MUTATIONAL ANALYSIS OF POLIOVIRUS PROTEIN 3AB TO IDENTIFY REGIONS CRITICAL TO NUCLEIC ACID CHAPERONE ACTIVITY
DeStefano, Jeffrey J
MetadataShow full item record
Poliovirus 3AB protein was the first picornavirus protein demonstrated to have nucleic acid chaperone activity. Current results demonstrate that chaperone activity requires the C-terminal 22 amino acid (3B region (also referred to as VPg), amino acid 88-109) of the protein as mutations in this region abrogated nucleic acid binding and chaperone activity. Protein 3B alone had no chaperone activity as determined by established assays testing chaperone activity including: the ability to stimulate nucleic acid hybridization in a primer-template annealing assay, or helix-destabilization in a nucleic acid unwinding assay, or aggregation of nucleic acids. In contrast, the putative 3AB C-terminal cytoplasmic domain (N terminal amino acid 81-109, 3B + the last 7 C-terminal amino acids of 3A, termed 3B+7 in this report) possessed strong activity in these assay, albeit at much higher concentrations than 3AB. Results from several mutations in 3B+7 are described and a model proposing that 3B+7 possesses the "intrinsic" chaperone activity of 3AB while the 3A N-terminal region (amino acid 1-58) and/or membrane anchor domain (amino acid 59-80) serve to increase the effective concentration of the 3B+7 region leading to the potent chaperone activity of 3AB. Two mutations with reduced chaperone activity in vitro, K81A and F83A in 3AB were tested in tissue culture. Viruses with these mutations produced near wildtype and minute plaques, respectively. F83A gave rise to revertants with either wildtype 3AB sequences or additional nearby compensatory mutations. Translation and polyprotein processing were not affected by these mutations but RNA synthesis compared to wildtype, was slightly lower for K81A and significantly lower for F83A. This data suggests that mutations that decrease chaperone activity of 3AB may lead to decreased RNA synthesis, although the exact steps that are affected need to be determined.