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. Res...

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Bibliographic Details
Published in:Journal of virology 2017, Vol.91 (1)
Main Authors: Cherwa, Jr, James E, Tyson, Joshua, Bedwell, Gregory J, Brooke, Dewey, Edwards, Ashton G, Dokland, Terje, Prevelige, Peter E, Fane, Bentley A
Format: Article
Language:English
Subjects:
Bacteriophage phi X 174 - chemistry
Bacteriophage phi X 174 - genetics
Bacteriophage phi X 174 - metabolism
Capsid - chemistry
Capsid - metabolism
Capsid Proteins - chemistry
Capsid Proteins - genetics
Capsid Proteins - metabolism
Directed Molecular Evolution
Gene Expression Regulation, Viral
Genes, Lethal
Genetic Fitness
Kinetics
Models, Molecular
Mutation
Protein Domains
Protein Multimerization
Protein Structure, Secondary
Spotlight
Structure and Assembly
Virus Assembly
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Summary: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. Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.
ISSN:0022-538X
1098-5514
DOI:10.1128/JVI.01878-16