Coxsackievirus A16 Replication Research Articles

EV71 3C protease cleaves host anti-viral factor OAS3 and enhances virus replication.

The global spread of enteroviruses (EVs) has become more frequent, severe and life-threatening. Intereron (IFN) I has been proved to control EVs by regulating IFN-stimulated genes (ISG) expression. 2′-5′-oligoadenylate synthetases 3 (OAS3) is an important ISG in the OAS/RNase L antiviral system. The relationship between OAS3 and EVs is still unclear. Here, we reveal that OAS3, superior to OAS1 and OAS2, significantly inhibited EV71 replication in vitro. However, EV71 utilized autologous 3C protease (3Cpro) to cleave intracellular OAS3 and enhance viral replication. Rupintrivir, a human rhinovirus 3C protease inhibitor, completely abolished the cleavage of EV71 3Cpro on OAS3. And the proteolytically deficient mutants H40G, E71A, and C147G of EV71 3Cpro also lost the ability of OAS3 cleavage. Mechanistically, the Q982-G983 motif in C-terminal of OAS3 was identified as a crucial 3Cpro cutting site. Further investigation indicated that OAS3 inhibited not only EV71 but also Coxsackievirus B3 (CVB3), Coxsackievirus A16 (CA16), Enterovirus D68 (EVD68), and Coxsackievirus A6 (CA6) subtypes. Notably, unlike other four subtypes, CA16 3Cpro could not cleave OAS3. Two key amino acids variation Ile36 and Val86 in CA16 3Cpro might result in weak and delayed virus replication of CA16 because of failure of OAS and 3AB cleavage. Our works elucidate the broad anti-EVs function of OAS3, and illuminate a novel mechanism by which EV71 use 3Cpro to escape the antiviral effect of OAS3. These findings can be an important entry point for developing novel therapeutic strategies for multiple EVs infection.

Lipid raft-associated PI3K/Akt/SREBP1 signaling regulates coxsackievirus A16 (CA16) replication

Coxsackievirus A16 (CA16) is one of predominant Enterovirus that possesses high pathogenicity. Lipid rafts, as cholesterol ­ and sphingolipid ­ enriched membrane nanodomains, are involved into many aspects of the virus life cycle. Our previous study found that lipid rafts integrity was essential for CA16 replication, but how lipid rafts regulate CA16 replication through activating downstream signaling remains largely unknown. Thus, in this study, we revealed that lipid rafts were required for activation of PI3K/Akt signaling at early stage of CA16 infection. Treatment with wortmannin significantly reduced the expression of virus protein, indicating PI3K/Akt signaling was beneficial for early stage of virus infection. In addition, lipid rafts integrity was also indispensable for PI3K/Akt activation during the late stage of CA16 infection, which played critical functions in mediating sterol regulatory element-binding proteins 1 (SREBP1) maturation. Whereas, over-expression of SREBP1 exhibited inhibition on virus replication, suggesting that PI3K/Akt signaling and SREBP1 might positively and negatively influence virus replication in two different stages of infection, respectively. Taken together, our study demonstrates an important role of the lipid raft-associated PI3K/Akt/SREBP1 signaling in modulating CA16 replication, which will deepen our understanding mechanism of CA16 infection.

Lipidomic Profiling Reveals Significant Perturbations of Intracellular Lipid Homeostasis in Enterovirus-Infected Cells.

Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are the most common causes of hand, foot, and mouth disease. Severe EV-A71 and CV-A16 infections may be associated with life-threatening complications. However, the pathogenic mechanisms underlying these severe clinical and pathological features remain incompletely understood. Lipids are known to play critical roles in multiple stages of the virus replication cycle. The specific lipid profile induced upon virus infection is required for optimal virus replication. The perturbations in the host cell lipidomic profiles upon enterovirus infection have not been fully characterized. To this end, we performed ultra-high performance liquid chromatography–electrospray ionization–quadrupole–time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS)-based lipidomics to characterize the change in host lipidome upon EV-A71 and CV-A16 infections. Our results revealed that 47 lipids within 11 lipid classes were significantly perturbed after EV-A71 and CV-A16 infection. Four polyunsaturated fatty acids (PUFAs), namely, arachidonic acid (AA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA), were consistently upregulated upon EV-A71 and CV-A16 infection. Importantly, exogenously supplying three of these four PUFAs, including AA, DHA, and EPA, in cell cultures significantly reduced EV-A71 and CV-A16 replication. Taken together, our results suggested that enteroviruses might specifically modulate the host lipid pathways for optimal virus replication. Excessive exogenous addition of lipids that disrupted this delicate homeostatic state could prevent efficient viral replication. Precise manipulation of the host lipid profile might be a potential host-targeting antiviral strategy for enterovirus infection.

Lycorine Derivative LY-55 Inhibits EV71 and CVA16 Replication Through Downregulating Autophagy.

Hand, foot, and mouth disease (HFMD) is a global health concern, especially in the Asia-Pacific region. HFMD caused by Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection is usually self-limited but occasionally leads to severe pulmonary edema, neurological complications, and even death. Unfortunately, no effective drugs are currently available in clinical practice for the prevention and treatment of HFMD. Thus, anti-HFMD drugs must be urgently developed. A previous study had reported that lycorine could inhibit EV71 replication. In the present study, we found that LY-55, a lycorine derivative, inhibited the replication of EV71 and CVA16 in vitro and provided partial protection to mice from EV71 infection, as indicated by the decreased viral load and protein expression levels in muscles, clinical scores, and increased survival rates of infected mice. Mechanistically, LY-55 was not directly viricidal. Instead, the LY-55-mediated inhibition of EV71 and CVA16 was found to be mechanistically possible, at least in part, through downregulating autophagy, which plays an important role for EV71 and CVA16 replication. These findings suggest that LY-55 could be a potential lead or supplement for the development of anti-HFMD agents in the future.

Autophagy triggered by MAVS inhibits Coxsackievirus A16 replication.

Mitochondrial antiviral signaling protein (MAVS), a crucial adaptor protein localized on mitochondria, plays vital roles in various biological processes. Autophagy and apoptosis are two independent and closely linked cell death pathways. But whether MAVS could induce apoptosis and autophagy in rhabdomyosarcoma cells (RD cells) and what is the relationship between autophagy and apoptosis still remains elusive. Here, we reveal that overexpression of MAVS could trigger both apoptosis and autophagy in RD cells. Interestingly, MAVS-induced apoptosis was dependent on the activation of the c-Jun N-terminal kinase (JNK) signaling pathway and inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway. Also, it was found that inhibition of autophagy by 3-methyladenine (3-MA) enhanced MAVS-induced apoptosis resulting in increased cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP). Meanwhile, autophagy induction by rapamycin resulted in decreased MAVS-induced apoptosis. In addition, we found that MAVS expression was inhibited upon Coxsackievirus A16 (CA16) infection and overexpression of MAVS could inhibit CA16 replication. Collectively, our study provides novel insights into the link between apoptosis and autophagy induced by MAVS overexpression in RD cells and gains a greater understanding of MAVS-induced antiviral functions, which provide new targets for CA16 treatment. Keywords: CA16; MAVS; apoptosis; autophagy.

Suppression of the toll-like receptor 7-dependent type I interferon production pathway by autophagy resulting from enterovirus 71 and coxsackievirus A16 infections facilitates their replication

Toll-like receptors (TLRs) act as molecular sentinels, detecting invading viral pathogens and triggering host innate immune responses, including autophagy. However, many viruses have evolved a series of strategies to manipulate autophagy for their own benefit. Enterovirus 71 (EV71) and coxsackievirus A16 (CA16), as the primary agents causing hand, foot and mouth disease (HFMD), can induce autophagy leading to their replication. Therefore, the objective of this study was to investigate whether enhanced viral replication caused by autophagy in EV71 and CA16 infections was associated with a TLR-related signaling pathway. Our results demonstrate that complete autophagy and incomplete autophagy were observed in human bronchial epithelial (16HBE) cells infected with EV71 and CA16. Moreover, suppression of autophagy by the pharmacological modulator 3-MA significantly and clearly decreased the survival rates and viral replication of EV71 and CA16 in 16HBE cells. Inhibition of autophagy also enhanced the expression of molecules related to the TLR7-dependent type I interferon (IFN-I) production pathway, such as TLR7, MyD88, IRF7 and IFN-α/β. Finally, immunofluorescence staining demonstrated that TLR7 endosome marker M6PR levels were clearly reduced in EV71- and CA16-infected cells, while they were markedly elevated in infected cells treated with 3-MA. These findings suggest that increased EV71 and CA16 replication meditated by autophagy in 16HBE cells might promote degradation of the endosome, leading to suppression of the TLR7-mediated IFN-I signaling pathway.

Type I Interferons Triggered through the Toll-Like Receptor 3-TRIF Pathway Control Coxsackievirus A16 Infection in Young Mice.

Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot, and mouth disease (HFMD) in children. The host defense mechanisms against CVA16 infection remain almost entirely unknown. Unlike previous observations with enterovirus 71 (EV71) infection, here we show that gamma interferon (IFN-γ) or invariant NK T cell deficiency does not affect disease development or the survival of CVA16-infected mice. In contrast, type I interferon receptor deficiency resulted in the development of more severe disease in mice, and the mice had a lower survival rate than wild-type mice. Similarly, a deficiency of Toll-like receptor 3 (TLR3) and TRIF, but not other pattern recognition receptors, led to the decreased survival of CVA16-infected mice. TLR3-TRIF signaling was indispensable for the induction of type I interferons during CVA16 infection in mice and protected young mice from disease caused by the infection. In particular, TRIF-mediated immunity was critical for preventing CVA16 replication in the neuronal system before disease occurred. IFN-β treatment was also found to compensate for TRIF deficiency in mice and decreased the disease severity in and mortality of CVA16-infected mice. Altogether, type I interferons induced by TLR3-TRIF signaling mediate protective immunity against CVA16 infection. These findings may shed light on therapeutic strategies to combat HFMD caused by CVA16 infection. Hand, foot, and mouth disease (HFMD) is a major threat to public health in the Asia-Pacific region. Both CVA16 and EV71 are major pathogens that are responsible for HFMD. The majority of research efforts have focused on the more virulent EV71, but little has been done with CVA16. Thus far, host immune responses to CVA16 infection have not yet been elucidated. The present study discovered an initial molecular mechanism underlying host protective immunity against CVA16 infection, providing the first explanation for why CVA16 and EV71 cause different clinical outcomes upon infection of humans. Therefore, different therapeutic strategies should be developed to treat HFMD cases caused by these two viruses.

Development and characterization of a clinical strain of Coxsackievirus A16 and an eGFP infectious clone.

Coxsackievirus A16 (CA16) is one of the major causes of hand, foot, and mouth disease (HFMD) worldwide, which is a common illness that affects children. The frequent occurrence of HFMD outbreaks has become a serious public health problem in Asia. Therefore, it is important to understand the pathogenesis and replication of CA16. In this study, a stable infectious cDNA clone of an epidemic strain of Coxsackievirus A16 (CA16) was assembled, and subsequently a reporter virus (eGFP-CA16) was constructed by inserting the eGFP gene between the 5'-UTR and the N-terminus of VP4, with the addition of a 2A protease cleavage site (ITTLG) at its C-terminus. This was transfected into Vero cells to generate infectious recombinant viruses. The growth characteristics and plaque morphology, in vitro, in mammalian cells were found to be indistinguishable between the parental and recombinant viruses. Although the eGFP-CA16 showed smaller plaque size as compared to recombinant CA16, both were found to exhibit similar growth trends and EC50 of NITD008. In summary, this stable infectious cDNA clone should provide a valuable experimental system to study CA16 infection and host response. The eGFP-CA16 is expected to provide a powerful tool to monitor eGFP expression in infected cells and to evaluate the antiviral activity of potential antiviral agents in the treatment of CA16 infections.

Glycyrrhizic acid as the antiviral component of Glycyrrhiza uralensis Fisch. against coxsackievirus A16 and enterovirus 71 of hand foot and mouth disease

Ethnopharmacological relevanceThe radices of Glycyrrhiza uralensis Fisch. and herbal preparations containing Glycyrrhiza spp. have been used for thousands of years as an herbal medicine for the treatment of viral induced cough, viral hepatitis, and viral skin diseases like ulcers in China. Glycyrrhizic acid (GA) is considered the principal component in Glycyrrhiza spp. with a wide spectrum of antiviral activity. AimThe present study attempt to validate the medicinal use of Glycyrrhiza uralensis for hand, foot and mouth disease (HFMD) and further to verify whether GA is an active antiviral component in the water extract of Glycyrrhiza uralensis. Materials and methodsRadices of Glycyrrhiza uralensis Fisch. were extracted with hot water. The chemical contents of the extract were profiled with HPLC analysis. The antiviral activity of the extract and the major components was evaluated against infection of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) on Vero cells. The cytopathic effect caused by the infection was measured with MTT assay. Infectious virion production was determined using secondary infection assays and viral protein expression by immunoblotting analysis. ResultsThe extract at 1000μg/ml suppressed EV71 replication by 1.0 log and CVA16 by 1.5 logs. The antiviral activity was associated with the content of GA in the extract since selective depletion of GA from the extract by acid precipitation resulted in loss of antiviral activity. In contrast, the acid precipitant retained antiviral activity. The precipitant at a concentration of 200μg/ml inhibited EV71 and CVA16 replication by 1.7 and 2.2 logs, respectively. Furthermore, GA dose-dependently blocked viral replication of EV71 and CVA16. At 3mM, GA reduced infectious CVA16 and EV71 production by 3.5 and 2.2 logs, respectively. At 5mM, CVA16 production was reduced by 6.0 logs and EV71 by 4.0 logs. Both EV71 and CVA16 are members of Enterovirus genus, time-of-drug addition studies however showed that GA directly inactivated CVA16, while GA anti-EV71 effect was associated with an event(s) post virus cell entry. ConclusionsThis study validated the medicinal usefulness of radices Glycyrrhiza uralensis against the etiological agents of HFMD. In addition to the identification of GA as the antiviral component of Glycyrrhiza uralensis against EV71 and CVA16 infection, this study also reveals that GA inhibits EV71 and CVA16 with distinct mechanisms.

Quick identification of effective small interfering RNAs that inhibit the replication of coxsackievirus A16

Coxsackievirus A16 (CA16) is a major causative agent of hand, foot, and mouth disease (HFMD). It can cause myocarditis, pericarditis and fatal shock. There is no effective therapy against CA16. RNA interference (RNAi) is a powerful tool to silence gene expression. The small interfering RNA (siRNA) that induces RNA degradation has recently been used as an anti-virus agent to inhibit virus replication. In this study, we established the complete nucleotide sequence of CA16 strain Shzh05-1, and then compared the nucleotide sequences of Shzh05-1 with sequences of other CA16 strains in GenBank. We chose conserved regions between Shzh05-1 and the two other CA16 strains to design 30 siRNAs and construct siRNA-encoding plasmids. Thirteen siRNAs targeting conserved regions of the virus could effectively block replication of CA16 in cultured cells. Combination transfection of these 13 effective siRNAs could also produce a high inhibitory effect. These strategies and results suggest that RNAi has potential therapeutic use for suppression of CA16 infection.

Stethoscope v. X Rays

ABSTRACT Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot, and mouth disease (HFMD) in children. The host defense mechanisms against CVA16 infection remain almost entirely unknown. Unlike previous observations with enterovirus 71 (EV71) infection, here we show that gamma interferon (IFN-γ) or invariant NK T cell deficiency does not affect disease development or the survival of CVA16-infected mice. In contrast, type I interferon receptor deficiency resulted in the development of more severe disease in mice, and the mice had a lower survival rate than wild-type mice. Similarly, a deficiency of Toll-like receptor 3 (TLR3) and TRIF, but not other pattern recognition receptors, led to the decreased survival of CVA16-infected mice. TLR3-TRIF signaling was indispensable for the induction of type I interferons during CVA16 infection in mice and protected young mice from disease caused by the infection. In particular, TRIF-mediated immunity was critical for preventing CVA16 replication in the neuronal system before disease occurred. IFN-β treatment was also found to compensate for TRIF deficiency in mice and decreased the disease severity in and mortality of CVA16-infected mice. Altogether, type I interferons induced by TLR3-TRIF signaling mediate protective immunity against CVA16 infection. These findings may shed light on therapeutic strategies to combat HFMD caused by CVA16 infection. IMPORTANCE Hand, foot, and mouth disease (HFMD) is a major threat to public health in the Asia-Pacific region. Both CVA16 and EV71 are major pathogens that are responsible for HFMD. The majority of research efforts have focused on the more virulent EV71, but little has been done with CVA16. Thus far, host immune responses to CVA16 infection have not yet been elucidated. The present study discovered an initial molecular mechanism underlying host protective immunity against CVA16 infection, providing the first explanation for why CVA16 and EV71 cause different clinical outcomes upon infection of humans. Therefore, different therapeutic strategies should be developed to treat HFMD cases caused by these two viruses.