Degradomics : a study of the cellular proteolytic landscape in enterovirus infections

Abstract

The common human pathogen, enteroviruses (EVs) are a genus of single stranded RNA viruses that include Coxsackie B virus (CVBs). The viral RNA encodes a polyprotein containing two viral proteases 2Apro and 3Cpro, that process the polyprotein into structural and non-structural proteins during virus replication. Previous research has shown that aside from their role in polyprotein processing, the proteases also cleave host proteins in order to modulate different cell functions such as translation, transcription and innate immunity. The innate immune response, and in particular the type 1 interferons (IFN), have an important role in controlling virus spread and protecting neighbouring cells from infection. Similarly, type III IFNs modulate the permissiveness of cells to CVBs. In paper I, we found that CVB3 has evolved a mechanism to evade the type III IFN response in a comparable manner to that previously shown for the type I IFNs. This perturbation is likely mediated via the proteolytic cleavage of the signal transduction proteins IPS-1 and TRIF by 2Apro.

When investigating virus-associated diseases, high quality reagents are essential especially when the aim is to detect virus in serum or tissue samples. We recognised a need for reagents capable of detecting CVBs and in paper II we described the development of CVB specific antibodies against 2Apro/3Cpro and show their utility in western blotting, confocal microscopy, immunohistochemistry and flow cytometry. Several observations have suggested a role for CVB infection in the development of type 1 diabetes (T1D). Interestingly, β-cells infected with CVBs in vitro have defective insulin secretion. The aim of paper III was to investigate whether β-cell dysfunction observed during CVB infection could be attributed to the activity of the viral proteases. We found that the viral proteins 2Apro, 3Cpro and 3A individually exert negative effects on glucose stimulated insulin release (GSIS) and voltage stimulated exocytosis in β-cells.

Based on the results in paper I and III, it is evident that 2Apro and 3Cpro have multifaceted roles in the viral replication cycle and in the modulation of the host cell. In paper IV we wanted to define the viral proteases specific targets in Coxsackeivirus B3 (CVB3) permissive cell lines of varied origin (HeLa, CaCo-2 and EndoC-βH1). By enriching for the N-terminal peptides using subtiligase labelling combined with LS-MS/MS, we identified and validated 81 host cell protease substrates. Among the substrates we identified, the protein TCF7L2 was a target of 2Apro mediated cleavage. TCF7L2 is an important transcription factor involved in maintaining β-cell functionality and glucose stimulated insulin secretion. This finding provides a potential mechanistic explanation for the observation that β-cells infected with CVBs in vitro are defective in insulin secretion. The studies presented in this thesis increase our understanding the molecular virology of enteroviruses. Moreover, they open a new chapter of research in examining how disease pathology might be caused by the activity of virally encoded proteases.