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).