title: ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors: Department of Biology, Central Alabama Community College, Alexander City, Alabama, USA jcherwajr@cacc.edu.
authors: James E
authors: JE
authors: Cherwa
authors: Department of Biology, Central Alabama Community College, Alexander City, Alabama, USA.
authors: Joshua
authors: J
authors: Tyson
authors: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors: Gregory J
authors: GJ
authors: Bedwell
authors: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors: Dewey
authors: D
authors: Brooke
authors: Jefferson County International Baccalaureate School, Birmingham, Alabama, USA.
authors: Ashton G
authors: AG
authors: Edwards
authors: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors: Terje
authors: T
authors: Dokland
authors: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors: Peter E
authors: PE
authors: Prevelige
authors: School of Plant Sciences and the BIO5 Institute, University of Arizona, Tucson, Arizona, USA.
authors: Bentley A
authors: BA
authors: Fane
mesh_terms: D010584:Bacteriophage phi X 174 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002213:Capsid / Q000737:chemistry* / Q000378:metabolism; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution; D015967:Gene Expression Regulation, Viral; D005804:Genes, Lethal; D056084:Genetic Fitness; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D000072417:Protein Domains; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins
keywords: Microviridae; bacteriophage ϕX174; microvirus; scaffolding protein; virus assembly