Bentley A Fane

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.

PMID: 19726521;PMCID: PMC2772694;Abstract:

Viruses often evolve resistance to antiviral agents. While resistant strains are able to replicate in the presence of the agent, they generally exhibit lower fitness than the wild-type strain in the absence of the inhibitor. In some cases, resistant strains become dependent on the antiviral agent. However, the agent rarely, if ever, elevates dependent strain fitness above the uninhibited wild-type level. This would require an adaptive mechanism to convert the antiviral agent into a beneficial growth factor. Using an inhibitory scaffolding protein that specifically blocks ΦX174 capsid assembly, we demonstrate that such mechanisms are possible. To obtain the quintuple-mutant resistant strain, the wild-type virus was propagated for approximately 150 viral life cycles in the presence of increasing concentrations of the inhibitory protein. The expression of the inhibitory protein elevated the strain's fitness significantly above the uninhibited wild-type level. Thus, selecting for resistance coselected for dependency, which was characterized and found to operate on the level of capsid nucleation. To the best of our knowledge, this is the first report of a virus evolving a mechanism to productively utilize an antiviral agent to stimulate its fitness above the uninhibited wild-type level. The results of this study may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

phiX174 utilizes two scaffolding proteins during morphogenesis, an internal protein (B) and an external protein (D). The B protein induces a conformational change in coat protein pentamers, enabling them to interact with both spike and external scaffolding proteins. While functions of the carboxyl terminus of protein B have been defined, the functions of the amino terminus remain obscure. To investigate the morphogenetic functions of the amino terminus, several 5' deleted genes were constructed and the proteins expressed in vivo. The DeltaNH(2) B proteins were assayed for the ability to complement an ochre B mutant and defects in the morphogenetic pathway were characterized. The results of the biochemical, genetic and second-site genetic analyses indicate that the amino terminus induces conformational changes in the viral coat protein and facilitates minor spike protein incorporation. Defects in conformational switching can be suppressed by substitutions in the external scaffolding protein, suggesting some redundancy of function between the two proteins.