calpain and Enterovirus-Infections

Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C's cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.

Topics: Animals; Calpain; Capsid; Capsid Proteins; Cells, Cultured; Enterovirus; Enterovirus Infections; Glycoproteins; Humans; Mass Spectrometry; Peptides; Polyproteins; Proteolysis; Rats; Viral Proteins

Enterovirus 71 (EV71) is a causative agent of an array of childhood diseases with severe neurological manifestations implicated. EV71 infection is known to induce caspase-dependent apoptosis in cell cultures and animal models. However, whether an alternative apoptotic pathway independent of caspase activation can be triggered by EV71 infection has not been explored. In this study, we showed that calcium (Ca²⁺)-activated calpains are capable of mediating caspase-independent pathway activation during EV71-induced apoptosis in HeLa cells. Results from subcellular fractionation analysis and confocal imaging indicated that during EV71 infection, apoptosis-inducing factor (AIF), a primary mediator of the caspase-independent pathway, became truncated and translocated from the mitochondrion to nucleus. This was accompanied by the release of cytochrome c, and sharply decreased mitochondrial membrane potential. AIF knockdown data indicated significant protection against apoptotic cell death, with greater protection provided by the addition of a pan-caspase inhibitor. The Ca²⁺-dependent, calpain isoforms 1 and 2, but not cathepsins, were proven crucial for the altered AIF behaviour as studied by the pharmacological inhibitor and the knockdown approaches. We then analysed Ca²⁺ dynamics in the infected cells and found elevated levels of mitochondrial Ca²⁺. Treatment with ruthenium red, a mitochondrial Ca²⁺ influx inhibitor, significantly blocked calpain activations and AIF cleavage. Our conclusion was that calpain activation via Ca²⁺ flux plays an essential role in eliciting an AIF-mediated, caspase-independent apoptotic pathway in EV71-infected cells. These findings should be useful for understanding the virus-induced cytopathology and the impact of Ca²⁺ homeostasis on EV71 infection.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Calcium; Calpain; Cell Line; Cell Nucleus; Chlorocebus aethiops; Enterovirus; Enterovirus Infections; HeLa Cells; Humans; Mitochondria; Vero Cells

Coxsackievirus B (CVB), a member of the enterovirus family, targets the polarized epithelial cells lining the intestinal tract early in infection. Although the polarized epithelium functions as a protective barrier, this barrier is likely exploited by CVB to promote viral entry and subsequent egress. Here we show that, in contrast to nonpolarized cells, CVB-infected polarized intestinal Caco-2 cells undergo nonapoptotic necrotic cell death triggered by inositol 1,4,5-trisphosphate receptor-dependent calcium release. We further show that CVB-induced cellular necrosis depends on the Ca(2+)-activated protease calpain-2 and that this protease is involved in CVB-induced disruption of the junctional complex and rearrangements of the actin cytoskeleton. Our study illustrates the cell signaling pathways hijacked by CVB, and perhaps other viral pathogens, to promote their replication and spread in polarized cell types.

Topics: Caco-2 Cells; Calcium Signaling; Calpain; Capsid Proteins; Caspase 3; Cell Polarity; Cytoskeleton; Endoplasmic Reticulum; Enterovirus B, Human; Enterovirus Infections; Enzyme Assays; HeLa Cells; Humans; Inositol 1,4,5-Trisphosphate Receptors; Intestinal Mucosa; Microscopy, Fluorescence; Necrosis; Tight Junctions; Type C Phospholipases; Virus Release