Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein

Abstract

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.

Enteroviruses and coronaviruses: similarities and therapeutic targets

“…An exciting development to inhibit protease activity has emerged from inhibiting the host cell calpain proteases. Calpain proteases are capable of proteolytically processing several target molecules, also including viral polyprotein that has been shown for enterovirus polyprotein [ 24 ]. Importantly, inhibitors against calpain proteases also efficiently inhibit the viral 3 C and M proteases [ 54 ].…”

“…The inhibitory action was earlier suggested targeting early replication/translation of echovirus 1 as well as vesicle trafficking, necrosis, and apoptosis during Coxsackievirus B3 and B4 infection [ 55 , 56 , 65 ]. The molecular mechanisms of action were not detailed until recently, Laajala et al showed that calpain proteases can participate in the proteolytic processing of the viral polyprotein [ 24 ]. Thus, calpains could contribute to the same action as what is performed by the 3 C viral protease.…”

“…Thus, calpains could contribute to the same action as what is performed by the 3 C viral protease. Indeed, in the study by Laajala et al [ 24 ], the polyprotein was processed by calpain proteases in vitro to deliver capsid proteins with very similar but not identical cutting site between the capsid proteins. The study also showed that the used calpain inhibitors I and II totally inhibited the action of expressed and purified viral 3 C protease.…”

“…In addition to the polyprotein cleavage, the viral proteases have been shown to cleave different host cell factors in order to promote the infection [ 23 ]. In addition to viral proteases, a recent paper suggests that cellular proteases, the calpains, may be important in polyprotein processing [ 24 ]. It was shown in vitro that the calpains could process the polyprotein of enteroviruses and release structural viral proteins 1 and 3.…”

Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

“…Unlike the Rac1 protein described above, calpains 1 and 2 are not required for the entry of the virus, but rather at later stages of the infection in RNA replication [14]. It was recently demonstrated that cellular calpains can cleave viral capsid proteins VP1 and VP3 from the enterovirus polyprotein [23], and that enterovirus infection upregulates the activity of calpains, suggesting that calpains are essential for the cleavage of the viral polyprotein. Laajala et al [23] also demonstrated high cross-reactivity of calpain inhibitor 1 with the viral proteases 2Apro and 3Cpro, making it a promising antiviral drug.…”