Exosomal let-7a-5p derived from human umbilical cord mesenchymal stem cells alleviates coxsackievirus B3-induced cardiomyocyte ferroptosis via the SMAD2/ZFP36 signal axis. / 人脐带间å 质干细胞来源的外泌体let-7a-5p通过SMAD2/ZFP36ä¿¡å·è½´å‡è½»æŸ¯è¨å¥‡ç— æ¯’B3诱导的心肌细胞铁死亡.

Viral myocarditis (VMC) is one of the most common acquired heart diseases in children and teenagers. However, its pathogenesis is still unclear, and effective treatments are lacking. This study aimed to investigate the regulatory pathway by which exosomes alleviate ferroptosis in cardiomyocytes (CMCs) induced by coxsackievirus B3 (CVB3). CVB3 was utilized for inducing the VMC mouse model and cellular model. Cardiac echocardiography, left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were implemented to assess the cardiac function. In CVB3-induced VMC mice, cardiac insufficiency was observed, as well as the altered levels of ferroptosis-related indicators (glutathione peroxidase 4 (GPX4), glutathione (GSH), and malondialdehyde (MDA)). However, exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exo) could restore the changes caused by CVB3 stimulation. Let-7a-5p was enriched in hucMSCs-exo, and the inhibitory effect of hucMSCs-exolet-7a-5p mimic on CVB3-induced ferroptosis was higher than that of hucMSCs-exomimic NC (NC: negative control). Mothers against decapentaplegic homolog 2 (SMAD2) increased in the VMC group, while the expression of zinc-finger protein 36 (ZFP36) decreased. Let-7a-5p was confirmed to interact with SMAD2 messenger RNA (mRNA), and the SMAD2 protein interacted directly with the ZFP36 protein. Silencing SMAD2 and overexpressing ZFP36 inhibited the expression of ferroptosis-related indicators. Meanwhile, the levels of GPX4, solute carrier family 7, member 11 (SLC7A11), and GSH were lower in the SMAD2 overexpression plasmid (oe-SMAD2)+let-7a-5p mimic group than in the oe-NC+let-7a-5p mimic group, while those of MDA, reactive oxygen species (ROS), and Fe2+ increased. In conclusion, these data showed that ferroptosis could be regulated by mediating SMAD2 expression. Exo-let-7a-5p derived from hucMSCs could mediate SMAD2 to promote the expression of ZFP36, which further inhibited the ferroptosis of CMCs to alleviate CVB3-induced VMC.

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.

Cardiac macrophages undergo dynamic changes after coxsackievirus B3 infection and promote the progression of myocarditis.

Although most patients with acute viral myocarditis recover spontaneously, some patients progress to heart failure. Perturbations in innate immunity may partially explain the heterogeneity of clinical outcomes. As the most abundant immune cells in the heart, cardiac macrophages have heterogeneous origins, including embryonic-derived resident macrophages (ResMϕs) and monocyte-derived macrophages (MoMFs). However, the time course change and role of cardiac macrophage subsets has not been fully explored. In the present study, we found that BALB/c mice had prolonged MoMF accumulation and low proportions of ResMϕs that could not be restored to normal levels. MoMFs of BALB/c mice generally exhibit an M1-dominant functional phenotype. Moreover, the preferential depletion of MoMF by a C-C chemokine receptor type 2 (CCR2) inhibitor resulted in improved acute myocarditis and chronic fibrosis, as well as the recovery of ResMϕs number and reduced CD4+ T cell expansion. Hence, immunomodulatory therapy that targets the balance among cardiac macrophages and modulates their function is expected to prevent the progression of cardiac injury to overt heart failure and improve adverse outcomes.

Coxsackievirus B3 infects and disrupts human induced-pluripotent stem cell derived brain-like endothelial cells.

Coxsackievirus B3 (CVB3) is a significant human pathogen that is commonly found worldwide. CVB3 among other enteroviruses, are the leading causes of aseptic meningo-encephalitis which can be fatal especially in young children. How the virus gains access to the brain is poorly-understood, and the host-virus interactions that occur at the blood-brain barrier (BBB) is even less-characterized. The BBB is a highly specialized biological barrier consisting primarily of brain endothelial cells which possess unique barrier properties and facilitate the passage of nutrients into the brain while restricting access to toxins and pathogens including viruses. To determine the effects of CVB3 infection on the BBB, we utilized a model of human induced-pluripotent stem cell-derived brain-like endothelial cells (iBECs) to ascertain if CVB3 infection may alter barrier cell function and overall survival. In this study, we determined that these iBECs indeed are susceptible to CVB3 infection and release high titers of extracellular virus. We also determined that infected iBECs maintain high transendothelial electrical resistance (TEER) during early infection despite possessing high viral load. TEER progressively declines at later stages of infection. Interestingly, despite the high viral burden and TEER disruptions at later timepoints, infected iBEC monolayers remain intact, indicating a low degree of late-stage virally-mediated cell death, which may contribute to prolonged viral shedding. We had previously reported that CVB3 infections rely on the activation of transient receptor vanilloid potential 1 (TRPV1) and found that inhibiting TRPV1 activity with SB-366791 significantly limited CVB3 infection of HeLa cervical cancer cells. Similarly in this study, we observed that treating iBECs with SB-366791 significantly reduced CVB3 infection, which suggests that not only can this drug potentially limit viral entry into the brain, but also demonstrates that this infection model could be a valuable platform for testing antiviral treatments of neurotropic viruses. In all, our findings elucidate the unique effects of CVB3 infection on the BBB and shed light on potential mechanisms by which the virus can initiate infections in the brain.

Exosomes mediate Coxsackievirus B3 transmission and expand the viral tropism.

Specific virus-receptor interactions are important determinants in viral host range, tropism and pathogenesis, influencing the location and initiation of primary infection as well as viral spread to other target organs/tissues in the postviremic phase. Coxsackieviruses of Group B (CVB) and its six serotypes (CVB1-6) specifically interact with two receptor proteins, coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor (DAF), and cause various lesions in most permissive tissues. However, our previous data and other studies revealed that virus receptor-negative cells or tissues can be infected with CVB type 3 (CVB3), which can also effectively replicate. To study this interesting finding, we explored the possibility that exosomes are involved in CVB3 tropism and that exosomes functionally enhance CVB3 transmission. We found that exosomes carried and delivered CVB3 virions, resulting in efficient infection in receptor-negative host cells. We also found that delivery of CVB3 virions attached to exosomes depended on the virus receptor CAR. Importantly, exosomes carrying CVB3 virions exhibited greater infection efficiency than free virions because they accessed various entry routes, overcoming restrictions to viral tropism. In vivo experiments demonstrated that inhibition of exosome coupling with virions attenuated CVB3-induced immunological system dysfunction and reduced mortality. Our study describes a new mechanism in which exosomes contribute to viral tropism, spread, and pathogenesis.

A Conserved Cysteine Residue in Coxsackievirus B3 Protein 3A with Implication for Elevated Virulence.

Enteroviruses (EV) are implicated in an extensive range of clinical manifestations, such as pancreatic failure, cardiovascular disease, hepatitis, and meningoencephalitis. We recently reported on the biochemical properties of the highly conserved cysteine residue at position 38 (C38) of enteroviral protein 3A and demonstrated a C38-mediated homodimerization of the Coxsackievirus B3 protein 3A (CVB3-3A) that resulted in its profound stabilization. Here, we show that residue C38 of protein 3A supports the replication of CVB3, a clinically relevant member of the enterovirus genus. The infection of HeLa cells with protein 3A cysteine 38 to alanine mutants (C38A) attenuates virus replication, resulting in comparably lower virus particle formation. Consistently, in a mouse infection model, the enhanced virus propagation of CVB3-3A wt in comparison to the CVB3-3A[C38A] mutant was confirmed and found to promote severe liver tissue damage. In contrast, infection with the CVB3-3A[C38A] mutant mitigated hepatic tissue injury and ameliorated the signs of systemic inflammatory responses, such as hypoglycemia and hypothermia. Based on these data and our previous report on the C38-mediated stabilization of the CVB3-3A protein, we conclude that the highly conserved amino acid C38 in protein 3A enhances the virulence of CVB3.

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.

Molecular basis of differential receptor usage for naturally occurring CD55-binding and -nonbinding coxsackievirus B3 strains.

Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and selectively utilizes the decay-accelerating factor (DAF; CD55) to infect cells. However, the differential receptor usage mechanism for CVB remains elusive. This study identified VP3-234 residues (234Q/N/V/D/E) as critical population selection determinants during CVB3 virus evolution, contributing to diverse binding affinities to CD55. Cryoelectron microscopy (cryo-EM) structures of CD55-binding/nonbinding isolates and their complexes with CD55 or CAR were obtained under both neutral and acidic conditions, and the molecular mechanism of VP3-234 residues determining CD55 affinity/specificity for naturally occurring CVB3 strains was elucidated. Structural and biochemical studies in vitro revealed the dynamic entry process of CVB3 and the function of the uncoating receptor CAR with different pH preferences. This work provides detailed insight into the molecular mechanism of CVB infection and contributes to an in-depth understanding of enterovirus attachment receptor usage.

Pathological Features of Echovirus-11-Associated Brain Damage in Mice Based on RNA-Seq Analysis.

Echovirus 11 (E11) is a neurotropic virus that occasionally causes fatal neurological diseases in infected children. However, the molecular mechanism underlying the disease and pathological spectrum of E11 infection remains unclear. Therefore, we modelled E11 infection in 2-day-old type I interferon receptor knockout (IFNAR-/-) mice, which are susceptible to enteroviruses, with E11, and identified symptoms consistent with the clinical signs observed in human cases. All organs of infected suckling mice were found to show viral replication and pathological changes; the muscle tissue showed the highest viral replication, whereas the brain and muscle tissues showed the most obvious pathological changes. Brain tissues showed oedema and a large number of dead nerve cells; RNA-Seq analysis of the brain and hindlimb muscle tissues revealed differentially expressed genes to be abundantly enriched in immune response-related pathways, with changes in the Guanylate-binding protein (GBP) and MHC class genes, causing aseptic meningitis-related symptoms. Furthermore, human glioma U251 cell was identified as sensitive target cells for E11 infection. Overall, these results provide new insights into the pathogenesis and progress of aseptic meningitis caused by E11.

Cellular Protein HuR Regulates the Switching of Genomic RNA Templates for Differential Functions during the Coxsackievirus B3 Life Cycle.

Coxsackievirus B3 (CVB3) is an enterovirus belonging to the family Picornaviridae. Its 5' untranslated region (UTR) contains a cloverleaf structure followed by an internal ribosome entry site (IRES). The cloverleaf forms an RNA-protein complex known to regulate virus replication, translation, and stability of the genome, and the IRES regulates virus RNA translation. For positive-strand RNA-containing viruses, such as members of the flaviviruses or enteroviruses, the genomic RNA is used for translation, replication, and encapsidation. Only a few regulatory mechanisms which govern the accessibility of genomic RNA templates for translation or replication have been reported. Here, we report the role of human antigen R (HuR) in regulating the fate of CVB3 positive-strand RNA into the replication cycle or translation cycle. We have observed that synthesis of HuR is induced during CVB3 infection, and it suppresses viral replication by displacing PCBP-2 (a positive regulator of virus replication) at the cloverleaf RNA. Silencing of HuR increases viral RNA replication and consequently reduces viral RNA translation in a replication-dependent manner. Furthermore, we have shown that HuR level is upregulated upon CVB3 infection. Moreover, HuR limits virus replication and can coordinate the availability of genomic RNA templates for translation, replication, or encapsidation. Our study highlights the fact that the relative abundance of translation factors and replication factors in the cell decides the outcome of viral infection. IMPORTANCE A positive-strand RNA virus must balance the availability of its genomic template for different viral processes at different stages of its life cycle. A few host proteins are shown to be important to help the virus in switching the usage of a template between these processes. These proteins inhibit translation either by displacing a stimulator of translation or by binding to an alternative site. Both mechanisms lead to ribosome clearance and availability of the genomic strand for replication. We have shown that HuR also helps in maintaining this balance by inhibiting replication and subsequently promoting translation and packaging.

Development of Group B Coxsackievirus as an Oncolytic Virus: Opportunities and Challenges.

Oncolytic viruses have emerged as a promising strategy for cancer therapy due to their dual ability to selectively infect and lyse tumor cells and to induce systemic anti-tumor immunity. Among various candidate viruses, coxsackievirus group B (CVBs) have attracted increasing attention in recent years. CVBs are a group of small, non-enveloped, single-stranded, positive-sense RNA viruses, belonging to species human Enterovirus B in the genus Enterovirus of the family Picornaviridae. Preclinical studies have demonstrated potent anti-tumor activities for CVBs, particularly type 3, against multiple cancer types, including lung, breast, and colorectal cancer. Various approaches have been proposed or applied to enhance the safety and specificity of CVBs towards tumor cells and to further increase their anti-tumor efficacy. This review summarizes current knowledge and strategies for developing CVBs as oncolytic viruses for cancer virotherapy. The challenges arising from these studies and future prospects are also discussed in this review.

Coxsackievirus B4 sewage-isolate induces pancreatitis after oral infection of mice.

Numerous serotypes which belong to the genus Enterovirus (EV) show variability in their virulence and clinical manifestations. They are also known to undergo changes caused by mutations and recombination during their circulation in the environment and the population. Various EV serotypes are prevalent in groundwater, wastewater and surface waters. Our previous studies showed that oral infection induces pancreatitis depending on specific conditions, such as gravidity, in an outbred murine model. Our aim in the present study was to further explore the pancreatic histopathology in an outbred mouse model following oral infection with clinical isolates from a patient who had aseptic meningitis and an isolate from a treated-sewage sample recovered from the residential area of the patient. The isolates were identified as coxsackievirus B4 (CVB4) in tissue culture. The CVB4 sewage-isolate induced pancreatitis after oral infection. In contrast, pancreatitis was absent following infection with the clinical isolates. Comparison of polyprotein sequences showed that the treated-sewage strains differed from the patient's isolates by 9 and 11 amino acids. We conclude that the isolates of clinical and environmental origin differed in their pathogenic properties and showed genetic variation.

Antiviral Peptides Targeting the Helicase Activity of Enterovirus Nonstructural Protein 2C.

Enteroviruses belong to the genus Enterovirus of the family Picornaviridae and include four human enterovirus groups (EV-A to -D): the epidemic of enteroviruses such as human enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) is a threat to global public health. Enteroviral protein 2C is the most conserved nonstructural protein among all enteroviruses and possesses RNA helicase activity that plays pivotal roles during enteroviral life cycles, which makes 2C an attractive target for developing antienterovirus drugs. In this study, we designed a peptide, named 2CL, based on the structure of EV-A71 2C. This peptide effectively impaired the oligomerization of EV-A71 2C protein and inhibited the RNA helicase activities of 2C proteins encoded by EV-A71 and CVA16, both of which belong to EV-A, and showed potent antiviral efficacy against EV-A71 and CVA16 in cells. Moreover, the 2CL treatment elicited a strong in vivo protective efficacy against lethal EV-A71 challenge. In addition, the antiviral strategy of targeting the 2C helicase activity can be applied to inhibit the replication of EV-B. Either 2CL or B-2CL, the peptide redesigned based on the 2CL-corresponding sequence of EV-Bs, could exert effective antiviral activity against two important EV-Bs, coxsackievirus B3 and echovirus 11. Together, our findings demonstrated that targeting the helicase activity of 2C with a rationally designed peptide is an efficient antiviral strategy against enteroviruses, and 2CL and B-2CL show promising clinical potential to be further developed as broad-spectrum antienterovirus drugs.IMPORTANCE Enteroviruses are a large group of positive-sense single-stranded RNA viruses and include numerous human pathogens, such as enterovirus A71 (EV-A71), coxsackieviruses, and echoviruses. However, no approved EV antiviral drugs are available. Enteroviral 2C is the most conserved nonstructural protein among all enteroviruses and contains the RNA helicase activity critical for the viral life cycle. Herein, according to the structure of EV-A71 2C, we designed a peptide that effectively inhibited the RNA helicase activities of EV-A71- and coxsackievirus A16 (CVA16)-encoded 2C proteins. Moreover, this peptide exerted potent antiviral effects against EV-A71 and CVA16 in cells and elicited therapeutic efficacy against lethal EV-A71 challenge in vivo Furthermore, we demonstrate that the strategy of targeting the 2C helicase activity can be used for other relevant enteroviruses, including coxsackievirus B3 and echovirus 11. In summary, our findings provide compelling evidence that the designed peptides targeting the helicase activity of 2C could be broad-spectrum antivirals for enteroviruses.

FUS/TLS Suppresses Enterovirus Replication and Promotes Antiviral Innate Immune Responses.

During viral infection, the dynamic virus-host relationship is constantly in play. Many cellular proteins, such as RNA-binding proteins (RBPs), have been shown to mediate antiviral responses during viral infection. Here, we report that the RBP FUS/TLS (fused in sarcoma/translocated in liposarcoma) acts as a host-restricting factor against infection with coxsackievirus B3 (CVB3). Mechanistically, we found that deletion of FUS leads to increased viral RNA transcription and enhanced internal ribosome entry site (IRES)-driven translation, with no apparent impact on viral RNA stability. We further demonstrated that FUS physically interacts with the viral genome, which may contribute to direct inhibition of viral RNA transcription/translation. Moreover, we identified a novel function for FUS in regulating host innate immune response. We show that in the absence of FUS, gene expression of type I interferons and proinflammatory cytokines elicited by viral or bacterial infection is significantly impaired. Emerging evidence suggests a role for stress granules (SGs) in antiviral innate immunity. We further reveal that knockout of FUS abolishes the ability to form SGs upon CVB3 infection or poly(I·C) treatment. Finally, we show that, to avoid FUS-mediated antiviral response and innate immunity, CVB3 infection results in cytoplasmic mislocalization and cleavage of FUS through the enzymatic activity of viral proteases. Together, our findings in this study identify FUS as a novel host antiviral factor which restricts CVB3 replication through direct inhibition of viral RNA transcription and protein translation and through regulation of host antiviral innate immunity.IMPORTANCE Enteroviruses are common human pathogens, including those that cause myocarditis (coxsackievirus B3 [CVB3]), poliomyelitis (poliovirus), and hand, foot, and mouth disease (enterovirus 71). Understanding the virus-host interaction is crucial for developing means of treating and preventing diseases caused by these pathogens. In this study, we explored the interplay between the host RNA-binding protein FUS/TLS and CVB3 and found that FUS/TLS restricts CVB3 replication through direct inhibition of viral RNA transcription/translation and through regulation of cellular antiviral innate immunity. To impede the antiviral role of FUS, CVB3 targets FUS for mislocalization and cleavage. Findings from this study provide novel insights into interactions between CVB3 and FUS, which may lead to novel therapeutic interventions against enterovirus-induced diseases.

Polyamine Analog Diethylnorspermidine Restricts Coxsackievirus B3 and Is Overcome by 2A Protease Mutation In Vitro.

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.

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.

Cardiomyocyte-specific deletion of Senp2 contributes to CVB3 viral replication and inflammation.

Viral myocarditis (VMC) is characterized by cardiac inflammation and excessive inflammatory responses after viral infection. SENP2, a deSUMO-specific protease, has been reported to regulate antiviral innate immunity. This study aimed to investigate whether SENP2 affects CVB3-induced VMC. We generated a CVB3-induced VMC mouse model in 6-week-old cardiomyocyte-specific Senp2 knockout mice. The mice were sacrificed at days 0, 2, 4 and 6 after CVB3 infection. The survival rate, body weight, myocardial histopathological changes, viral load, cytokine levels and antiviral gene expression in cardiac tissues of both groups were investigated. Our study indicated that the expression of Senp2 in primary cardiomyocytes was upregulated by CVB3 infection. Moreover, deletion of Senp2 in the heart exacerbated CVB3 infection-induced myocarditis, facilitated CVB3 viral replication and downregulated the expression of antiviral proteins. In conclusion, our findings suggest a protective role for SENP2 in CVB3-induced VMC.

Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells.

Enteroviruses manipulate host membranes to form replication organelles, which concentrate viral and host factors to allow for efficient replication. However, this process has not been well-studied in living cells throughout the course of infection. To define the dynamic process of enterovirus membrane remodeling of major secretory pathway organelles, we have developed plasmid-based reporter systems that utilize viral protease-dependent release of a nuclear-localized fluorescent protein from the endoplasmic reticulum (ER) membrane during infection, while retaining organelle-specific fluorescent protein markers such as the ER and Golgi. This system thus allows for the monitoring of organelle-specific changes induced by infection in real-time. Using long-term time-lapse imaging of living cells infected with coxsackievirus B3 (CVB), we detected reporter translocation to the nucleus beginning ~4 h post-infection, which correlated with a loss of Golgi integrity and a collapse of the peripheral ER. Lastly, we applied our system to study the effects of a calcium channel inhibitor, 2APB, on virus-induced manipulation of host membranes. We found that 2APB treatment had no effect on the kinetics of infection or the percentage of infected cells. However, we observed aberrant ER structures in CVB-infected cells treated with 2APB and a significant decrease in viral-dependent cell lysis, which corresponded with a decrease in extracellular virus titers. Thus, our system provides a tractable platform to monitor the effects of inhibitors, gene silencing, and/or gene editing on viral manipulation of host membranes, which can help determine the mechanism of action for antivirals.

Structures and Functions of Viral 5' Non-Coding Genomic RNA Domain-I in Group-B Enterovirus Infections.

Group-B enteroviruses (EV-B) are ubiquitous naked single-stranded positive RNA viral pathogens that are responsible for common acute or persistent human infections. Their genome is composed in the 5' end by a non-coding region, which is crucial for the initiation of the viral replication and translation processes. RNA domain-I secondary structures can interact with viral or cellular proteins to form viral ribonucleoprotein (RNP) complexes regulating viral genomic replication, whereas RNA domains-II to -VII (internal ribosome entry site, IRES) are known to interact with cellular ribosomal subunits to initiate the viral translation process. Natural 5' terminally deleted viral forms lacking some genomic RNA domain-I secondary structures have been described in EV-B induced murine or human infections. Recent in vitro studies have evidenced that the loss of some viral RNP complexes in the RNA domain-I can modulate the viral replication and infectivity levels in EV-B infections. Moreover, the disruption of secondary structures of RNA domain-I could impair viral RNA sensing by RIG-I (Retinoic acid inducible gene I) or MDA5 (melanoma differentiation-associated protein 5) receptors, a way to overcome antiviral innate immune response. Overall, natural 5' terminally deleted viral genomes resulting in the loss of various structures in the RNA domain-I could be major key players of host-cell interactions driving the development of acute or persistent EV-B infections.

Exosomes derived from umbilical cord mesenchymal stem cells alleviate viral myocarditis through activating AMPK/mTOR-mediated autophagy flux pathway.

Human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-exosomes) have been implicated as a novel therapeutic approach for tissue injury repair and regeneration, but the effects of hucMSC-exosomes on coxsackievirus B3 (CVB3)-induced myocarditis remain unknown. The object of the present study is to investigate whether hucMSC-exosomes have therapeutic effects on CVB3-induced myocarditis (VMC). HucMSC-exosomes were identified using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot. The purified hucMSC-exosomes tagged with PKH26 were tail intravenously injected into VMC model mice in vivo and used to administrate CVB3-infected human cardiomyocytes (HCMs) in vitro, respectively. The effects of hucMSC-exosomes on myocardial pathology injury, proinflammatory cytokines and cardiac function were evaluated through haematoxylin and eosin (H&E) staining, quantitative polymerase chain reaction (qPCR) and Doppler echocardiography. The anti-apoptosis role and potential mechanism of hucMSC-exosomes were explored using TUNEL staining, flow cytometry, immunohistochemistry, Ad-mRFP-GFP-LC3 transduction and Western blot. In vivo results showed that hucMSC-exosomes (50 µg iv) significantly alleviated myocardium injury, shrank the production of proinflammatory cytokines and improved cardiac function. Moreover, in vitro data showed that hucMSC-exosomes (50 µg/mL) inhibited the apoptosis of CVB3-infected HCM through increasing pAMPK/AMPK ratio and up-regulating autophagy proteins LC3II/I, BECLIN-1 and anti-apoptosis protein BCL-2 as well as decreasing pmTOR/mTOR ratio, promoting the degradation of autophagy flux protein P62 and down-regulating apoptosis protein BAX. In conclusion, hucMSC-exosomes could alleviate CVB3-induced myocarditis via activating AMPK/mTOR-mediated autophagy flux pathway to attenuate cardiomyocyte apoptosis, which will be benefit for MSC-exosome therapy of myocarditis in the future.