Proteolytic Activities of Enterovirus 2A Do Not Depend on Its Interaction with SETD3

Enterovirus 2A^pro is a protease that proteolytically processes the viral polyprotein and cleaves several host proteins to antagonize host responses during enteroviral infection. Recently, the host protein actin histidine methyltransferase SET domain containing 3 (SETD3) was identified to interact with 2A^pro and to be essential for virus replication. The role of SETD3 and its interaction with 2A^pro remain unclear. In this study, we investigated the potential involvement of SETD3 in several functions of 2A^pro. For this, we introduced the 2A^pro from coxsackievirus B3 (CVB3) in a mutant of encephalomyocarditis virus (EMCV) containing an inactivated Leader protein (EMCV-L^zn) that is unable to shut down host mRNA translation, to trigger nucleocytoplasmic transport disorder (NCTD), and to suppress stress granule (SG) formation and type I interferon (IFN) induction. Both in wt HeLa cells and in HeLa SETD3 knockout (SETD3^KO) cells, the virus containing active 2A^pro (EMCV-2A^pro) efficiently cleaved eukaryotic translation initiation factor 4 gamma (eIF4G) to shut off host mRNA translation, cleaved nucleoporins to trigger NCTD, and actively suppressed SG formation and IFN gene transcription, arguing against a role of SETD3 in these 2A^pro-mediated functions. Surprisingly, we observed that the catalytic activity of enteroviral 2A is not crucial for triggering NCTD, as a virus containing an inactive 2A^pro (EMCV-2A^m) induced NCTD in both wt and SETD3^KO cells, albeit delayed, challenging the idea that the NCTD critically depends on nucleoporin cleavage by this protease. Taken together, our results do not support a role of SETD3 in the proteolytic activities of enterovirus 2A^pro.