Investigation of role of enterovirus A71 nonstructural 3A protein and interacting protein in viral replication

Enterovirus A71 (EV-A71) is a positive-sense single-stranded RNA (+ssRNA) virus in the Picornaviridae family that causes hand, foot, and mouth disease (HFMD) associated with neurological complications in young children. As yet, there is no specific treatment for EV-A71 infection due to inadequate information on viral neuropathogenesis. Generally, the +ssRNA viruses have strategies for establishing viral replication by remodeling host cell membranes to construct distinct platforms for viral RNA synthesis and virion assembly, termed as replication organelles (ROs). In enterviruses, the nonstructural 3A protein has been reported as a key player in RO formation. In this study, the cellular factor in human SH-SY5Y neuronal cells interacting with the EV-A71 3A protein was searched for understanding structure and function as well as the role of the 3A protein or its interacting counterpart in neuropathogenesis. Transient expression of recombinant 3A protein in fusion with FLAG and mCherry protein (FLAG-3A-mCherry) was successfully established in human neuronal SH-SY5Y cells. By FLAG pull-down, proteins in neuronal cell co-purified with FLAG-3A-mCherry were identified by LC-MS/MS. The potential identified EV-A71 3A interacting protein was human PRSS3 (mesotrypsinogen, also known as brain trypsinogen). The transcript variants of PRSS3 were further identified in SH-SY5Y cells. It was shown that only PRSS3 variant 3 (PRSS3-v3) was detected. Thus, full-length cDNA of PRSS3-v3 molecularly fused with the coding sequence of Myc tag (FL-PRSS3-Myc) was constructed. The interaction of FLAG-3A-mCherry and FL-PRSS3-Myc was confirmed by co-immunoprecipitation assay using anti-Myc magnetic beads. The role of PRSS3 in EV-A71 replication was subsequently investigated by overexpressing FL-PRSS3-Myc or silencing PRSS3 by siRNA in EV-A71 infected cells. The results have shown that PRSS3 had a positive effect on EV-A71 replication. The detailed mechanism of human PRSS3 involved in EV-A71 replication and neuropathogenesis warrants further experimental elucidation. In conclusion, this study has discovered a novel EV-A71 3A interacting protein that offers the opportunity to study the neuropathogenesis of EV-A71 infection which paves the way for developing a specific treatment for EV-A71 infection.