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Viruses cleave cellular proteins to remodel the host proteome. The study of these cleavages has revealed mechanisms of immune evasion, resource exploitation, and pathogenesis. However, the full extent of virus-induced proteolysis in infected cells is unknown, mainly because until recently the technology for a global view of proteolysis within cells was lacking. Here, we report the first comprehensive catalog of proteins cleaved upon enterovirus infection and identify the sites within proteins where the cleavages occur. We employed multiple strategies to confirm protein cleavages and assigned them to one of the two enteroviral proteases. Detailed characterization of one substrate, LSM14A, a p body protein with a role in antiviral immunity, showed that cleavage of this protein disrupts its antiviral function. This study yields a new depth of information about the host interface with a group of viruses that are both important biological tools and significant agents of disease.

Increasing evidence highlights the importance of the antiviral activities of the type III interferons (IFNλs; IL-28A, IL-28B, IL29, and IFNλ4) in the intestine. However, many viruses have developed strategies to counteract these defense mechanisms by preventing the production of IFNs. Here we use infection models, a clinical virus isolate, and several molecular biology techniques to demonstrate that both type I and III IFNs induce an antiviral state and attenuate Coxsackievirus group B (CVB) replication in human intestinal epithelial cells (IECs). While treatment of IECs with a viral mimic (poly (I:C)) induced a robust expression of both type I and III IFNs, no such up-regulation was observed after CVB infection. The blunted IFN response was paralleled by a reduction in the abundance of proteins involved in the induction of interferon gene transcription, including TIR-domain-containing adapter-inducing interferon-β (TRIF), mitochondrial antiviral-signaling protein (MAVS), and the global protein translation initiator eukaryotic translation initiation factor 4G (eIF4G). Taken together, this study highlights a potent anti-Coxsackieviral effect of both type I and III IFNs in cells located at the primary site of infection. Furthermore, we show for the first time that the production of type I and III IFNs in IECs is blocked by CVBs. These findings suggest that CVBs evade the host immune response in order to successfully infect the intestine.

Coxsackievirus B (CVB) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type 1 diabetes. The licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. Even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (VLPs), can offer important advantages in the manufacturing and epitope engineering. We have previously produced VLPs for CVB3 and CVB1 in insect cells. Here, we describe the production of CVB3-VLPs with enhanced production yield and purity using an improved purification method consisting of tangential flow filtration and ion exchange chromatography, which is compatible with industrial scale production. We also resolved the CVB3-VLP structure by Cryo-Electron Microscopy imaging and single particle reconstruction. The VLP diameter is 30.9 nm on average, and it is similar to Coxsackievirus A VLPs and the expanded enterovirus cell-entry intermediate (the 135s particle), which is ~2 nm larger than the mature virion. High neutralizing and total IgG antibody levels, the latter being a predominantly Th2 type (IgG1) phenotype, were detected in C57BL/6J mice immunized with non-adjuvanted CVB3-VLP vaccine. The structural and immunogenic data presented here indicate the potential of this improved methodology to produce highly immunogenic enterovirus VLP-vaccines in the future.