φX174 procapsid assembly: Effects of an inhibitory external scaffolding protein and resistant coat proteins in vitro

During φX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro. In previous studies, φX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context.