A Single Amino Acid Substitution in Poliovirus Nonstructural Protein 2C ATPase Causes Conditional Defects in Encapsidation and Uncoating

The specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2C(ATPase) In particular, residue N252 of poliovirus 2C(ATPase) interacts with VP3 of coxsackievirus A20, in the context of a chimeric virus. Poliovirus 2C(ATPase) has important roles both in RNA replication and encapsidation. In this study, we searched for additional sites in 2C(ATPase), near N252, that are required for encapsidation. Accordingly, segments adjacent to N252 were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2C(ATPase), caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wild-type (wt) or temperature-sensitive (ts) growth phenotypes or had a lethal growth phenotype due to defective RNA replication. The K259A mutation, mapping to an α helix in the predicted structure of 2C(ATPase), resulted in a cold-sensitive virus. In vivo protein synthesis and virus production were strikingly delayed at 33°C relative to the wt, suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33°C. Cell imaging confirmed a much-reduced production of K259A mature virus at 33°C relative to the wt. In conclusion, we have for the first time linked a cold-sensitive encapsidation defect in 2C(ATPase) (K259A) to a subsequent delay in uncoating of the virus particle at 33°C during the next cycle of infection.