Major Group-B Enterovirus populations deleted in the noncoding 5' region of genomic RNA modulate activation of the type I interferon pathway in cardiomyocytes and induce myocarditis.

Major 5'-terminally deleted (5'TD) RNA forms of group-B coxsackievirus (CVB-5'TD) has been associated with myocarditis in both mice and humans. Although it is known that interferon-ß (IFN-ß) signaling is critical for an efficient innate immune response against CVB-induced myocarditis, the link between CVB-5'TD RNA forms and type I IFN signaling in cardiomyocytes remains to be explored. In a mouse model of CVB3/28-induced myocarditis, major early-emerging forms of CVB-5'TD RNA have been characterized as replicative viral populations that impair IFN-ß production in the heart. Synthetic CVB3/28 RNA forms mimicking each of these major 5'TD virus populations were transfected in mice and have been shown to modulate innate immune responses in the heart and to induce myocarditis in mice. Remarkably, transfection of synthetic viral RNA with deletions in the secondary structures of the 5'-terminal CVB3 RNA domain I, modifying stem-loops "b", "c" or "d", were found to impair IFN-ß production in human cardiomyocytes. In addition, the activation of innate immune response by Poly(I:C), was found to restore IFN-ß production and to reduce the burden of CVB-5'TD RNA-forms in cardiac tissues, thereby reducing the mortality rate of infected mice. Overall, our results indicate that major early-emerging CVB3 populations deleted in the domain I of genomic RNA, in the 5' noncoding region, modulate the activation of the type I IFN pathway in cardiomyocytes and induce myocarditis in mice. These findings shed new light on the role of replicative CVB-5'TD RNA forms as key pathophysiological factors in CVB-induced human myocarditis.

ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection.

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.

MicroRNA-22-3p displaces critical host factors from the 5' UTR and inhibits the translation of Coxsackievirus B3 RNA.

Coxsackievirus B3 (CVB3) is known to cause acute myocarditis and pancreatitis in humans. We investigated the microRNAs (miRNAs) that can potentially govern the viral life cycle by binding to the untranslated regions (UTRs) of CVB3 RNA. MicroRNA-22-3p was short-listed, as its potential binding site overlapped with the region crucial for recruiting internal ribosome entry site trans-acting factors (ITAFs) and ribosomes. We demonstrate that miR-22-3p binds CVB3 5' UTR, hinders recruitment of key ITAFs on viral mRNA, disrupts the spatial structure required for ribosome recruitment, and ultimately blocks translation. Likewise, cells lacking miR-22-3p exhibited heightened CVB3 infection compared to wild type, confirming its role in controlling infection. Interestingly, miR-22-3p level was found to be increased at 4 hours post-infection, potentially due to the accumulation of viral 2A protease in the early phase of infection. 2Apro enhances the miR-22-3p level to dislodge the ITAFs from the SD-like sequence, rendering the viral RNA accessible for binding of replication factors to switch to replication. Furthermore, one of the cellular targets of miR-22-3p, protocadherin-1 (PCDH1), was significantly downregulated during CVB3 infection. Partial silencing of PCDH1 reduced viral replication, demonstrating its proviral role. Interestingly, upon CVB3 infection in mice, miR-22-3p level was found to be downregulated only in the small intestine, the primary target organ, indicating its possible role in influencing tissue tropism. It appears miR-22-3p plays a dual role during infection by binding viral RNA to aid its life cycle as a viral strategy and by targeting a proviral protein to restrict viral replication as a host response.IMPORTANCECVB3 infection is associated with the development of end-stage heart diseases. Lack of effective anti-viral treatments and vaccines for CVB3 necessitates comprehensive understanding of the molecular players during CVB3 infection. miRNAs have emerged as promising targets for anti-viral strategies. Here, we demonstrate that miR-22-3p binds to 5' UTR and inhibits viral RNA translation at the later stage of infection to promote viral RNA replication. Conversely, as host response, it targets PCDH1, a proviral factor, to discourage viral propagation. miR-22-3p also influences CVB3 tissue tropism. Deciphering the multifaced role of miR-22-3p during CVB3 infection unravels the necessary molecular insights, which can be exploited for novel intervening strategies to curb infection and restrict viral pathogenesis.

Complement components regulates ferroptosis in CVB3 viral myocarditis by interatction with TFRC.

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.

The lncRNA MEG3/miRNA-21/P38MAPK axis inhibits coxsackievirus 3 replication in acute viral myocarditis.

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.

Macrophage CAPN4 regulates CVB3-induced cardiac inflammation and injury by promoting NLRP3 inflammasome activation and phenotypic transformation to the inflammatory subtype.

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 (CAPN4flox/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.

Critical roles of parkin and PINK1 in coxsackievirus B3-induced viral myocarditis.

Viral myocarditis is an inflammatory disease of the myocardium, often leads to cardiac dysfunction and death. PARKIN (PRKN) and PINK1, well known as Parkinson's disease-associated genes, have been reported to be involved in innate immunity and mitochondrial damage control. Therefore, we investigated the role of parkin and PINK1 in coxsackievirus B3 (CVB3)-induced viral myocarditis because the etiology of myocarditis is related to abnormal immune response to viral infection and mitochondrial damage. After viral infection, the survival was significantly lower and myocardial damage was more severe in parkin knockout (KO) and PINK1 KO mice compared to wild-type (WT) mice. Parkin KO and PINK1 KO showed defective immune cell recruitment and impaired production of antiviral cytokines such as interferon-gamma, allowing increased viral replication. In addition, parkin KO and PINK1 KO mice were more susceptible to CVB3-induced mitochondrial damage than WT mice, resulting in susceptibility to viral-induced cardiac damage. Finally, using publicly available RNA-seq data, we found that pathogenic mutants of the PRKN gene are more common in patients with dilated cardiomyopathy and myocarditis than in controls or the general population. This study will help elucidate the molecular mechanism of CVB3-induced viral myocarditis.

Role of RNA Domain Structure and Orientation in the Coxsackievirus B3 Virulence Phenotype.

Coxsackievirus B3 (CVB3) is an enterovirus that causes diseases such as pancreatitis and myocarditis in humans. Approximately 10% of the CVB3 RNA genome consists of a highly structured 5' untranslated region (5' UTR) that is organized into six domains and contains a type I internal ribosome entry site (IRES). These features are common to all enteroviruses. Each RNA domain plays a vital role in translation and replication during the viral multiplication cycle. We used SHAPE-MaP chemistry to generate secondary structures of the 5' UTR from the avirulent strain CVB3/GA and the virulent strain CVB3/28. Our comparative models show how key nucleotide substitutions cause major restructuring of domains II and III of the 5' UTR in CVB3/GA. Despite these structural shifts, the molecule maintains several well-characterized RNA elements, which allows persistence of the unique avirulent strain. The results shed light on the 5' UTR regions serving as virulence determinants and those required for fundamental viral mechanisms. We used the SHAPE-MaP data to produce theoretical tertiary models using 3dRNA v2.0. These models suggest a compact conformation of the 5' UTR from the virulent strain CVB3/28 that brings critical domains into close contact. In contrast, the model of the 5' UTR from the avirulent strain CVB3/GA suggests a more extended conformation where the same critical domains are more separated. Our results suggest that the structure and orientation of RNA domains in the 5' UTR are responsible for low-efficiency translation, low viral titers, and absence of virulence observed during infection by CVB3/GA. IMPORTANCE Human enteroviruses, which include five different species and over 100 serotypes, are responsible for diseases ranging from mild respiratory infections to serious infections of pancreas, heart, and neural tissue. All enteroviral RNA genomes have a long and highly structured 5' untranslated region (5' UTR) containing an internal ribosome entry site (IRES). Major virulence determinants are located in the 5' UTR. We present RNA structure models that directly compare the 5' UTR derived from virulent and avirulent strains of the enterovirus coxsackievirus B3 (CVB3). The secondary-structure models show rearrangement of RNA domains known to be virulence determinants and conservation of structure in RNA elements known to be vital for translation and replication in the avirulent strain CVB3/GA. The tertiary-structure models reveal reorientation of RNA domains in CVB3/GA. Identifying the details of structure in these critical RNA domains will help direct antiviral approaches to this major human pathogen.

Macrophage-Specific Coxsackievirus and Adenovirus Receptor Deletion Enhances Macrophage M1 Polarity in CVB3-Induced Myocarditis.

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-1ß 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-1ß, 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 ß) 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 ß 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.

LncGBP9 knockdown alleviates myocardial inflammation and apoptosis in mice with acute viral myocarditis via suppressing NF-κB signaling pathway.

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-1ß) 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.

TFRC upregulation promotes ferroptosis in CVB3 infection via nucleus recruitment of Sp1.

CVB3 is a single positive-strand enterovirus, and a common pathogen in myocarditis etiology. Although a number of antiviral candidates are under development, specific targeted therapy is not available for CVB3. Ferroptosis is a new type of regulatory cell death discovered in recent years. In this study, our team provided the first evidence that ferroptosis existed in CVB3 infection in vivo and in vitro by iron overload, and massive accumulation of lipid peroxides. Mechanistically, we construct a classical model of HeLa cells following a time-course infection (6, 12, 24, 36, 48 h) with CVB3 (MOI = 10). We demonstrated that the TFRC gene plays an important role in promoting ferroptosis in CVB3 infection and downregulation of TFRC attenuated the ferroptosis. Interestingly, we observed that TFRC was nuclear translocation induced by the CVB3, which was predominantly localized in the cell membrane, but redistributed to the nucleus during CVB3 infection. Moreover, we found that the transcription factor Sp1 was an essential factor that could bind to the TFRC promoter and upregulate the TFRC transcription. Collectively, these results suggest that the Sp1/TFRC/Fe axis may provide a new target for the development of therapies against CVB3 infection.

Poly(rC) binding protein 1 benefits coxsackievirus B3 infection via suppressing the translation of p62/SQSTM1.

Coxsackievirus B3 (CVB3) is a positive single-strand RNA virus causing myocarditis, pancreatitis and meningitis. During CVB3 infection, various host cellular components, including proteins and non-coding RNAs, interact with the virus and affect viral infection. Poly(rC) binding protein 1 (PCBP1) is a multifunctional RNA binding protein regulating transcription, translation and mRNA stability of a variety of genes. In this study, we observed a significant reduction of PCBP1 protein during CVB3 infection. By bioinformatic prediction and luciferase-assay verification, we confirmed that the expression of PCBP1 was directly inhibited by miR-21, a microRNA upregulated during CVB3 infection. Furthermore, we found that overexpression of PCBP1 promoted CVB3 infection and knocking down of PCBP1 inhibited it. In the subsequent mechanism study, our results revealed that PCBP1 blocked the translation of p62/SQSTM1 (sequestosome 1), an autophagy-receptor protein suppressing CVB3 replication, by interacting with the cis-element in the 5' untranslational region (5' UTR) of p62/SQSTM1. In summary, our studies have identified PCBP1 as a beneficial factor for CVB3 infection. These findings may deepen the understanding of host-virus interactions and provide a potential target for intervention of CVB3 infection.

Ligand-of-Numb protein X1 controls the coxsackievirus B3-induced myocarditis via regulating the stability of coxsackievirus and adenovirus receptor.

Group B coxsackieviruses (CVBs) are the main cause of virus-induced myocarditis. CVBs use coxsackievirus and adenovirus receptor (CAR) for infection and targeting CAR has been shown to ameliorate CVBs-induced myocarditis. Ligand-of-Numb protein X1 (LNX1) is an E3 ubiquitin ligase that was shown to interact with CAR. However, the precise effect of LNX1 on CAR and the roles of LNX1 on CVBs-induced myocarditis remain unknown. In the present study, we generated mice deficient in LNX1 in the heart and evaluated the symptoms of myocarditis after CVB3 infection. We also monitored the expression and ubiquitination of CAR in LNX1-deficient cardiomyocytes after CVBs infection. We found that CVBs infection decreased CAR expression while promoted the expression of LNX1. Mice with deficiency of LNX1 in the heart had normal myocardial development while had deteriorated myocarditis symptoms after CVB3 infection. In LNX1-deficient cardiomyocytes, decreased ubiquitination of CAR and upregulation of CAR were observed after CVB3 infection. In summary, LNX1 controls CVB3-induced myocarditis by regulating the expression of CAR.

The Disruption of the Endothelial Barrier Contributes to Acute Lung Injury Induced by Coxsackievirus A2 Infection in Mice.

Sporadic occurrences and outbreaks of hand, foot, and mouth disease (HFMD) caused by Coxsackievirus A2 (CVA2) have frequently reported worldwide recently, which pose a great challenge to public health. Epidemiological studies have suggested that the main cause of death in critical patients is pulmonary edema. However, the pathogenesis of this underlying comorbidity remains unclear. In this study, we utilized the 5-day-old BALB/c mouse model of lethal CVA2 infection to evaluate lung damage. We found that the permeability of lung microvascular was significantly increased after CVA2 infection. We also observed the direct infection and apoptosis of lung endothelial cells as well as the destruction of tight junctions between endothelial cells. CVA2 infection led to the degradation of tight junction proteins (e.g., ZO-1, claudin-5, and occludin). The gene transcription levels of von Willebrand factor (vWF), endothelin (ET), thrombomodulin (THBD), granular membrane protein 140 (GMP140), and intercellular cell adhesion molecule-1 (ICAM-1) related to endothelial dysfunction were all significantly increased. Additionally, CVA2 infection induced the increased expression of inflammatory cytokines (IL-6, IL-1ß, and MCP-1) and the activation of p38 mitogen-activated protein kinase (MAPK). In conclusion, the disruption of the endothelial barrier contributes to acute lung injury induced by CVA2 infection; targeting p38-MAPK signaling may provide a therapeutic approach for pulmonary edema in critical infections of HFMD.

Comparison of Monoclonal Gammopathies Linked to Poliovirus or Coxsackievirus vs. Other Infectious Pathogens.

Chronic stimulation by infectious pathogens or self-antigen glucosylsphingosine (GlcSph) can lead to monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). Novel assays such as the multiplex infectious antigen microarray (MIAA) and GlcSph assays, permit identification of targets for >60% purified monoclonal immunoglobulins (Igs). Searching for additional targets, we selected 28 purified monoclonal Igs whose antigen was not represented on the MIAA and GlcSph assays; their specificity of recognition was then analyzed using microarrays consisting of 3760 B-cell epitopes from 196 pathogens. The peptide sequences PALTAVETG and PALTAAETG of the VP1 coat proteins of human poliovirus 1/3 and coxsackievirus B1/B3, respectively, were specifically recognized by 6/28 monoclonal Igs. Re-analysis of patient cohorts showed that purified monoclonal Igs from 10/155 MGUS/SM (6.5%) and 3/147 MM (2.0%) bound to the PALTAVETG or PALTAAETG epitopes. Altogether, PALTAV/AETG-initiated MGUS are not rare and few seem to evolve toward myeloma.

A Crucial Role of ACBD3 Required for Coxsackievirus Infection in Animal Model Developed by AAV-Mediated CRISPR Genome Editing Technique.

Genetic screens using CRISPR/Cas9 have been exploited to discover host-virus interactions. These screens have identified viral dependencies on host proteins during their life cycle and potential antiviral strategies. The acyl-CoA binding domain containing 3 (ACBD3) was identified as an essential host factor for the Coxsackievirus B3 (CVB3) infection. Other groups have also investigated the role of ACBD3 as a host factor for diverse enteroviruses in cultured cells. However, it has not been tested if ACBD3 is required in the animal model of CVB3 infection. Owing to embryonic lethality, conventional knockout mice were not available for in vivo study. As an alternative approach, we used adeno-associated virus (AAV)-mediated CRISPR genome editing to generate mice that lacked ACBD3 within the pancreas, the major target organ for CVB3. Delivery of sgRNAs using self-complementary (sc) AAV8 efficiently induced a loss-of-function mutation in the pancreas of the Cas9 knock-in mice. Loss of ACBD3 in the pancreas resulted in a 100-fold reduction in the CVB3 titer within the pancreas and a noticeable reduction in viral protein expression. These results indicate a crucial function of ACBD3 in CVB3 infection in vivo. AAV-mediated CRISPR genome editing may be applicable to many in vivo studies on the virus-host interaction and identify a novel target for antiviral therapeutics.

MicroRNA-30a-5p silencing polarizes macrophages toward M2 phenotype to alleviate cardiac injury following viral myocarditis by targeting SOCS1.

Viral myocarditis (VMC) is a life-threatening disease characterized by severe cardiac inflammation generally caused by coxsackievirus B3 (CVB3) infection. Several microRNAs (miRNAs or miRs) are known to play crucial roles in the pathogenesis of VMC. The study aimed to decipher the role of miR-30a-5p in the underlying mechanisms of VMC pathogenesis. We first quantified miR-30a-5p expression in a CVB3-induced mouse VMC model. The physiological characteristics of mouse cardiac tissues were then detected by hematoxylin and eosin (HE) and Picrosirius red staining. We established the correlation between miR-30a-5p and SOCS1, using dual-luciferase gene assay and Pearson's correlation coefficient. The expression of inflammatory factors (IFN-γ, IL-6, IL-10, and IL-13), M1 polarization markers [TNF-α, inducible nitric oxide synthase (iNOS)], M2 polarization markers (Arg-1, IL-10), and myocardial hypertrophy markers [atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)] was detected by RT-qPCR and Western blot analysis. miR-30a-5p was found to be highly expressed in VMC mice. Silencing of miR-30a-5p improved the cardiac function index and reduced heart weight-to-body weight ratio, myocardial tissue pathological changes and fibrosis degree, serological indexes, as well as proinflammatory factor levels, while enhancing anti-inflammatory factor levels in VMC mice. Furthermore, silencing of miR-30a-5p inhibited M1 polarization of macrophages while promoting M2 polarization in vivo and in vitro. SOCS1 was a target gene of miR-30a-5p, and the aforementioned cardioprotective effects of miR-30a-5p silencing were reversed upon silencing of SOCS1. Overall, this study shows that silencing of miR-30a-5p may promote M2 polarization of macrophages and improve cardiac injury following VMC via SOCS1 upregulation, constituting a potential therapeutic target for VMC treatment.NEW & NOTEWORTHY We found in this study that microRNA (miR)-30a-5p inhibition might improve cardiac injury following viral myocarditis (VMC) by accelerating M2 polarization of macrophages via SOCS1 upregulation. Furthermore, the anti-inflammatory mechanisms of miR-30a-5p inhibition may contribute to the development of new therapeutic strategies for VMC.

Coxsackievirus B3 Infection of Human iPSC Lines and Derived Primary Germ-Layer Cells Regarding Receptor Expression.

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.

Two Natural Recombination gave rise to the Coxsackievirus B3 GV that triggered outbreaks in China in 2006 - 2012.

Coxsackievirus B3 serotype GV caused the epidemic of Coxsackievirus B3 infection in China from 2006 to 2012. To study the evolution and recombination of Coxsackievirus B3 serotype GV, we performed recombination and phylogenetic analysis of 499 complete genomes of Enterovirus B available in GenBank, dated April 2019. Results indicated that most of the strains of Coxsackievirus B3 GV in P1 region were derived from a Coxsackievirus B3 GVI parent, and in P2-3 region from EchoV E25 strain, with nucleotide identities of 97.2% and 94.7%, respectively. Other strains of Coxsackievirus B3 GV-C1 in P1-P2 regions were derived from Coxsackievirus B3 GV-C3, whereas those in P3 regions were from CVB5. These naturally occurring recombination events were confirmed by phylogenetic analysis. This study indicates that two naturally occurring recombination gave rise to the coxsackievirus B3 GV that triggered outbreaks in China in 2006 - 2012.

Coxsackievirus B Type 4 Infection in ß Cells Downregulates the Chaperone Prefoldin URI to Induce a MODY4-like Diabetes via Pdx1 Silencing.

Enteroviruses are suspected to contribute to insulin-producing ß 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 ß cell function and identity. Genetic URI ablation in the mouse pancreas causes PDX1 depletion in ß cells. Importantly, diabetic PDX1 heterozygous mice overexpressing URI in ß 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 ß 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.