Bentley A Fane

Although bacteriophage φX174 is easy to propagate and genetically tractable, it is use as a peptide display platform has not been explored. One region within the φX174 major spike protein G tolerated 13 of 16 assayed insertions, ranging from 10 to 75 amino acids. The recombinant proteins were functional and incorporated into infectious virions. In the folded protein, the peptides would be icosahedrally displayed within loops that extend from the protein׳s β-barrel core. The well-honed genetics of φX174 allowed permissive insertions to be quickly identified by the cellular phenotypes associated with cloned gene expression. The cloned genes were easily transferred from plasmids to phage genomes via recombination rescue. Direct ELISA validated several recombinant virions for epitope display. Some insertions conferred a temperature-sensitive (ts) protein folding defect, which was suppressed by global suppressors in protein G, located too far away from the insertion to directly alter peptide display.

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.

PMID: 11813297;Abstract:

The sensitivity of in vivo transgenic mutation assays benefits from the sequencing of mutations, although the large number of possible mutations hinders high throughput sequencing. A forward mutational assay exists for ΦX174 that requires on altered, functionol ΦX174 protein and therefore should have fewer torgets (sense, base-pair substitutions) than forward assays that inactivate a protein. We investigated this assay to determine the number of targets and their suitability for detecting o known mutagen, N-ethyl-N-nitrosourea (ENU). We identified 25 target sites and 33 different mutations in ΦX174 gene A after sequencing over 350 spontaneous and ENU-induced mutants, mostly from mouse embryonic cell line PX-2 isolated from mice transgenic for ΦX174 am3, cs70 (line 54). All six types of base-pair substitution were represented omong both the spontaneous and ENU-treated mutant spectra. The mutant spectra from cells treated with 200 and 400 μg/ml ENU were both highly different from the spontaneous spectrum (P 0.000001) but not from each other. The dose trend was significant (P 0.0001) for a linear regression of mutant frequencies (R² = 0.79), with a ninefold increase in mutant frequency at the 400 μg/ml dose. The spontaneous mutant frequency was 1.9 × 10^-5 and the spontaneous spectrum occurred at 11 target base pairs with 15 different mutations. Thirteen mutations at 12 torgets were identified only from ENU-treated cells. Seven mutations had highly significant increases with ENU treotment (P 0.0001) and 15 showed significant increases. The results suggest that the ΦX174 forward assay might be developed into a sensitive, inexpensive in vivo mutagenicity assay.

The phiX174 DNA pilot protein H forms an oligomeric DNA-translocating tube during penetration. However, monomers are incorporated into 12 pentameric assembly intermediates, which become the capsid's icosahedral vertices. The protein's N terminus, a predicted transmembrane helix, is not represented in the crystal structure. To investigate its functions, a series of absolute and conditional lethal mutations were generated. The absolute lethal proteins, a deletion and a triple substitution, were efficiently incorporated into virus-like particles lacking infectivity. The conditional lethal mutants, bearing cold-sensitive (cs) and temperature-sensitive (ts) point mutations, were more amenable to further analyses. Viable particles containing the mutant protein can be generated at the permissive temperature and subsequently analyzed at the restrictive temperature. The characterized cs defect directly affected host cell attachment. In contrast, ts defects were manifested during morphogenesis. Particles synthesized at permissive temperature were indistinguishable from wild-type particles in their ability to recognize host cells and deliver DNA. One mutation conferred an atypical ts synthesis phenotype. Although the mutant protein was efficiently incorporated into virus-like particles at elevated temperature, the progeny appeared to be kinetically trapped in a temperature-independent, uninfectious state. Thus, substitutions in the N terminus can lead to H protein misincorporation, albeit at wild-type levels, and subsequently affect particle function. All mutants exhibited recessive phenotypes, i.e., rescued by the presence of the wild-type H protein. Thus, mixed H protein oligomers are functional during DNA delivery. Recessive and dominant phenotypes may temporally approximate H protein functions, occurring before or after oligomerization has gone to completion.

In an earlier study, protein-based barriers to horizontal gene transfer were investigated by placing the bacteriophage G4 G gene, encoding the major spike protein, into the φX174 genome. The foreign G protein promoted off-pathway assembly reactions, resulting in a lethal phenotype. After three targeted genetic selections, one of two foreign spike proteins was productively integrated into the φX174 system: the complete G4 or a recombinant G4/φX174 protein (94% G4:6% φX174). However, strain fitness was very low. In this study, the chimeras were characterized and experimentally evolved. Inefficient assembly was the primary contributor to low fitness: accordingly, mutations affecting assembly restored fitness. The spike protein preference of the ancestral and evolved strains was determined in competition experiments between the foreign and φX174G proteins. Before adaptation, both G proteins were incorporated into virions; afterwards, the foreign proteins were strongly preferred. Thus, a previously inhibitory protein became the preferred substrate during assembly.