Site-saturation mutagenesis of the PALTAVETG motif in coxsackievirus A9 capsid protein VP1 reveals evidence of conservation of a periodic hydrophobicity profile

Enteroviruses possess a highly conserved 9 amino acid stretch of mainly hyd rophobic character in the capsid protein VP1. A novel strategy, combining s ite-saturation mutagenesis and a single-tube cloning and transfection proce dure, has been developed for the analysis of this motif in coxsackievirus A 9 (CAV-9). Four individual amino acids were separately mutated. Mutagenesis of three of the four positions in CAV-9 resulted in a number of viable but impaired mutant strains, each containing a single amino acid substitution. In contrast, no mutants with amino acid substitutions at leucine 31 were i solated, although three different leucine codons were found among the virus es recovered, Small plaque size was regularly associated with reduced yield s of infectious virus and an amino acid substitution at the target site in the viruses isolated from the site-saturated virus pools. From the range of amino acids observed in viable mutants, it was possible to estimate the ch aracteristics that are required at individual amino acid positions. It seem s that in the motif studied here, a periodic hydrophobicity profile needs t o be conserved. The constraints observed on the ranges of acceptable amino acids presumably reflect the structural-functional requirements that have r esulted in the conservation of the motif.