Characterization and function of putative substrate specificity domain in microvirus external scaffolding proteins.
Microviruses (canonical members are bacteriophages phiX174, G4, and alpha3) are T=1 icosahedral virions with an assembly pathway mediated by two scaffolding proteins. The external scaffolding protein D plays a major role during morphogenesis, particularly in icosahedral shell formation. The results of previous studies, conducted with a cloned chimeric external scaffolding gene, suggest that the first alpha-helix acts as a substrate specificity domain, perhaps mediating the initial coat-external scaffolding protein interaction. However, the expression of a cloned gene could lead to protein concentrations higher than those found in typical infections. Moreover, its induction before infection could alter the timing of the protein's accumulation. Both of these factors could drive or facilitate reactions that may not occur under physiological conditions or before programmed cell lysis. In order to elucidate a more detailed mechanistic model, a chimeric external scaffolding gene was placed directly in the phiX174 genome under wild-type transcriptional and translational control, and the chimeric virus, which was not viable on the level of plaque formation, was characterized. The results of the genetic and biochemical analyses indicate that alpha-helix 1 most likely mediates the nucleation reaction for the formation of the first assembly intermediate containing the external scaffolding protein. Mutants that can more efficiently use the chimeric scaffolding protein were isolated. These second-site mutations appear to act on a kinetic level, shortening the lag phase before virion production, perhaps lowering the critical concentration of the chimeric protein required for a nucleation reaction.
Uchiyama
Asako
A
Department of Veterinary Sciences and Microbiology, University of Arizona, Tucson, AZ 85721-0090, USA.
Chen
Min
M
Fane
Bentley A
BA
D000595:Amino Acid Sequence; D010584:Bacteriophage phi X 174 / Q000737:chemistry / Q000235:genetics / Q000254:growth & development*; D005814:Genes, Viral; D016679:Genome, Viral; D007700:Kinetics; D017915:Microvirus / Q000737:chemistry / Q000235:genetics / Q000254:growth & development*; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins