Enteroviral proteases: structure, host interactions and pathogenicity

Abstract

Enteroviruses are common human pathogens, and infections are particularly frequent in children. Severe infections can lead to a variety of diseases, including poliomyelitis, aseptic meningitis, myocarditis and neonatal sepsis. Enterovirus infections have also been implicated in asthmatic exacerbations and type 1 diabetes. The large disease spectrum of the closely related enteroviruses may be partially, but not fully, explained by differences in tissue tropism. The molecular mechanisms by which enteroviruses cause disease are poorly understood, but there is increasing evidence that the two enteroviral proteases, 2A(pro) and 3C(pro) , are important mediators of pathology. These proteases perform the post-translational proteolytic processing of the viral polyprotein, but they also cleave several host-cell proteins in order to promote the production of new virus particles, as well as to evade the cellular antiviral immune responses. Enterovirus-associated processing of cellular proteins may also contribute to pathology, as elegantly demonstrated by the 2A(pro) -mediated cleavage of dystrophin in cardiomyocytes contributing to Coxsackievirus-induced cardiomyopathy. It is likely that improved tools to identify targets for these proteases will reveal additional host protein substrates that can be linked to specific enterovirus-associated diseases. Here, we discuss the function of the enteroviral proteases in the virus replication cycle and review the current knowledge regarding how these proteases modulate the infected cell in order to favour virus replication, including ways to avoid detection by the immune system. We also highlight new possibilities for the identification of protease-specific cellular targets and thereby a way to discover novel mechanisms contributing to disease. Copyright © 2016 John Wiley & Sons, Ltd.

Figure 1

Proposed model of the enterovirus replication cycle. (1) Entry. After attachment to host‐cell surface receptors virus is internalized and uncoated, leading to the release of viral RNA into the cytoplasm. (2) Translation. Viral polyprotein is translated and then processed by the 2A^pro and 3C^pro proteases. Host‐cell translation is also perturbed as a component of the translation machinery (eIF4G) cleaved by 2A^pro. (3) Immune evasion. Host‐cell immune response is blunted by proteolysis mediated by viral proteases 2A^pro and 3C^pro as intracellular receptors (MDA5/RIG‐1), and proteins relaying innate signalling (IPS‐1) are targeted, blocking the production of interferons and cytokines. (4) Replication. Viral proteins, in orchestration with host‐cell factors, replicate the viral RNA at membrane‐associated replication sites. (5) Release. Enteroviral positive‐stranded RNA genomes are encapsidated by the viral structural proteins, and the new viral progeny are released either by cell lysis or in extracellular vesicles.

Figure 2

Sequence alignments of 2A^pro and 3C^pro, their topological structure presentations and 3‐dimensional tertiary structures. Orange and cyan colourings are used for 2A^pro, and red and blue colourings are used for 3C^pro. Panels (a) and (b) show the primary amino acid sequence alignment of 2A^pro and 3C^pro within the Enterovirus family. The residues of the catalytic triad, as well as the ion‐binding residues, are highlighted with arrows underneath the sequences. The secondary structure elements are shown above the alignments (cylinder = alpha‐helical structure; arrow = beta‐sheet structure; turns in purple; 3/10 helices in pink). Secondary structure assignment was made using DSSP 66. Panels (c) and (d) show a topological schematic of the proteases. The same visual secondary structure representations are used as in panels (a) and (b). Panel (e) shows a cartoon representation of EV71 2A^pro (PDBID: 4FVB). The side chains of the amino acids of the catalytic triad are shown as sticks. Similarly to panel (e), panel (f) shows a cartoon representation of CVB3 3C^pro (PDBID: 2VB0). Panels (g) and (h) show surface representations of the proteases, with their active sites highlighted with yellow. In comparison, the active site of 2A^pro is more confined and restricted by surrounding structures than the active site of 3C^pro.

Figure 3

Primary amino acid sequence percentage identity matrix of the enteroviral proteases. The top‐right half of the matrix shows sequence identities between the different enteroviral species and coxsackievirus B3 for 2A^pro, and the lower‐left half for 3C^pro. The average sequence conservation between the different species is 53% for 2A^pro and 56% for 3C^pro. The rhinoviruses show the most sequence divergence with around 35–50% percentage identities for 2A^pro and 45–55% for 3C^pro.

Figure 4

The substrate sequence LOGOs of the enteroviral proteases published by Blom et al. 1996 76 (upper panels) (reprinted with permission from John Wiley and Sons) and new logos based on a larger substrate pool (lower panels; Nurminen et al. manuscript) show the most conserved positions of the substrate sequences around the cleavage site. Hydrophilic residues are shown in green colour, and hydrophobic residues are shown in black colour. Negatively charged residues are coloured red. The lower panel logos were created using all currently available enteroviral polyprotein sequences in the Uniprot database 77. Duplicate sequences were removed to avoid bias towards sequences with multiple entries. The logos were generated using WebLogo 78. Left panels: The most important recognition sites for 2A^pro in order of lowest variability are at locations P1′, P2, P2′, P4 and P3. The relatively low variability to the right of P2′ can be a result of the sequence being a functional part of 2A^pro itself, as the protease cleaves its own N‐terminal end free from the polyprotein by in cis cleavage. Right panels: The most important recognition sites for 3C^pro in order of lowest variability are at locations P1, P1′, P4 and P2′.

Figure 5

Infection of HeLa cells with coxsackievirus B3 (CVB3) results in the proteolytic cleavage of Ras GTPase‐activating protein‐binding protein 1 (G3BP1). HeLa cells were infected at a MOI of 20 with CVB3, (mock control sample treated with media alone). At each time‐point, the cells were lysed and the expression of G3BP1 and viral proteins VP1 and 3C^pro were analysed by Western blot. The arrow indicates an accumulation of G3BP1 cleavage product 6 h post infection. Actin was used as a loading control.