Bentley A Fane

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.

Approximately 20% of the data in this manuscript was generated by students enrolled in the 2012 Summer Virology Lab Course.

PMID: 1833267;PMCID: PMC1204541;Abstract:

This study describes the isolation of second-site suppressors which correct for the defects associated with cold-sensitive (cs) prohead accessory proteins of bacteriophage ΦX174. Five phenotypically different suppressors were isolated. Three of these suppressors confer novel temperature-sensitive (ts) phenotypes. They were unable to complement a ts mutation in gene F which encodes the major coat protein of the phage. All five suppressor mutations confer nucleotide changes in the gene F DNA sequence. These changes define four amino acid sites in the gene F protein. Three suppressor mutations placed into an otherwise wild-type background display a cold resistant phenotype in liquid culture infections when compared to a wild-type ΦX174 control.

PMID: 1532571;PMCID: PMC205913;Abstract:

The functions of the small DNA-binding protein, gpJ, of bacteriophages φX174 and G4 were examined by in vivo cross-complementation and sucrose gradient sedimentation. The morphogenetic roles of the two proteins may differ. The φX174 J protein may be required for the formation or stabilization of the φX174 prohead.