Bentley A Fane

PMID: 22297520;Abstract:

For a machine to function, it must first be assembled. The morphogenesis of the simplest icosahedral virus would require only 60 copies of a single capsid protein to coalesce. If the capsid protein's structure could be entirely dedicated to this endeavor, the morphogenetic mechanism would be relatively uncomplicated. However, capsid proteins have had to evolve other functions, such as receptor recognition, immune system evasion, and the incorporation of other structure proteins, which can detract from efficient assembly. Moreover, evolution has mandated that viruses obtain additional proteins that allow them to adapt to their hosts or to more effectively compete in their respective niches. Consequently, genomes have increased in size, which has required capsids to do likewise. This, in turn, has lead to more complex icosahedral geometries. These challenges have driven the evolution of scaffolding proteins, which mediate, catalyze, and promote proper virus assembly. The mechanisms by which these proteins perform their functions are discussed in this review. © 2012 Springer Science+Business Media, LLC.

PMID: 1825987;PMCID: PMC1204354;Abstract:

Within the amino acid sequences of polypeptide chains little is known of the distribution of sites and sequences critical for directing chain folding and assembly. Temperature-sensitive folding (tsf) mutations identifying such sites have been previously isolated and characterized in gene 9 of phage P22 encoding the tailspike endorhamnosidase. We report here the isolation of a set of second-site conformational suppressors which alleviate the defect in such folding mutants. The suppressors were selected for their ability to correct the defects of missense tailspike polypeptide chains, generated by growth of gene 9 amber mutants on Salmonella host strains inserting either tyrosine, serine, glutamine or leucine at the nonsense codons. Second-site suppressors were recovered for 13 of 22 starting sites. The suppressors of defects at six sites mapped within gene 9. (Suppressors for seven other sites were extragenic and distant from gene 9.) The missense polypeptide chains generated from all six suppressible sites displayed ts phenotypes. Temperature-sensitive alleles were isolated at these amber sites by pseudoreversion. The intragenic suppressors restored growth at the restrictive temperature of these presumptive tsf alleles. Characterization of protein maturation in cells infected with mutant phages carrying the intragenic suppressors indicates that the suppression is acting at the level of polypeptide chain folding and assembly.

PMID: 14972548;Abstract:

The øX174 DNA binding protein contains two DNA binding domains, containing a series of DNA binding basic amino acids, separated by a proline-rich linker region. Within each DNA binding domain, there is a conserved glycine residue. Glycine and proline residues were mutated and the effects on virion structure were examined. Substitutions for glycine residues yield particles with similar properties to previously characterized mutants with substitutions for DNA binding residues. Both sets of mutations share a common extragenic second-site suppressor, suggesting that the defects caused by the mutant proteins are mechanistically similar. Hence, glycine residues may optimize DNA-protein contacts. The defects conferred by substitutions for proline residues appear to be fundamentally different. The properties of the mutant particles along with the atomic structure of the virion suggest that the proline residues may act to guide the packaged DNA to the adjacent fivefold related asymmetric unit, thus preventing a chaotic packaging arrangement. © 2003 Elsevier Inc. All rights reserved.

By acquiring resistance to an inhibitor, viruses can become dependent on that inhibitor for optimal fitness. However, inhibitors rarely, if ever, stimulate resistant strain fitness to values that equal or exceed the uninhibited wild-type level. This would require an adaptive mechanism that converts the inhibitor into a beneficial replication factor. Using a plasmid-encoded inhibitory external scaffolding protein that blocks ϕX174 assembly, we previously demonstrated that such mechanisms are possible. The resistant strain, referred to as the evolved strain, contains four mutations contributing to the resistance phenotype. Three mutations confer substitutions in the coat protein, whereas the fourth mutation alters the virus-encoded external scaffolding protein. To determine whether stimulation by the inhibitory protein coevolved with resistance or whether it was acquired after resistance was firmly established, the strain temporally preceding the previously characterized mutant, referred to as the intermediary strain, was isolated and characterized. The results of the analysis indicated that the mutation in the virus-encoded external scaffolding protein was primarily responsible for stimulating strain fitness. When the mutation was placed in a wild-type background, it did not confer resistance. The mutation was also placed in cis with the plasmid-encoded dominant lethal mutation. In this configuration, the stimulating mutation exhibited no activity, regardless of the genotype (wild type, evolved, or intermediary) of the infecting virus. Thus, along with the coat protein mutations, stimulation required two external scaffolding protein genes: the once inhibitory gene and the mutant gene acquired during evolution.

Bacteriophage øX174 morphogenesis requires two scaffolding proteins: an internal species, similar to those employed in other viral systems, and an external species, which is more typically associated with satellite viruses. The current model of øX174 assembly is based on structural and in vivo data. During morphogenesis, 240 copies of the external scaffolding protein mediate the association of 12 pentameric particles into procapsids. The hypothesized pentameric intermediate, the 12S⁎ particle, contains 16 proteins: 5 copies each of the coat, spike and internal scaffolding proteins and 1 copy of the DNA pilot protein. Assembly naïve 12S⁎ particles and external scaffolding oligomers, most likely tetramers, formed procapsid-like particles in vitro, suggesting that the 12S⁎ particle is a bona fide assembly intermediate and validating the current model of procapsid morphogenesis. The in vitro system required a crowding agent, was influenced by the ratio of the reactants and was most likely driven by hydrophobic forces. While the system reported here shared some characteristics with other in vitro internal scaffolding protein-mediated systems, it displayed unique features. These features most likely reflect external scaffolding protein-mediated morphogenesis and the øX174 procapsid structure, in which external scaffolding-scaffolding protein interactions, as opposed to coat-coat protein interactions between pentamers, constitute the primary lattice-forming contacts.