Coat protein mutations that alter the flux of morphogenetic intermediates through the ϕ X174 early assembly pathway

Brody J. Blackburn, Shuaizhi Li, Aaron P. Roznowski, Alexis R. Perez, Rodrigo H. Villarreal, Curtis J. Johnson, Margaret Hardy, Edward C. Tuckerman, April D. Burch, Bentley A Fane

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Two scaffolding proteins orchestrate ϕ X174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine, and methionine substitutions were phenotypically indistinguishable from the wild type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperaturesensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, clonedgene expression compensated for inefficient B protein binding, as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wildtype levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability.

Original languageEnglish (US)
Article numbere01384-17
JournalJournal of Virology
Volume91
Issue number24
DOIs
StatePublished - Dec 1 2017

Fingerprint

scaffolding proteins
Capsid Proteins
coat proteins
mutation
Mutation
Protein Binding
lethal genes
Proteins
protein binding
mutants
Genes
Phenotype
Aromatic Amino Acids
phenotype
amino acids
genes
Phenylalanine
Morphogenesis
Codon
Tryptophan

Keywords

  • Bacteriophage phiX174
  • Coat protein
  • Scaffolding protein
  • Virus assembly

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Coat protein mutations that alter the flux of morphogenetic intermediates through the ϕ X174 early assembly pathway. / Blackburn, Brody J.; Li, Shuaizhi; Roznowski, Aaron P.; Perez, Alexis R.; Villarreal, Rodrigo H.; Johnson, Curtis J.; Hardy, Margaret; Tuckerman, Edward C.; Burch, April D.; Fane, Bentley A.

In: Journal of Virology, Vol. 91, No. 24, e01384-17, 01.12.2017.

Research output: Contribution to journalArticle

Blackburn, BJ, Li, S, Roznowski, AP, Perez, AR, Villarreal, RH, Johnson, CJ, Hardy, M, Tuckerman, EC, Burch, AD & Fane, BA 2017, 'Coat protein mutations that alter the flux of morphogenetic intermediates through the ϕ X174 early assembly pathway', Journal of Virology, vol. 91, no. 24, e01384-17. https://doi.org/10.1128/JVI.01384-17
Blackburn, Brody J. ; Li, Shuaizhi ; Roznowski, Aaron P. ; Perez, Alexis R. ; Villarreal, Rodrigo H. ; Johnson, Curtis J. ; Hardy, Margaret ; Tuckerman, Edward C. ; Burch, April D. ; Fane, Bentley A. / Coat protein mutations that alter the flux of morphogenetic intermediates through the ϕ X174 early assembly pathway. In: Journal of Virology. 2017 ; Vol. 91, No. 24.
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AU - Villarreal, Rodrigo H.

AU - Johnson, Curtis J.

AU - Hardy, Margaret

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