The critical role of enterovirus 2A protease in viral translation, replication, and antagonism of host antiviral responses.

Schipper, J.; Aloise, C.; Sutter, S. O.; Zwaagstra, M.; van Vliet, A. L. W.; Abdelnabi, R.; Ignacio, B.; Bonger, K. M.; Roelofs, D.; van den Brand, J. M. A.; Wubbolts, R.; Bruurs, L. J. M.; Thibaut, H. J.; Neyts, J.; Tanenbaum, M. E.; van Kuppeveld, F. J. M.

doi:10.1101/2025.02.20.639337

bioRxiv · 10.1101/2025.02.20.639337

The critical role of enterovirus 2A protease in viral translation, replication, and antagonism of host antiviral responses.

Schipper, J.·Aloise, C.·Sutter, S. O.·Zwaagstra, M.·van Vliet, A. L. W.·Abdelnabi, R.·Ignacio, B.·Bonger, K. M.·Roelofs, D.·van den Brand, J. M. A.·Wubbolts, R.·Bruurs, L. J. M.·Thibaut, H. J.·Neyts, J.·Tanenbaum, M. E.·van Kuppeveld, F. J. M.

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Abstract

Enteroviruses dramatically remodel the cellular infrastructure for efficient replication and curtailing host antiviral responses. Proteases 2Apro and 3Cpro have been implicated in these processes based on in vitro studies, ectopic overexpression, and surrogate infection systems, but their relative contributions are unknown. Here, we replace the essential 2A cleavage site at the P1-P2 junction with an internal ribosome entry site (IRES), 3CD cleavage site, or T2A sequence, allowing us to catalytically inactivate 2Apro. Viruses with an inactive 2Apro are hampered in replication in cell lines and are severely attenuated in a Coxsackievirus B3 (CVB3) mouse pancreatitis infection model. We show that 2Apro, but not 3Cpro, is essential during infection for disturbing nucleocytoplasmic transport, shutting down host mRNA translation, suppressing stress granule formation, cleaving retinoic acid-inducible gene-1 (RIG-I)-like receptor (RLR) signaling pathway proteins and suppressing interferon-/{beta} (IFN-/{beta}) transcription, and overcoming the antiviral action of IFN-induced restriction factors. Moreover, using an advanced single-molecule live cell imaging approach, we reveal that 2Apro is important for the initial round of replication of the incoming viral RNA, which is a bottleneck for efficient infection. In conclusion, we establish that 2Apro plays a critical role in subverting antiviral responses and establishing a favorable host environment to expedite enterovirus replication.

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Schipper, J., Aloise, C., Sutter, S. O., Zwaagstra, M., van Vliet, A. L. W., Abdelnabi, R., Ignacio, B., Bonger, K. M., Roelofs, D., van den Brand, J. M. A., Wubbolts, R., Bruurs, L. J. M., Thibaut, H. J., Neyts, J., Tanenbaum, M. E., van Kuppeveld, F. J. M.. 2025-02-20. The critical role of enterovirus 2A protease in viral translation, replication, and antagonism of host antiviral responses.. https://doi.org/10.1101/2025.02.20.639337

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