Bentley A Fane

PMID: 17468245;PMCID: PMC1913433;Abstract:

The obligate intracellular nature of chlamydiae presents challenges to the characterization of its phages, which are potential tools for a genetic transfer system. An assay for phage infectivity is described, and the infectious properties of phage Chp2 were determined. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

PMID: 9018136;Abstract:

The X174 J protein is 37 amino acids in length and contains 12 basic residues. There are no acidic amino acids in the protein. The basic residues are concentrated in two clusters in the N-terminus which are separated by a proline-rich region. To investigate the morphogenetic functions of the J protein and possible mechanisms by which it may bind DNA, a genetic analysis was conducted. Lysine→leucine and arginine→leucine substitutions were generated within the basic amino acid clusters. At least three substitutions were required to eliminate viability in vivo. Lethal mutants with three or four substitutions exhibit dominant lethal phenotypes, indicating that the mutant proteins retain enough function to interfere with productive assembly. In cells infected with a dominant lethal mutant, noninfectious packaged particles were produced. Infectivity can be restored by second-site suppressors in the viral coat protein which disrupt polar interactions atop the threefold axis of symmetry in the capsid. The viability of strains containing compensating frameshift mutations within the proline-rich region suggests that only the proline residues in this segment are critical for efficient function.

The Journal of Virology invited us to submit this article in the GEM platform, which features key developments in virology. Although these articles are peer-reviewed and can report data, they are really more review-like than a manuscript reporting primary data.

PMID: 11000202;PMCID: PMC112362;Abstract:

Viral assembly is an ideal system in which to investigate the transient recognition and interplay between proteins. During morphogenesis, scaffolding proteins temporarily associate with structural proteins, stimulating conformational changes that promote assembly and inhibit off-pathway reactions. Microviridae morphogenesis is dependent on two scaffolding proteins, an internal and an external species. The external scaffolding protein is the most conserved protein within the Microviridae, whose canonical members are φX174, G4, and α3. However, despite 70% homology on the amino acid level, overexpression of a foreign Microviridae external scaffolding protein is a potent cross-species inhibitor of morphogenesis. Mutants that are resistant to the expression of a foreign scaffolding protein cannot be obtained via one mutational step. To define the requirements for and constraints on scaffolding protein interactions, chimeric external scaffolding proteins have been constructed and analyzed for effects on in vivo assembly. The results of these experiments suggest that at least two cross-species inhibitory domains exist within these proteins; one domain most likely blocks procapsid formation, and the other allows procapsid assembly but blocks DNA packaging. A mutation conferring resistance to the expression of a chimeric protein (chiD(r)) that inhibits DNA packaging was isolated. The mutation maps to gene A, which encodes a protein essential for packaging. The chiD(r) mutation confers resistance only to a chimeric D protein; the mutant is still inhibited by the expression of foreign D proteins. The results presented here demonstrate how closely related proteins could be developed into antiviral agents that specifically target virion morphogenesis.

During the final stages of phi X174 morphogenesis, there is an 8.5-A radial collapse of coat proteins around the packaged genome, which is tethered to the capsid's inner surface by the DNA-binding protein. Two approaches were taken to determine whether protein-DNA interactions affect the properties of the mature virion and thus the final stages of morphogenesis. In the first approach, genome-capsid associations were altered with mutant DNA-binding proteins. The resulting particles differed from the wild-type virion in density, native gel migration, and host cell recognition. Differences in native gel migration were especially pronounced. However, no differences in protein stoichiometries were detected. An extragenic second-site suppressor of the mutant DNA-binding protein restores all assayed properties to near wild-type values. In the second approach, phi X174 was packaged with foreign, single-stranded, covalently closed, circular DNA molecules identical in length to the phi X174 genome. The resulting particles exhibited native gel migration rates that significantly differed from the wild type. The results of these experiments suggest that the structure of the genome and/or its association with the capsid's inner surface may perform a scaffolding-like function during the procapsid-to- virion transition.