.)

The 5'UTR of the genomic RNA of CVB3, unusually long and rich on highly structured secondary structure, contains a conserved cis acting RNA element named the cryptic AUG (cAUG), where the cellular 48S complex is formed. In this study, we investigate the role of this cAUG in CVB3 translation, replication, and virulence. Mutant viral sub-genomic replicon RNA was constructed by site-directed mutagenesis. We characterize in vitro translation and replication efficiencies and in vivo virulence of a cAUG mutant in comparison with wild-type strain. UV-cross-linking assay and Real-Time PCR were used, respectively, to detect binding host proteins and to quantify viral production. Secondary structures of domain containing the cAUG site were studied and compared. The results suggest that introduced mutation in the CVB3 5'UTR affects in vitro and ex vivo viral translation which cannot be rescued by compensatory mutations. A reduced interaction of the La and PCBP2 translation initiation factors with cAUG residue of mutant was revealed. Decreasing production of viral mutant RNA was also demonstrated. Furthermore, secondary structure prediction reveals changes in the ribosome binding sites of the cAUG moiety of mutant sense strand RNA and no alterations in the structure of wild type, suggesting that cAUG mutation specifically affects the secondary structure of the sense RNA strand. Taken together, AUG integrity influences the efficiency of ribosome recruitment through IRES element and the capacity of replication., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

RNA viruses in the Picornaviridae family express a large 250 kDa viral polyprotein that is processed by virus-encoded proteinases into mature functional proteins with specific functions for virus replication. One of these proteins is the highly conserved enteroviral transmembrane protein 3A that assists in reorganizing cellular membranes associated with the Golgi apparatus. Here, we studied the molecular properties of the Coxsackievirus B3 (CVB3) protein 3A with regard to its dimerization and its functional stability. By applying mutational analysis and biochemical characterization, we demonstrate that protein 3A forms DTT-sensitive disulfide-linked dimers via a conserved cytosolic cysteine residue at position 38 (Cys38). Homodimerization of CVB3 protein 3A via Cys38 leads to profound stabilization of the protein, whereas a C38A mutation promotes a rapid proteasome-dependent elimination of its monomeric form. The lysosomotropic agent chloroquine (CQ) exerted only minor stabilizing effects on the 3A monomer but resulted in enrichment of the homodimer. Our experimental data demonstrate that disulfide linkages via a highly conserved Cys-residue in enteroviral protein 3A have an important role in the dimerization of this viral protein, thereby preserving its stability and functional integrity., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Hand, foot and mouth disease (HFMD) caused by Coxsackievirus Group B5 (CVB5) is one of the most common herpetic diseases in human infants and children. The pathogenesis of CVB5 remains unknown. Circular RNAs (CircRNAs), as novel noncoding RNAs, have been shown to play a key role in many pathogenic processes in different species; however, their functions during the process of CVB5 infection remain unclear. In the present study, we investigated the expression profiles of circRNAs using RNA sequencing technology in CVB5-infected and mock-infected human rhabdomyosarcoma cells (CVB5 virus that had been isolated from clinical specimens). In addition, several differentially expressed circRNAs were validated by RT-qPCR. Moreover, the innate immune responses related to circRNA-miRNA-mRNA interaction networks were constructed and verified. A total of 5461 circRNAs were identified at different genomic locations in CVB5 infections and controls, of which 235 were differentially expressed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis demonstrated that the differentially expressed circRNAs were principally involved in specific signaling pathways related to ErbB, TNF, and innate immunity. We further predicted that novel_circ_0002006 might act as a molecular sponge for miR-152-3p through the IFN-I pathway to inhibit CVB5 replication, and that novel_circ_0001066 might act as a molecular sponge for miR-29b-3p via the NF-κB pathway and for the inhibition of CVB5 replication. These findings will help to elucidate the biological functions of circRNAs in the progression of CVB5-related HFMD and identify prospective biomarkers and therapeutic targets for this disease.

The longevity and reusability of N95-grade filtering facepiece respirators (N95 FFRs) are limited by consecutive donning and disinfection treatments. Herein, we developed stable N97 nanofibrous respirators based on chemically modified surface to enable remarkable filtration characteristics via polarity driven interaction. This was achieved by a thin-film coated polyacrylonitrile nanofibrous membrane (TFPNM), giving an overall long-lasting filtration performance with high quality factor at 0.42 Pa -1 (filtration efficiency: over 97 %; pressure drop: around 10 Pa), which is higher than that of the commercial N95 FFRs (0.10-0.41 Pa -1 ) tested with a flow rate of 5 L min -1 and the 0.26 μm NaCl aerosol. A coxsackie B4 virus filtration test demonstrated that TFPNM also had strong virus capture capacity of 97.67 %. As compared with N95 FFRs, the TFPNM was more resistant to a wider variety of disinfection protocols, and the overall filtration characteristics remained N97 standard., (© 2021 Wiley-VCH GmbH.)

Coxsackievirus B3 (CVB3), an enterovirus (EV) in the family of Picornaviridae , is a global human pathogen for which effective antiviral treatments and vaccines are lacking. Previous research demonstrated that EV-D68 downregulated the membrane fusion protein SNAP47 (synaptosome associated protein 47) and SNAP47 promoted EV-D68 replication via regulating autophagy. In the current study, we investigated the interplay between CVB3 and cellular SNAP47 using HEK293T/HeLa cell models. We showed that, upon CVB3 infection, protein levels of SNAP47 decreased independent of the activity of virus-encoded proteinase 3C. We further demonstrated that the depletion of SNAP47 inhibited CVB3 infection, indicating a pro-viral function of SNAP47. Moreover, we found that SNAP47 co-localizes with the autophagy-related protein ATG14 on the cellular membrane fractions together with viral capsid protein VP1, and expression of SNAP47 or ATG14 enhanced VP1 conjugation. Finally, we revealed that disulfide interactions had an important role in strengthening VP1 conjugation. Collectively, our study elucidated a mechanism by which SNAP47 and ATG14 promoted CVB3 propagation through facilitating viral capsid assembly.

Infection by RNA viruses causes extensive cellular reorganization, including hijacking of membranes to create membranous structures termed replication organelles, which support viral RNA synthesis and virion assembly. In this study, we show that infection with coxsackievirus B3 entails a profound impairment of the protein homeostasis at virus-utilized membranes, reflected by an accumulation of ubiquitinylated proteins, including K48-linked polyubiquitin conjugates, known to direct proteins to proteasomal degradation. The enrichment of membrane-bound ubiquitin conjugates is attributed to the presence of the non-structural viral proteins 2B and 3A, which are known to perturb membrane integrity and can cause an extensive rearrangement of cellular membranes. The locally increased abundance of ubiquitinylated proteins occurs without an increase of oxidatively damaged proteins. During the exponential phase of replication, the oxidative damage of membrane proteins is even diminished, an effect we attribute to the recruitment of glutathione, which is known to be required for the formation of infectious virus particles. Furthermore, we show that the proteasome contributes to the processing of viral precursor proteins. Taken together, we demonstrate how an infection with coxsackievirus B3 affects the cellular protein and redox homeostasis locally at the site of viral replication and virus assembly.

Protease-activated receptor 1 (PAR1) is widely expressed in humans and mice, and is activated by a variety of proteases, including thrombin. Recently, we showed that PAR1 contributes to the innate immune response to viral infection. Mice with a global deficiency of PAR1 expressed lower levels of CXCL10 and had increased Coxsackievirus B3 (CVB3)-induced myocarditis compared with control mice. In this study, we determined the effect of cell type-specific deletion of PAR1 in cardiac myocytes (CMs) and cardiac fibroblasts (CFs) on CVB3-induced myocarditis. Mice lacking PAR1 in either CMs or CFs exhibited increased CVB3 genomes, inflammatory infiltrates, macrophages and inflammatory mediators in the heart and increased CVB3-induced myocarditis compared with wild-type controls. Interestingly, PAR1 enhanced poly I:C induction of CXCL10 in rat CFs but not in rat neonatal CMs. Importantly, activation of PAR1 reduced CVB3 replication in murine embryonic fibroblasts and murine embryonic cardiac myocytes. In addition, we showed that PAR1 reduced autophagy in murine embryonic fibroblasts and rat H9c2 cells, which may explain how PAR1 reduces CVB3 replication. These data suggest that PAR1 on CFs protects against CVB3-induced myocarditis by enhancing the anti-viral response whereas PAR1 on both CMs and fibroblasts inhibits viral replication., (© 2021. The Author(s).)

Enterovirus uncoating receptors bind at the surface depression ("canyon") that encircles each capsid vertex causing the release of a host-derived lipid called "pocket factor" that is buried in a hydrophobic pocket formed by the major viral capsid protein, VP1. Coxsackievirus and adenovirus receptor (CAR) is a universal uncoating receptor of group B coxsackieviruses (CVB). Here, we present five high-resolution cryoEM structures of CVB representing different stages of virus infection. Structural comparisons show that the CAR penetrates deeper into the canyon than other uncoating receptors, leading to a cascade of events: collapse of the VP1 hydrophobic pocket, high-efficiency release of the pocket factor and viral uncoating and genome release under neutral pH, as compared with low pH. Furthermore, we identified a potent therapeutic antibody that can neutralize viral infection by interfering with virion-CAR interactions, destabilizing the capsid and inducing virion disruption. Together, these results define the structural basis of CVB cell entry and antibody neutralization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

An understanding of how viruses interact with their receptors is vital as this step is a major determinant of host susceptibility and disease. The enterovirus coxsackievirus A9 (CVA9) is an important pathogen responsible for respiratory infections, myocarditis, infections of the central nervous system, chronic dilated cardiomyopathy and possibly type I diabetes. CVA9 harbours an integrin- recognition motif, RGD (Arg-Gly-Asp), in the capsid protein VP1 and this motif is believed to be primarily responsible for binding to integrins αvβ6 and/or αvβ3 during cell entry. Despite the consistent conservation of RGD-flanking amino acids in multiple RGD-containing picornaviruses, the significance of these amino acids to cell tropism has not been thoroughly investigated. In this study we used 10 CVA9 mutants and a panel of cells to analyse cell tropism. We showed that CVA9 infection proceeds by either an RGD- dependent or an apparently RGD- independent pathway. Differences in the amino acid found at the +1 position of the RGD motif affect the cell tropism of CVA9 when an RGD- dependent pathway is used. Naturally occurring CVA9 isolates have either the sequence RGDM and RGDL and we found that the corresponding viruses in our panel infected cells most efficiently. There was also a strong selection pressure for RGDL in adaptation experiments. However, there was also an unexpected selection of an RGDL variant in an apparently RGD- independent cell line. There was also no simple relationship between infection of cells and expression of integrins αvβ3 and αvβ6. The results obtained have greatly improved our understanding of how CVA9 infects cells. This will be useful in the design of antivirus drugs and also gives a framework for the modification of CVA9 or other RGD containing picornaviruses for specific targeting of cancer cells for oncolytic therapy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Enteroviruses pose a persistent and widespread threat to human physical health, with no specific treatments available. Small molecule capsid binders have the potential to be developed as antivirals that prevent virus attachment and entry into host cells. To aid with broad-range drug development, we report here structures of coxsackieviruses B3 and B4 bound to different interprotomer-targeting capsid binders using single-particle cryo-EM. The EM density maps are beyond 3 Å resolution, providing detailed information about interactions in the ligand-binding pocket. Comparative analysis revealed the residues that form a conserved virion-stabilizing network at the interprotomer site, and showed the small molecule properties that allow anchoring in the pocket to inhibit virus disassembly.

Enteroviruses, including Coxsackievirus B3 (CVB3), are pervasive pathogens that cause significant disease, including cardiomyopathies. Unfortunately, no treatments or vaccines are available for infected individuals. We identified the host polyamine pathway as a potential drug target, as inhibiting polyamine biosynthesis significantly reduces enterovirus replication in vitro and in vivo. Here, we show that CVB3 is sensitive to polyamine depletion through the polyamine analog diethylnorspermidine (DENSpm), which enhances polyamine catabolism through induction of polyamine acetylation. We demonstrate that CVB3 acquires resistance to DENSpm via mutation of the 2A protease, which enhances proteolytic activity in the presence of DENSpm. Resistance to DENSpm occurred via mutation of a non-catalytic site mutation and results in decreased fitness. These data demonstrate that potential for targeting polyamine catabolism as an antiviral target as well as highlight a potential mechanism of resistance.

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence.

Viral myocarditis is caused by a viral infection and characterized by the inflammation of the myocardium. Coxsackievirus B3 (CVB3) infection is one of the most common among the infections caused by this virus. The host's early innate immune response to CVB3 infection particularly depends on the functions of type I interferons (IFNs). In this study, we report that a host microRNA, miR-30a, was upregulated by CVB3 to facilitate its replication. We demonstrated that miR-30a was a potent negative regulator of IFN-I signaling by targeting tripartite motif protein 25 (TRIM25). In addition, we found that TRIM25 overexpression significantly suppressed CVB3 replication, whereas TRIM25 knockdown increased viral titer and VP1 protein expression. MiR-30a inhibits the expression of TRIM25 and TRIM25-mediated retinoic acid-inducible gene (RIG)-I ubiquitination to suppress IFN-β activation and production, thereby resulting in the enhancement of CVB3 replication. These results indicate the proviral role of miR-30a in modulating CVB3 infection for the first time. This not only provides a new strategy followed by CVB3 in order to modulate IFN-I-mediated antiviral immune responses by engaging host miR-30a but also improves our understanding of its pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Xie, Li, Li, Shen, Gong and Zhang.)

C1q/TNF-related protein 9 (CTRP9) is implicated in diverse cardiovascular diseases, but its role in viral myocarditis (VMC) is not well explored. This study is aimed at investigating the role and potential mechanism of CTRP9 in VMC. Herein, we found that the peripheral blood collected from children with VMC had lower CTRP9 levels than that from children who had recovered from VMC. H9c2 cardiomyocytes treated with coxsackievirus B3 (CVB3) were applied to establish a VMC model in vitro , and the expression of CTRP9 was significantly decreased in CVB3-induced H9c2 cells. The overexpression of CTRP9 attenuated CVB3-induced apoptosis, inflammation, and fibrosis reactions in H9c2 cells by promoting cell proliferation, reducing the cell apoptosis rate, and inhibiting inflammatory cytokine levels and fibrosis-related gene expression. Moreover, we found that thrombospondin 1 (THBS1) levels were increased in children with VMC, and CTRP9 negatively regulated THBS1 expression by interacting with THBS1. The downregulation of THBS1 inhibited CVB3-induced apoptosis, inflammation, and fibrosis in H9c2 cells. In addition, our mechanistic investigation indicated that the overexpression of THBS1 impaired the inhibitory effect of CTRP9 on CVB3-induced H9c2 cells. The results further revealed that the CVB3-induced NF- κ B and TGF- β 1/Smad2/3 signaling pathways of H9c2 cells were blocked by CTRP9 yet activated by THBS1. In conclusion, CTRP9 protected H9c2 cells from CVB3-induced injury via the NF- κ B and TGF- β 1/Smad2/3 signaling pathways by modulating THBS1., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Kebei Liu et al.)

Enteroviruses are suspected to contribute to insulin-producing β cell loss and hyperglycemia-induced diabetes. However, mechanisms are not fully defined. Here, we show that coxsackievirus B type 4 (CVB4) infection in human islet-engrafted mice and in rat insulinoma cells displays loss of unconventional prefoldin RPB5 interactor (URI) and PDX1, affecting β cell function and identity. Genetic URI ablation in the mouse pancreas causes PDX1 depletion in β cells. Importantly, diabetic PDX1 heterozygous mice overexpressing URI in β cells are more glucose tolerant. Mechanistically, URI loss triggers estrogen receptor nuclear translocation leading to DNA methyltransferase 1 (DNMT1) expression, which induces Pdx1 promoter hypermethylation and silencing. Consequently, demethylating agent procainamide-mediated DNMT1 inhibition reinstates PDX1 expression and protects against diabetes in pancreatic URI-depleted mice . Finally, the β cells of human diabetes patients show correlations between viral protein 1 and URI, PDX1, and DNMT1 levels. URI and DNMT1 expression and PDX1 silencing provide a causal link between enterovirus infection and diabetes., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Coxsackie B viruses (CVB) cause a wide spectrum of diseases, ranging from mild respiratory syndromes and hand, foot, and mouth disease to life-threatening conditions, such as pancreatitis, myocarditis, and encephalitis. Previously, we and others found that the soluble virus receptor trap sCAR-Fc strongly attenuates CVB3 infection in mice. In this study, we investigated whether treatment with sCAR-Fc results in development of resistance by CVB3. Two CVB3 strains (CVB3-H3 and CVB3 Nancy) were passaged in HeLa cells in the presence of sCAR-Fc. The CVB3-H3 strain did not develop resistance, whereas two populations of CVB3 Nancy mutants emerged, one with complete (CVB3 M ) and one with partial (CVB3 K ) resistance. DNA sequence alignment of the resistant virus variant CVB3 M with CVB3 Nancy revealed an amino acid exchange from Asn(N) to Ser(S) at position 139 of the CVB3 capsid protein VP2 (N2139S), an amino acid predicted to be involved in the virus's interaction with its cognate receptor CAR. Insertion of the N2139S mutation into CVB3-H3 by site-directed mutagenesis promoted resistance of the engineered CVB3-H3 N2139S to sCAR-Fc. Interestingly, development of resistance by CVB3-H3 N2139S and the exemplarily investigated CVB3 M -clone 2 (CVB3 M2 ) against soluble CAR did not compromise the use of cellular CAR for viral infection. Infection of HeLa cells showed that sCAR-Fc resistance, however, negatively affected both virus stability and viral replication compared to that of the parental strains. These data demonstrate that during sCAR-Fc exposure, CVB3 can develop resistance against sCAR-Fc by single-amino-acid exchanges within the virus-receptor binding site, which, however, come at the expense of viral fitness. IMPORTANCE The emergence of resistant viruses is one of the most frequent obstacles preventing successful therapy of viral infections, representing a significant threat to human health. We investigated the emergence of resistant viruses during treatment with sCAR-Fc, a well-studied, highly effective antiviral molecule against CVB infections. Our data show the molecular aspects of resistant CVB3 mutants that arise during repetitive sCAR-Fc usage. However, drug resistance comes at the price of lower viral fitness. These results extend our knowledge of the development of resistance by coxsackieviruses and indicate potential limitations of antiviral therapy using soluble receptor molecules., (Copyright © 2020 Pinkert et al.)

Echovirus 30 (E30), a member of the enterovirus B species, is a major cause of viral meningitis, targeting children and adults alike. While it is a frequently isolated enterovirus and the cause of several outbreaks all over the world, surprisingly little is known regarding its entry and replication strategy within cells. In this study, we used E30 strain Bastianni (E30B) generated from an infectious cDNA clone in order to study early entry events during infection in human RD cells. E30B required the newly discovered Fc echovirus receptor (FcRn) for successful infection, but not the coxsackievirus and adenovirus receptor (CAR) or decay-accelerating factor (DAF), although an interaction with DAF was observed. Double-stranded RNA replication intermediate was generated between 2 and 3 h postinfection (p.i.), and viral capsid production was initiated between 4 and 5 h p.i. The drugs affecting Rac1 (NSC 23766) and cholesterol (filipin III) compromised infection, whereas bafilomycin A1, dyngo, U-73122, wortmannin, and nocodazole did not, suggesting the virus follows an enterovirus-triggered macropinocytic pathway rather than the clathrin pathway. Colocalization with early endosomes and increased infection due to constitutively active Rab5 expression suggests some overlap and entry to classical early endosomes. Taken together, these results suggest that E30B induces an enterovirus entry pathway, leading to uncoating in early endosomes. IMPORTANCE Echovirus 30 (E30) is a prevalent enterovirus causing regular outbreaks in both children and adults in different parts of the world. It is therefore surprising that relatively little is known of its infectious entry pathway. We set out to generate a cDNA clone and gradient purified the virus in order to study the early entry events in human cells. We have recently studied other enterovirus B group viruses, like echovirus 1 (EV1) and coxsackievirus A9 (CVA9), and found many similarities between those viruses, allowing us to define a so-called "enterovirus entry pathway." Here, E30 is reminiscent of these viruses, for example, by not relying on acidification for infectious entry. However, despite not using the clathrin entry pathway, E30 accumulates in classical early endosomes., (Copyright © 2020 American Society for Microbiology.)

Human enteroviruses (EVs), including coxsackieviruses, the numbered enteroviruses, and echoviruses, cause a wide range of diseases, such as hand, foot, and mouth disease (HFMD), encephalitis, myocarditis, acute flaccid myelitis (AFM), pneumonia, and bronchiolitis. Therefore, broad-spectrum anti-EV drugs are urgently needed to treat EV infection. Here, we demonstrate that FNC (2'-deoxy-2'-β-fluoro-4'-azidocytidine), a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs, including enterovirus 71 (EV71), coxsackievirus A16 (CA16), CA6, EVD68, and coxsackievirus B3 (CVB3), at the nanomolar level. The antiviral mechanism of FNC involves mainly positive- and negative-strand RNA synthesis inhibition by targeting and competitively inhibiting the activity of EV71 viral RNA-dependent RNA polymerase (3D pol ), as demonstrated through quantitative real-time reverse transcription-PCR (RT-qPCR), in vitro 3D pol activity, and isothermal titration calorimetry (ITC) experiments. We further demonstrated that FNC treatment every 2 days with 1 mg/kg of body weight in EV71 and CA16 infection neonatal mouse models successfully protected mice from lethal challenge with EV71 and CA16 viruses and reduced the viral load in various tissues. These findings provide important information for the clinical development of FNC as a broad-spectrum inhibitor of human EV pathogens. IMPORTANCE Human enterovirus (EV) pathogens cause various contagious diseases such as hand, foot, and mouth disease, encephalitis, myocarditis, acute flaccid myelitis, pneumonia, and bronchiolitis, which have become serious health threats. However, except for the EV71 vaccine on the market, there are no effective strategies to prevent and treat other EV pathogen infections. Therefore, broad-spectrum anti-EV drugs are urgently needed. In this study, we demonstrated that FNC, a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs at the nanomolar level. Further investigation revealed that FNC inhibits positive- and negative-strand RNA synthesis of EVs by interacting and interfering with the activity of EV71 viral RNA-dependent RNA polymerase (3D pol ). Our findings demonstrate for the first time that FNC is an effective broad-spectrum inhibitor for human EV pathogens., (Copyright © 2020 Xu et al.)

Coxsackievirus B4 (CV-B4) is suspected to be an environmental factor that has the intrinsic capacity to damage the pancreatic beta cells and therefore causes insulitis and type 1 diabetes (T1D). Although vaccination against CV-B4 could reduce the incidence of this chronic auto-immune disease, there is currently no therapeutic reagent or vaccine in clinical use. By the employment of the Bac-to-Bac® vector system to express the major viral capsid protein, we contributed towards the development of a CV-B4 vaccine by producing CV-B4 virus-like particles (VLPs) from recombinant baculovirus in infected insect cells. In fact Western blot and Immunofluorescence analysis detected the viral protein 1 (VP1) in the cells resulting from the construction of a recombinant bacmid DNA carrying the key immunogenic protein then transfected in the insect cells. Sucrose gradient ultracentrifugation fractions of the infected cell lysates contained the recombinant protein and the electron microscopy demonstrated the presence of VLPs in these sucrose fractions. This study clearly shows for the first time the expression of CVB4 VP1 structure protein alone can form VLPs in the baculovirus-infected insect cell keeping conserved both characteristics and morphology.

Protein modification with ISG15 (ISGylation) represents a major type I IFN-induced antimicrobial system. Common mechanisms of action and species-specific aspects of ISGylation, however, are still ill defined and controversial. We used a multiphasic coxsackievirus B3 (CV) infection model with a first wave resulting in hepatic injury of the liver, followed by a second wave culminating in cardiac damage. This study shows that ISGylation sets nonhematopoietic cells into a resistant state, being indispensable for CV control, which is accomplished by synergistic activity of ISG15 on antiviral IFIT1/3 proteins. Concurrent with altered energy demands, ISG15 also adapts liver metabolism during infection. Shotgun proteomics, in combination with metabolic network modeling, revealed that ISG15 increases the oxidative capacity and promotes gluconeogenesis in liver cells. Cells lacking the activity of the ISG15-specific protease USP18 exhibit increased resistance to clinically relevant CV strains, therefore suggesting that stabilizing ISGylation by inhibiting USP18 could be exploited for CV-associated human pathologies., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Three strains of coxsackievirus B3 (CVB3) differ by single mutations in capsid protein VP1 or VP3 and also differ in stability at 37°C in tissue culture medium. Among these strains, the CVB3/28 parent strain has been found to be uniquely sensitive to a component in fetal bovine serum (FBS) identified as serum albumin. In cell culture medium, serum increased the rate of CVB3/28 conversion to noninfectious particles at least 2-fold. The effect showed a saturable dose response. Rates of conversion to noninfectious virus with high concentrations of soluble coxsackievirus and adenovirus receptor (sCAR) were similar with and without FBS, but FBS amplified the catalytic effect of 100 nM sCAR nearly 3-fold. Such effects in other systems are due to nonessential activating cofactors. IMPORTANCE A factor other than the virus receptor expressed by target cells has been found to accelerate the loss of an enterovirus (CVB3/28) infectious titer, with little effect on nearly identical mutant strains. The destabilizing factor in fetal bovine serum, identified as albumin, does not interfere with the catalytic activity of soluble receptor at saturating receptor concentrations and amplifies the catalytic activity of the soluble receptor at a concentration that otherwise produces about one-third the saturated receptor-catalyzed rate of virus decay. This finding evidences the possibility that other virus-"priming" ligands may also be nonessential activating cofactors that serve to accelerate receptor-catalyzed viral eclipse., (Copyright © 2020 American Society for Microbiology.)

We report that several viruses from the human enterovirus group B cause massive vimentin rearrangements during lytic infection. Comprehensive studies suggested that viral protein synthesis was triggering the vimentin rearrangements. Blocking the host cell vimentin dynamics with β, β'-iminodipropionitrile (IDPN) did not significantly affect the production of progeny viruses and only moderately lowered the synthesis of structural proteins such as VP1. In contrast, the synthesis of the nonstructural proteins 2A, 3C, and 3D was drastically lowered. This led to attenuation of the cleavage of the host cell substrates PABP and G3BP1 and reduced caspase activation, leading to prolonged cell survival. Furthermore, the localization of the proteins differed in the infected cells. Capsid protein VP1 was found diffusely around the cytoplasm, whereas 2A and 3D followed vimentin distribution. Based on protein blotting, smaller amounts of nonstructural proteins did not result from proteasomal degradation but from lower synthesis without intact vimentin cage structure. In contrast, inhibition of Hsp90 chaperone activity, which regulates P1 maturation, lowered the amount of VP1 but had less effect on 2A. The results suggest that the vimentin dynamics regulate viral nonstructural protein synthesis while having less effect on structural protein synthesis or overall infection efficiency. The results presented here shed new light on differential fate of structural and nonstructural proteins of enteroviruses, having consequences on host cell survival. IMPORTANCE A virus needs the host cell in order to replicate and produce new progeny viruses. For this, the virus takes over the host cell and modifies it to become a factory for viral proteins. Irrespective of the specific virus family, these proteins can be divided into structural and nonstructural proteins. Structural proteins are the building blocks for the new progeny virions, whereas the nonstructural proteins orchestrate the takeover of the host cell and its functions. Here, we have shown a mechanism that viruses exploit in order to regulate the host cell. We show that viral protein synthesis induces vimentin cages, which promote production of specific viral proteins that eventually control apoptosis and host cell death. This study specifies vimentin as the key regulator of these events and indicates that viral proteins have different fates in the cells depending on their association with vimentin cages., (Copyright © 2020 Turkki et al.)

Background: Insulin-dependent Diabetes Mellitus Type 1 (T1D) also referred to as autoimmune diabetes. T1D is a chronic disease which is characterized by way of insulin deficiency. The deficiency is due to the loss of pancreatic β cells and leads to hyperglycemia. There are many factors which play a significant role in T1D disease pathogenicity including genetic predisposition, the immune system, and environmental factors. The environmental factors may include Coxsackie B4 virus, a small RNA virus., Objective: The objective of current in silico study is to identify active lead compounds against Coxsackie B4 virus, a small RNA virus which has been reported in diabetic patients after PCR. There is a need to predict inhibitors against TID caused by Coxsackie B4 viral protein that may be used as a drug against TID in the future., Methods: For this purpose, different bioinformatics databases and tools were used. The protein structure generation and validation, retrieval of ligands and their properties analysis were performed by different databases, web servers, and software tools. Moreover, the docking tools were used to identify the target site of the protein and interaction of different inhibitors with the target protein molecule., Results: Based on the analysis, two lead compounds ZINC00034488 and ZINC00034585 were selected as potential drugs. These compounds are non-toxic and have best interaction energy and fulfill Lipinski rule, Veber rule, Ghose Rule, Weighted QED, Unweighted QED and BBB likeness parameters., Conclusion: Our work will help researchers to get an idea about the understanding of chemicals against Coxsackie B4 Viruses and helpful to design a drug and test these chemicals to overcome Diabetes Mellitus Type 1 caused by Coxsackie B4 virus., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

During enteroviral infections, the canonical translation factor eukaryotic translation initiation factor 4 γ I (eIF4GI) is cleaved by viral protease 2A. The resulting C-terminal fragment is recruited by the viral internal ribosome entry site (IRES) for efficient translation of the viral RNA. However, the 2A protease is not present in the viral capsid and is synthesized only after the initial round of translation. This presents the conundrum of how the initial round of translation occurs in the absence of the C-terminal eIF4GI fragment. Interestingly, the host protein DAP5 (also known as p97, eIF4GIII, and eIF4G2), an isoform of eIF4GI, closely resembles the eIF4GI C-terminal fragment produced after 2A protease-mediated cleavage. Using the Coxsackievirus B3 (CVB3) IRES as a model system, here we demonstrate that DAP5, but not the full-length eIF4GI, is required for CVB3 IRES activity for translation of input viral RNA. Additionally, we show that DAP5 is specifically required by type I IRES but not by type II or type III IRES, in which cleavage of eIF4GI has not been observed. We observed that both DAP5 and C-terminal eIF4GI interact with CVB3 IRES in the same region, but DAP5 exhibits a lower affinity for CVB3 IRES compared with the C-terminal eIF4GI fragment. It appears that DAP5 is required for the initial round of viral RNA translation by sustaining a basal level of CVB3 IRES activity. This activity leads to expression of 2A protease and consequent robust CVB3 IRES-mediated translation by the C-terminal eIF4GI fragment., (© 2019 Dave et al.)

Coxsackievirus B3 is an enterovirus, with positive-sense single-stranded RNA genome containing 'Internal Ribosome Entry Site' (IRES) in the 5'UTR. Once sufficient viral proteins are synthesized in the cell from the input RNA, viral template switches from translation to replication to synthesize negative-strand RNA. Inhibition of translation is a key step in regulating this switch as the positive-strand RNA template should be free of ribosomes to enable polymerase movement. In this study, we show how a host protein hnRNP C1/C2 inhibits viral RNA translation. hnRNP C1/C2 interacts with stem-loop V in the IRES and displaces poly-pyrimidine tract binding protein, a positive regulator of translation. We further demonstrate that hnRNP C1/C2 induces translation to replication switch, independently from the already known role of the ternary complex (PCBP2-3CD-cloverleaf RNA). These results suggest a novel function of hnRNP C1/C2 in template switching of positive-strand from translation to replication by a new mechanism. Using mathematical modelling, we show that the differential affinity of hnRNP C1/C2 for positive and negative-strand RNAs guides the final ± RNA ratio, providing first insight in the regulation of the positive to negative-strand RNA ratio in enteroviruses.

Cell autonomous immunity is the ability of individual cells to initiate a first line of host defense against invading microbes, such as viruses. Autophagy receptors, a diverse family of multivalent proteins, play a key role in this host response by detecting, sequestering, and eliminating virus in a process termed virophagy. To counteract this, positive-stranded RNA viruses, such as enteroviruses, have evolved strategies to circumvent the host autophagic machinery in an effort to promote viral propagation; however, the underlying mechanisms remain largely unclear. Here we studied the interaction between coxsackievirus B3 (CVB3) and the autophagy receptor SQSTM1 (sequestosome 1)/p62 and CALCOCO2/NDP52 (calcium binding and coiled-coil domain-containing protein 2/nuclear dot 10 protein 52). We demonstrated that SQSTM1 and CALCOCO2 differentially regulate CVB3 infection. We showed that knockdown of SQSTM1 causes increased viral protein production and elevated viral titers, whereas depletion of CALCOCO2 results in a significant inhibition of viral growth. Both receptors appear to have a role in virophagy through direct interaction with the viral capsid protein VP1 that undergoes ubiquitination during infection. Further investigation of the proviral mechanism of CALCOCO2 revealed that CALCOCO2, but not SQSTM1, suppresses the antiviral type I interferon signaling by promoting autophagy-mediated degradation of the mitochondrial antiviral signaling (MAVS) protein. Moreover, we demonstrated that viral proteinase 2A-mediated cleavage of SQSTM1 at glycine 241 impairs its capacity to associate with viral capsid, whereas cleavage of CALCOCO2 by viral proteinase 3C at glutamine 139, generates a stable C-terminal fragment that retains the proviral function of full-length CALCOCO2. Altogether, our study reveals a mechanism by which CVB3 targets selective autophagy receptors to evade host virophagy.

Enterovirus B (EV-B), a major proportion of the genus Enterovirus in the family Picornaviridae, is the causative agent of severe human infectious diseases. Although cellular receptors for coxsackievirus B in EV-B have been identified, receptors mediating virus entry, especially the uncoating process of echovirus and other EV-B remain obscure. Here, we found that human neonatal Fc receptor (FcRn) is the uncoating receptor for major EV-B. FcRn binds to the virus particles in the "canyon" through its FCGRT subunit. By obtaining multiple cryo-electron microscopy structures at different stages of virus entry at atomic or near-atomic resolution, we deciphered the underlying mechanisms of enterovirus attachment and uncoating. These structures revealed that different from the attachment receptor CD55, binding of FcRn to the virions induces efficient release of "pocket factor" under acidic conditions and initiates the conformational changes in viral particle, providing a structural basis for understanding the mechanisms of enterovirus entry., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Infection by enteroviruses can cause severe neurological complications in humans. The interactions between the enteroviral and host proteins may facilitate the virus replication and be involved in the pathogenicity of infected individuals. It has been shown that human enteroviruses possess various mechanisms to suppress host innate immune responses in infected cells. Previous studies showed that infection by enterovirus 71 (EV71) causes the degradation of MDA5, which is a critical cytoplasmic pathogen sensor in the recognition of picornaviruses for initiating transcription of type I interferons. In the present study, we demonstrated that the RNA-dependent RNA polymerase (RdRP; also denoted 3D pol ) encoded by EV71 interacts with the caspase activation and recruitment domains (CARDs) of MDA5 and plays a role in the inhibition of MDA5-mediated beta interferon (IFN-β) promoter activation and mRNA expression. In addition, we found that the 3D pol protein encoded by coxsackievirus B3 also interacted with MDA5 and downregulated the antiviral signaling initiated by MDA5. These findings indicate that enteroviral RdRP may function as an antagonist against the host antiviral innate immune response. IMPORTANCE Infection by enteroviruses causes severe neurological complications in humans. Human enteroviruses possess various mechanisms to suppress the host type I interferon (IFN) response in infected cells to establish viral replication. In the present study, we found that the enteroviral 3D pol protein (or RdRP), which is a viral RNA-dependent RNA polymerase for replicating viral RNA, plays a role in the inhibition of MDA5-mediated beta interferon (IFN-β) promoter activation. We further demonstrated that enteroviral 3D pol protein interacts with the caspase activation and recruitment domains (CARDs) of MDA5. These findings indicate that enteroviral RdRP functions as an antagonist against the host antiviral response., (Copyright © 2019 American Society for Microbiology.)

The aim of this study was to determine the role of the human neonatal receptor for the Fc fragment of IgG (hFcRn) as a common uncoating cellular receptor for echoviruses and coxsackievirus A9 during infection of human rhabdomyosarcoma (RD) cells., Material and Methods: The protective effect of the human serum albumin, purified from globulins, (HSAGF) and antibodies to hFcRn was studied in RD cells infected with several strains and clones of species B enteroviruses possessing different receptor specificity (echoviruses 3, 9, 11, 30 and coxsackieviruses A9, B4, B5)., Results: It was shown that HSA-GF at concentrations of 4% or less protected RD cells from infection with echoviruses 3, 9, 11 and coxsackievirus A9. The antibodies to hFcRn at concentrations of 2.5 ug/mL or less demonstrated the similar spectrum of protective activity in RD cells against infection with echoviruses 3, 9, 11, 30 and coxsackievirus A9. The protective effect of HSA-GF or the antibodies to hFcRn was not observed in RD cells infected with coxsackieviruses B4 and B5 that need coxsackievirus-adenovirus receptor for uncoating., Discussion: The usage of the previously characterized echovirus 11 clonal variants with different receptor specificity allowed us to define the function of hFcRn as a canyon-binding uncoating receptor in RD cells. The kinetics and magnitude of the observed protective effects correlated with receptor specificity of the enteroviruses used in this work supporting the two-step interaction of DAF-dependent echoviruses with the cellular receptors., Conclusions: In this study, the function of hFcRn was defined in RD cells as a canyon-binding and uncoating receptor for echoviruses and coxsackievirus A9. The two-step interaction of DAF-dependent echoviruses during entry into the cells was confirmed: initially with the binding receptor DAF and subsequently with the uncoating receptor hFcRn., Competing Interests: The authors declare no conflict of interest.

Coxsackievirus B3 (CVB3) is the predominant pathogen of viral myocarditis. In our previous study, we found that CVB3 caused abnormal lipid accumulation in host cells. However, the underlying mechanisms by which CVB3 disrupts and exploits the host lipid metabolism are not well understood. Sterol regulatory element binding protein 1 (SREBP1) is the major transcriptional factor in lipogenic genes expression. In this study, we demonstrated that CVB3 infection and nonstructural 2A protein upregulated and activated SREBP1a at the transcriptional level. Deletion analysis of SREBP1a promoter revealed that two regions, -1821/-1490 and -312/+217, in this promoter were both required for its activation by 2A. These promoter regions possessed several binding motifs for transcription factor SP1. Next, we used SP1-specific small interfering RNAs (siRNAs) to confirm that SP1 might be the essential factor in SREBP1a upregulation by 2A. Furthermore, we showed that MEK/ERK pathway was involved in the activation of SREBP1a by 2A and that blocking this signaling pathway with the specific inhibitor U0126 attenuated SREBP1a activation and lipid accumulation by 2A. Finally, we showed that inhibition of SREBP1 with siRNAs attenuated lipid accumulation induced by CVB3 infection and reduced virus replication. Moreover, inhibition of the MEK/ERK pathway also led to reduction of SREBP1a activation, lipid accumulation, and virus replication during CVB3 infection. Taken together, these data demonstrate that CVB3 nonstructural 2A protein activates SREBP1a at the transcription level through a mechanism involving MEK/ERK signaling pathway and SP1 transcription factor, which promotes cellular lipid accumulation and benefits virus replication. IMPORTANCE Coxsackievirus B3 (CVB3) infection is the leading cause of viral myocarditis, but effective vaccines and antiviral therapies against CVB3 infection are still lacking. It is important to understand the precise interactions between host and virus for the rational design of effective therapies. During infection, CVB3 disrupts and exploits host lipid metabolism to promote excessive lipid accumulation, which benefits virus replication. SREBP1 is the master regulator of cellular lipid metabolism. Here, we report that one of the viral nonstructural proteins, 2A, upregulates and activates SREBP1a. Furthermore, we find that inhibition of SREBP1 decreases CVB3 virus replication. These results reveal the regulation of SREBP1a expression by 2A and the roles of SREBP1 in lipid accumulation and viral replication during CVB3 infection. Our findings provide a new insight into CVB3 host interactions and inform a potential novel therapeutic target for this important pathogen., (Copyright © 2018 American Society for Microbiology.)

Replicative ability of 5 oncolytic enterovirus strains was evaluated on a panel of 18 human normal and tumor cells. The capacity of each cell line to support replication of enterovirus strains varied. Cell lines weakly replicating one virus could be highly sensitive to another viral strain. Differences in the expression of CXADR cell receptor did not correlate with susceptibility to infection and replication of Coxsackie B virus, but complete inactivation of CXADR gene and poliovirus receptor gene (PVR) led to loss of the sensitivity to Coxsackie B5 and poliovirus, respectively. Detection of additional expression markers will contribute to understanding the causes of different sensitivity of tumor cells to viruses.

Coxsackievirus B3 (CVB3), a single-stranded RNA virus of the picornavirus family, has been described as a novel oncolytic virus. However, the CVB3 strain used induced hepatitis and myocarditis in vivo. It was hypothesized that oncolytic activity and safety of CVB3 depends on the virus strain and its specific receptor tropism. Different laboratory strains of CVB3 (Nancy, 31-1-93, and H3), which use the coxsackievirus and adenovirus receptor (CAR), and the strain PD, which uses N- and 6-O-sulfated heparan sulfate (HS) for entry into the cells, were investigated for their potential to lyse tumor cells and for their safety profile. The investigations were carried out in colorectal carcinoma. In vitro investigations showed variable infection efficiency and lysis of colorectal carcinoma cell lines by the CVB3 strains. The most efficient strain was PD, which was the only one that could lyse all investigated colorectal carcinoma cell lines. Lytic activity of CAR-dependent CVB3 did not correlate with CAR expression on cells, whereas there was a clear correlation between lytic activity of PD and its ability to bind to HS at the cell surface of colorectal carcinoma cells. Intratumoral injection of Nancy, 31-1-93, or PD into subcutaneous colorectal DLD1 cell tumors in BALB/c nude mice resulted in strong inhibition of tumor growth. The effect was seen in the injected tumor, as well as in a non-injected, contralateral tumor. However, all animals treated with 31-1-93 and Nancy developed systemic infection and died or were moribund and sacrificed within 8 days post virus injection. In contrast, five of the six animals treated with PD showed no signs of a systemic viral infection, and PD was not detected in any organ. The data demonstrate the potential of PD as a new oncolytic virus and HS-binding of PD as a key feature of oncolytic activity and improved safety.

Viral myocarditis is a cardiovascular disease that seriously affects human health. Its mechanism is not clear. Coxsackievirus B3 (CVB3) is a member of the picornavirus family and is the leading cause of viral myocarditis. Our group tested the genes in a mouse model of CVB3 virus infection and confirmed that the NADPH oxidase gene had a high expression trend in the acute phase of infection. Whether Nox4, the homologue of NADPH oxidase, participates in the process of viral myocarditis has not been reported. In this study, we found increased expression of Nox4 in viral myocarditis in vivo and in vitro. DPI is a non-specific inhibitor of Nox4 that improved CVB3-induced myocarditis after injection in vivo. DPI also inhibited intracellular ROS release and apoptosis in vitro. Our data indicated that Nox4-dependent ROS production was involved in CVB3-induced myocardial apoptosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Thymomas are associated with autoantibody formation. The most common are anti-acetylcholine receptor antibodies, which correspond to myasthenia gravis (MG). Other autoantibodies, such as antistriational antibodies, can occur, but their relation to clinical syndromes is frequently uncertain. The etiology of antistriational antibodies is also poorly understood. In this case, a 61-year-old man with a history of thymoma was admitted with respiratory failure. The patient was positive for anti-acetylcholine receptor antibodies and antistriational antibodies. He developed cardiogenic shock and died within 2 days despite aggressive therapy. Laboratory studies revealed elevated cardiac enzymes and marked IgG elevation against Coxsackie A virus serotypes 9 and 24. Subclinical IgG elevations against additional Coxsackie A and Coxsackie B virus serotypes were also noted. Autopsy revealed lymphohistiocytic infiltrates with multinucleated giant cells in the myocardium and skeletal muscles, including the diaphragm. Giant cell polymyositis and myocarditis is a rare, lethal complication in patients with thymoma and MG. The pathogenesis is uncertain. An autoimmune process, possibly elicited by antistriational antibodies, has been suggested. The coexistence of antistriational antibodies and Coxsackie viral serologies has not been reported. This case may suggest that giant cell polymyositis and myocarditis in patients with thymoma and MG is a postviral autoimmune process.

Coxsackie B Virus (CBV) infection has been linked to the aetiology of type 1 diabetes (T1D) and vaccination has been proposed as prophylaxis for disease prevention. Serum neutralising antibodies and the presence of viral protein and RNA in tissues have been common tools to examine this potential disease relationship, whilst the role of anti-CBV cytotoxic T cell responses and their targets have not been studied. To address this knowledge gap, we augmented conventional HLA-binding predictive algorithm-based epitope discovery by cross-referencing epitopes with sites of positive natural selection within the CBV3 viral genome, identified using mixed effects models of evolution. Eight epitopes for the common MHC class I allele HLA-A*0201 occur at sites that appear to be positively selected. Furthermore, such epitopes span the viral genome, indicating that effective anti-viral responses may not be restricted to the capsid region. To assess the spectrum of IFNy responses in non-diabetic subjects and recently diagnosed type 1 diabetes (T1D) patients, we stimulated PBMC ex vivo with pools of synthetic peptides based on component-restricted sequences identified in silico. We found responders were more likely to recognize multiple rather than a single CBV peptide pool, indicating that the natural course of infection results in multiple targets for effector memory responses, rather than immunodominant epitopes or viral components. The finding that anti-CBV CD8 T cell immunity is broadly targeted has implications for vaccination strategies and studies on the pathogenesis of CBV-linked diseases., Competing Interests: The authors have declared that no competing interests exist.

In this study, we investigated the roles of RIP1/RIP3 mediated cardiomyocyte necroptosis in CVB3-induced acute myocarditis. Serum concentrations of creatinine kinase (CK), CK-MB, and cardiac troponin I were detected using a Hitachi Automatic Biochemical Analyzer in a mouse model of acute VMC. Histological changes in cardiac tissue were observed by light microscope and expression levels of RIP1/RIP3 in the cardiac tissue were detected via Western blot and immunohistochemistry. The data showed that RIP1/RIP3 was highly expressed in cardiomyocytes in the acute VMC mouse model and that the necroptosis pathway specific blocker, Nec-1, dramatically reduced the myocardial damage by downregulating the expression of RIP1/RIP3. These findings provide evidence that necroptosis plays a significant role in cardiomyocyte death and it is a major pathway for cell death in acute VMC. Blocking the necroptosis pathway may serve as a new therapeutic option for the treatment of acute viral myocarditis.

Background: Echovirus (E) 30 (E-30) meningitis is characterized by neuroinflammation involving immune cell pleocytosis at the protective barriers of the central nervous system (CNS). In this context, infection of the blood-cerebrospinal fluid barrier (BCSFB), which has been demonstrated to be involved in enteroviral CNS pathogenesis, may affect the tight junction (TJ) and adherens junction (AJ) function and morphology., Methods: We used an in vitro human choroid plexus epithelial (HIBCPP) cell model to investigate the effect of three clinical outbreak strains (13-311, 13-759, and 14-397) isolated in Germany in 2013, and compared them to E-30 Bastianni. Conducting transepithelial electrical resistance (TEER), paracellular dextran flux measurement, quantitative real-time polymerase chain reaction (qPCR), western blot, and immunofluorescence analysis, we investigated TJ and AJ function and morphology as well as strain-specific E-30 infection patterns. Additionally, transmission electron and focused ion beam microscopy electron microscopy (FIB-SEM) was used to evaluate the mode of leukocyte transmigration. Genome sequencing and phylogenetic analyses were performed to discriminate potential genetic differences among the outbreak strains., Results: We observed a significant strain-dependent decrease in TEER with strains E-30 Bastianni and 13-311, whereas paracellular dextran flux was only affected by E-30 Bastianni. Despite strong similarities among the outbreak strains in replication characteristics and particle distribution, strain 13-311 was the only outbreak isolate revealing comparable disruptive effects on TJ (Zonula Occludens (ZO) 1 and occludin) and AJ (E-cadherin) morphology to E-30 Bastianni. Notwithstanding significant junctional alterations upon E-30 infection, we observed both para- and transcellular leukocyte migration across HIBCPP cells. Complete genome sequencing revealed differences between the strains analyzed, but no explicit correlation with the observed strain-dependent effects on HIBCPP cells was possible., Conclusion: The findings revealed distinct E-30 strain-specific effects on barrier integrity and junctional morphology. Despite E-30-induced barrier alterations leukocyte trafficking did not exclusively occur via the paracellular route.