Identification And Characterization Of Enterovirus 71 Nonstructural Protein 3AB

Enterovirus 71 (EV71), a member of the genus Enterovirus in the family Picornaviridae, is the major causative pathogen for hand-foot-and-mouth disease (HFMD) in children, and can also cause neurological syndromes, such as aseptic meningitis, encephalitis, poliomyelitis-like paralysis, and even death. Following the mostly complete eradication of poliovirus and poliomyelitis inmost regions around the world, EV71 hasemerged as the most important human neuron-tropic enterovirus that causeserious and even fatal infectious disease in a wide range. So far, no effective vaccine or antiviraltherapy is available for EV71. Despite of the importance of EV71, the nonstructural protein 3AB from this virus is little understood for its function during EV71 replication.Using Bac-to-Bac baculovirus expression system and maltose fusion protein affinity chromatography purification, we expressed 3AB protein as recombinant proteinwith function and activity. Here we found that 3AB possesses a nucleic acid helix-destabilizing activity. We acknowledged that it could unwind RNA-RNA, RNA-DNA and DNA-DNA duplexes all with single-stranded overhangs. Meanwhile, 3AB was able to destabilize both 3'-tailed and 5'-tailed RNA helices, but not the duplex with blunt-ended. This suggests that helix-destabilizing activity of EV713AB has no directionality or polarity.We also found that Mg2+is not essential for the helix-destabilizing activity of EV713AB. The helix-destabilizing activity of EV713AB was insensitive to Mg2+ when its concentration is lower than 5 mM, while high Mg2+concentration (10 mM) is even moderate inhibitory. Our results also showed that the inhibition action of Zn2+ was sensitive, when at lower (0.5 mM) Zn2+concentrations, the helix-destabilizing activity of MBP-3AB was moderately inhibited.We detected that the helix destabilizing activity of 3AB was ATP independent and that 3AB do not possess the ATPase activity, which declared that MBP-3AB is not helicase.Further research found that EV71 3AB possesses an RNAchaperone activity that is able to destabilize structured RNA strands and stimulate RNA strand annealing. Moreover, we determined that 3B plus the last 7 amino acids at the C-terminal of 3A (termed 3B+7) possess the RNA chaperone activity, and five amino acids, i.e. Lys-80, Phe-82, Phe-85, Tyr-89, and Arg-103 are critical and probably the active sites of 3AB for its RNA chaperone activity, maybe the ideal targets for antiviral therapy.This report reveals that EV713AB displays an RNA chaperone activity, adds a new member to the growing list of virus-encoded RNA chaperones. This research fills in the blank in the functional research of the nonstructural protein 3 AB, a thorough understanding to its roles in EV71 virology would be desired for combatingthis emerging virus, and may serve as a theoretical basis for future studies.