The twenty-nine amino acid C-terminal cytoplasmic domain of poliovirus 3AB is critical for nucleic acid chaperone activity

Abstract

Poliovirus 3AB protein is the first picornavirus protein demonstrated to have nucleic acid chaperone activity. Further characterization of 3AB demonstrates that the C-terminal 22 amino acids (3B region (also referred to as VPg), amino acid 88–109) of the protein is required for chaperone activity, as mutations in this region abrogate nucleic acid binding and chaperone function. Protein 3B alone has no chaperone activity as determined by established assays that include the ability to stimulate nucleic acid hybridization in a primer-template annealing assay, helix-destabilization in a nucleic acid unwinding assay or aggregation of nucleic acids. In contrast, the putative 3AB C-terminal cytoplasmic domain (C terminal amino acids 81–109, 3B + the last 7 C-terminal amino acids of 3A, termed 3B+7 in this report) possesses strong activity in these assays, albeit at much higher concentrations than 3AB. The characteristics of several mutations in 3B+7 are described here, as well as a model proposing that 3B+7 is the site of the “intrinsic” chaperone activity of 3AB while the 3A N-terminal region (amino acids 1–58) and/or membrane anchor domain (amino acids 59–80) serve to increase the effective concentration of the 3B+7 region leading to the potent chaperone activity of 3AB.

The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB

“…Despite of the numerous commonalities between poliovirus and EV71, the 3AB proteins from these two enteroviruses still exhibited some difference. For example, poliovirus 3AB has been reported to be much more potent than 3B þ7 for RNA chaperone activities (Gangaramani et al, 2010); however, we observed that for EV71, the RNA chaperone activity of wild-type 3AB is only moderately stronger than that of 3Bþ 7 (Fig. 8D).…”

“…During the life cycle of EV71, the RNA chaperone activity of 3AB may participate in diverse processes, such as coating and protecting viral genomic RNA, facilitating the formation and/or deformation of vRNA cis-acting elements, vRNA synthesis by RdRp, and translation (Gangaramani et al, 2010). However, due to technical limitations, Fig.…”

“…To further characterize and verify the RNA chaperone activity of EV71 3AB, a classic assay for measuring helix destabilization and annealing stimulation of RNA chaperones (Cheng et al, 2013;DeStefano and Titilope, 2006;Gangaramani et al, 2010;Heath et al, 2003;Yang et al, 2014) was adapted for EV71 3AB. As illustrated in Fig.…”

“…Previous study of poliovirus 3AB has reported that the 3B region plus the C-terminal last 7 a.a. of 3A is the core region for the RNA chaperone activity of 3AB (Gangaramani et al, 2010). To determine which region of EV71 3AB is required for its RNA chaperone activity, we expressed EV71 3A (1-86 a.a.), 3B (87-108 a.a.) and 3Bþ 7 (80-108 a.a., termed 3Bþ 7) ( Fig.…”

“…Since 3Bþ 7 contains most RNA chaperone activity of 3AB but either 3A or 3B does not, it is reasonable to speculate that the residues within the 3Bþ 7 region and nearby the junction between 3A and 3B are critical for the RNA chaperone activity. Based on the amino acid sequence alignment analysis of 3AB proteins between EV71 and poliovirus, five residues K80, F82, F85, Y89 and R103, whose corresponding residues have been reported to be critical for the RNA chaperone activity of poliovirus 3AB (Gangaramani et al, 2010), were selected; and then, each residue was point mutated to Alanine (A) in 3Bþ 7 (as indicated in Fig. 8A).…”