Bentley A Fane

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.

PMID: 10792987;Abstract:

Microviridae morphogenesis is dependent on two scaffolding proteins, an internal and external species. Both structural and genetic analyses suggest that the COOH-terminus of the internal protein is critical for coat protein recognition and specificity. To test this hypothesis, chimeric internal scaffolding genes between Microviridae members φX174, G4, and α3 were constructed and the proteins expressed in vivo. All of the chimetic proteins were functional in complementation assays. However, the efficient complementation was observed only when the vital coat protein and COOH- terminus of internal scaffolding were of the same origin. Genes with 5' deletions of the φX174 internal scaffolding gene were also constructed and expressed in vivo. Proteins lacking the first 10 amino acids, which self- associate across the twofold axes of symmetry in the atomic structure, efficiently complement φX174 am(B) mutants at temperatures above 24°C. These results suggest that internal scaffolding protein self-associations across the twofold axes of symmetry are required only at lower temperatures. (C) 2000 Academic Press.

PMID: 8642594;Abstract:

Bacteriophage G4 and ∅X174 are members of the Microviridae family. The degree of similarity of the structural proteins ranges from 66% identity of the F protein to 40% identity of the G protein. The atomic structure of the ∅X174 virion had previously been determined by X-ray crystallography. Bacteriophage G4 procapsids, consisting of the structural proteins F, G, D, B, H, and small traces of J but no DNA, were set up for crystallization. However, the resultant crystals were of degraded procapsid particles, which had lost the assembly scaffolding proteins D and B, resulting in particles that resembled empty virions. The structure of the degraded G4 procapsid has been determined to 3.0 Å resolution. The particles crystallized in the hexagonal space group P6322 with unit cell dimensions a = b = 414.2(5) Å and c = 263.0(3) Å. The diffraction data were collected at the Cornell High Energy Synchrotron Source (CHESS) on film and image plates using oscillation photography. Packing considerations indicated there were two particles per unit cell. A self-rotation function confirmed that the particles were positioned on 32 point group special positions in the unit cell. Initial phases were calculated to 6 Å resolution, based on the known ∅X174 virion model. Phase information was then extended in steps to 3.0 Å resolution by molecular replacement electron density modification and particle envelope generation. The resulting electron density map was readily interpretable in terms of the F and G polypeptides, as occur in the mature capsid of ∅X174. In a few regions of the electron density map there were inconsistencies between the density and the published amino acid sequence. Redetermining the amino acid sequence confirmed that the density was correct. The r.m.s. deviation between the C(α) backbone of the mature capsid of ∅X174 and the degraded G4 procapsid was 0.36 Å for the F protein and 1.38 Å for the G protein. This is consistent with the greater conservation of the F protein compared to the G protein sequences among members of the Microviridae family. Functionally important features between ∅X174 and G4 had greater conservation. Calcium ions (Ca²⁺) were shown to bind to G4 at a general site located near the icosahedral 3-fold axis on the F protein capsid, equivalent to sites found previously in ∅X174. Binding of Ca²⁺ also caused the ordering of the conserved region of the DNA binding protein J, which was present in the degraded procapsid particle in the absence of DNA.