Mutation in enterovirus 71 nonstructural protein 3A increases genome replication fidelity and exhibits attenuated...

Increasing numbers of studies have highlighted the important implications of the replication fidelity of RNA viruses for virus replication, pathogenesis, and the development of antiviral drugs and live-attenuated vaccines. However, most of the research has focused on viral polymerase, and little information is available about the potential role of other viral replicase proteins. Here, we demonstrated that the mutations of 3A_V75A and 3D_V63A+M393L in enterovirus 71 (EV71) conferred high-fidelity phenotypes through deep sequencing of virus populations in cell culture and animal models of infection. The 3A_V75A, 3D_V63A+M393L, and 3A_V75A-3D_V63A+M393L high-fidelity variants were highly attenuated in immunocompetent suckling mice. The RdRP enzymology data indicated that the V63A + M393L mutation of 3D^pol increased the fidelity of RNA synthesis. The addition of the purified 3AB, either WT or V75A, as previously reported, was able to stimulate the RNA synthesis; interestingly, it effectively enhanced the replication fidelity of WT 3D^pol. Moreover, the RNA chaperone activity assay showed that V75A 3AB had reduced helix unwinding activity compared with WT 3AB, implying the potential correlation between chaperone activity and fidelity regulation. Together, our results reveal a novel role of the 3A protein in fidelity, providing a basis for the development of antiviral inhibitors and live-attenuated EV71 vaccines.IMPORTANCENumerous viral polymerases in various RNA viruses, such as EV71 3D^pol, have been reported to be involved in the regulation of replication fidelity, while the role of other viral replicases in this process is poorly understood. In this study, we demonstrate that the 3A_V75A variant of EV71 confers increased fidelity and attenuated virulence, and the addition of 3AB, either WT or V75A mutant, can enhance the replication fidelity of WT 3D^pol during RNA synthesis. Collectively, this work identifies EV71 nonstructural protein 3A as a previously unrecognized fidelity determinant.