Bentley A Fane

PMID: 1828803;Abstract:

Suppressor mutations which alleviate the defects in folding mutants of the P22 gene 9 tailspike protein have recently been isolated (Fane, B. and King, J. (1991) Genetics 127, 263-277). The starting folding defects were in missense polypeptide chains generated by host amino acid insertions at different amber mutant sites. Fragments of genes carrying the amber mutations with and without their independently isolated suppressor mutations were cloned and sequenced. The parental nonsense mutations were located at Q45, K122, E156, W202, W207, Y232, and W365. Their conformational suppressors were single amino acid substitutions at a limited set of sites, V84>A, V331>A, and A334>V. The V331>A or A334>V suppressors were independently recovered starting with different mutant sites suggesting that they acted by some global or general mechanism. When the V331>A and A334>V mutations were crossed into well-characterized temperature-sensitive folding (tsf) mutants at various sites in the tailspike protein, they suppressed all of the eight tsf mutants tested. Since the tsf defects destabilize folding intermediates rather than the native conformation, this result implies that the suppressors act in the folding pathway. Strains carrying the isolated suppressor mutations displayed no obvious phenotypic defect and formed native biologically active tailspikes. Thus, these single amino acid substitutions have striking influences on the efficiency of intracellular chain folding, without causing functional defects in the native protein.

C-terminal, aromatic amino acids in the phiX174 internal scaffolding protein B mediate conformational switches in the viral coat protein. These switches direct the coat protein through early assembly. In addition to the aromatic amino acids, two acidic residues, D111 and E113, form salt bridges with basic, coat protein side chains. Although salt bridge formation did not appear to be critical for assembly, the substitution of an aromatic amino acid for D111 produced a lethal phenotype. This side chain is uniquely oriented toward the center of the coat-scaffolding binding pocket, which is heavily dominated by aromatic ring-ring interactions. Thus, the D111Y substitution may restructure pocket contacts. Previously characterized B(-) mutants blocked assembly before procapsid formation. However, the D111Y mutant produced an assembled particle, which contained the structural and external scaffolding proteins but lacked protein B and DNA. A suppressor within the external scaffolding protein, which mediates the later stages of particle morphogenesis, restored viability. The unique formation of a postprocapsid particle and the novel suppressor may be indicative of a novel B protein function. However, genetic data suggest that the particle represents the delayed manifestation of an early assembly error. This seemingly late-acting defect was rescued by previously characterized suppressors of early, preprocapsid, B(-) assembly mutations, which act on the level of coat protein flexibility. Likewise, the newly isolated suppressor in the external scaffolding protein also exhibited a global suppressing phenotype. Thus, the off-pathway product isolated from infected cells may not accurately reflect the temporal nature of the initial defect.

PMID: 12788631;Abstract:

Putative conformational switching and inhibitory regions in the Microviridae external scaffolding protein were investigated. Substitutions for glycine 61, hypothesized to promote a postdimerization conformational switch, have dominant lethal phenotypes. In previous studies, chimeric α3/φX174 proteins for structures α-helix 1 and loop 6/α-helix 7 inhibited φX174 morphogenesis when expressed from high copy number plasmids. To determine if inhibition was due to overexpression, chimeric genes were constructed into the φX174 genome. In coinfections with wild-type, protein ratios would be 1:1. The helix 1 chimera has a recessive lethal phenotype; thus, overexpression confers inhibition. In single infections, the mutant cannot form procapsids, suggesting that helix 1 mediates the initial recognition of structural proteins. The lethal chimeric helix 7 protein has a dominant phenotype. Alone, the mutant forms defective procapsids, suggesting a later morphogenetic defect. The results of second-site genetic analyses indicate that the capsid-external scaffolding protein interface is larger than revealed in the crystal structure. © 2003 Elsevier Science (USA). All rights reserved.

PMID: 9931252;Abstract:

The assembly of the viral structural proteins into infectious virions is often mediated by scaffolding proteins. These proteins are transiently associated with morphogenetic intermediates but not found in the mature particle. The genes encoding three Microviridae (∅X174, G4 and α3) internal scaffolding proteins (B proteins) have been cloned, expressed in vivo and assayed for the ability to complement null mutations of different Microviridae species. Despite divergence as great as 70% in amino acid sequence over the aligned length, cross-complementation was observed, indicating that these proteins are capable of directing the assembly of foreign structural proteins into infectious particles. These results suggest that the Microviridae internal scaffolding proteins may be inherently flexible. There was one condition in which a B protein could not cross-function. The ∅X174 B protein cannot productively direct the assembly of the G4 capsid at temperatures above 21°C. Under these conditions, assembly is arrested early in the morphogenetic pathway, before the first B protein mediated reaction. Two G4 mutants, which can productively utilize the ∅X174 B protein at elevated temperatures, were isolated. Both mutations confer amino acid substitutions in the viral coat protein but differ in their relative abilities to utilize the foreign scaffolding protein. The more efficient substitution is located in a region where coat-scaffolding interactions have been observed in the atomic structure and may emphasize the importance of interactions in this region.

PMID: 16142226;Abstract:

Biochemical and genetic data defining the assembly pathway and structural biology of the T4 tail apparatus are merging to create a four-dimensional image reconstruction. Human inventions seem to be large-scale replicas of molecular devices honed by evolution. © 2005 Nature Publishing Group.