Bentley A Fane

PMID: 1648264;Abstract:

Amino acid substitutions at a site in the center of the bacteriophage protein P22 tailspike polypeptide chain suppress temperature-sensitive folding mutations at many sites throughout the chain. Characterization of the intracellular folding and chain assembly process reveals that the suppressors act in the folding pathway, inhibiting the aggregation of an early folding intermediate into the kinetically trapped inclusion body state. The suppressors alone increase the folding efficiency of the otherwise wild-type polypeptide chain without altering the stability or activity of the native state. These amino acid substitutions identify an unexpected aspect of the protein folding grammar-sequences within the chain that carry information inhibiting unproductive off-pathway conformations. Such mutations may serve to increase the recovery of protein products of cloned genes.

PMID: 18400861;PMCID: PMC2395140;Abstract:

In the φX174 procapsid crystal structure, 240 external scaffolding protein D subunits form 60 pairs of asymmetric dimers, D1D 2 and D3D4, in a non-quasi-equivalent structure. To achieve this arrangement, α-helix 3 assumes two different conformations: (i) kinked 30° at glycine residue 61 in subunits D 1 and D3 and (ii) straight in subunits D2 and D4. Substitutions for G61 may inhibit viral assembly by preventing the protein from achieving its fully kinked conformation while still allowing it to interact with other scaffolding and structural proteins. Mutations designed to inhibit conformational switching in α-helix 3 were introduced into a cloned gene, and expression was demonstrated to inhibit wild-type morphogenesis. The severity of inhibition appears to be related to the size of the substituted amino acid. For infections in which only the mutant protein is present, morphogenesis does not proceed past the first step that requires the wild-type external scaffolding protein. Thus, mutant subunits alone appear to have little or no morphogenetic function. In contrast, assembly in the presence of wild-type and mutant subunits is blocked prematurely, before D protein is required in a wild-type infection, or channeled into an off-pathway reaction. These data suggest that the wild-type protein transports the inhibitory protein to the pathway. Viruses resistant to the lethal dominant proteins were isolated, and mutations were mapped to the coat and internal scaffolding proteins. The affected amino acids cluster in the atomic structure and may act to exclude mutant subunits from occupying particular positions atop pentamers of the viral coat protein. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

PMID: 15046981;Abstract:

Packaging of viral genomes into their respective capsids requires partial neutralization of the highly negatively charged RNA or DNA. Many viruses, including the Microviridae bacteriophages φX174, G4, and α3, have solved this problem by coding for a highly positively charged nucleic acid-binding protein that is packaged along with the genome. The φX174 DNA-binding protein, J, is 13 amino acid residues longer than the α3 and G4 J proteins by virtue of an additional nucleic acid-binding domain at the amino terminus. Chimeric φX174 particles containing the smaller DNA-binding protein cannot be generated due to procapsid instability during DNA packaging. However, chimeric α3 and G4 phages, containing the φX174 DNA-binding protein in place of the endogenous J protein, assemble and are infectious, but are less dense than the respective wild-type species. In addition, host cell attachment and native gel migration assays indicate surface variations of these viruses that are controlled by the nature of the J protein. The structure of α3 packaged with φX174 J protein was determined to 3.5Å resolution and compared with the previously determined structures of φX174 and α3. The structures of the capsid and spike proteins in the chimeric particle remain unchanged within experimental error when compared to the wild-type α3 virion proteins. The amino-terminal region of the φX174 J protein, which is missing from wild-type α3 virions, is mostly disordered in the α3 chimera. The differences observed between solution properties of wild-type φX174, wild-type α3, and α3 chimera, including their ability to attach to host cells, correlates with the degree of order in the amino-terminal domain of the J protein. When ordered, this domain binds to the interior of the viral capsid and, thus, might control the flexibility of the capsid. In addition, the properties of the φX174 J protein in the chimera and the results of mutational analyses suggest that an evolutionary correlation may exist between the size of the J protein and the stoichiometry of the DNA pilot protein H, required in the initial stages of infection. Hence, the function of the J protein is to facilitate DNA packaging, as well as to mediate surface properties such as cell attachment and infection. © 2004 Elsevier Ltd. All rights reserved.

PMID: 2822533;PMCID: PMC1203193;Abstract:

Amber mutations have been isolated and mapped to more than 60 sites in gene 9 of P22 encoding the thermostable phage tailspike protein. Gene 9 is the locus of over 30 sites of temperature sensitive folding (tsf) mutations, which affect intermediates in the chain folding and subunit association pathway. The phenotypes of the amber missense proteins produced on tRNA suppressor hosts inserting serine, glutamine, tryosine and leucine have been determined at different temperatures. Thirty-three of the sites are tolerant, producing functional proteins with any of the four amino acids inserted at the sites, independent of temperature. Tolerant sites are concentrated at the N-terminal end of the protein indicating that this region is not critical for conformation or function. Sixteen of the sites yield temperature sensitive missense proteins on at least one nonsense suppressing host. Most of the sites with ts phenotypes map to the central region of the gene which is also the region where most of the tsf mutations map. Mutations at 15 of the sites have a lethal phenotype on at least one tRNA suppressor host. For nine out of ten sites tested with at least one lethal phenotype, the primary defect was in the folding or subunit association of the missense polypeptide chain. This analysis of the tailspike missense proteins distinguishes three classes of amino acid sites in the polypeptide chain; residues whose side chains contribute little to folding, subunit assembly or function; residues critical for maintaining the folding and subunit assembly pathway at the high end of the temperature range of phage growth; and residues critical over the entire temperature range of growth.

Shared host cells can serve as melting pots for viral genomes, giving many phylogenies a web-like appearance due to horizontal gene transfer. However, not all virus families exhibit web-like phylogenies. Microviruses form three distinct clades, represented by φX174, G4, and α3. Here, we investigate protein-based barriers to horizontal gene transfer between clades. We transferred gene G, which encodes a structural protein, between φX174 and G4, and monitored the evolutionary recovery of the resulting chimeras. In both cases, particle assembly was the major barrier after gene transfer. The G4φXG chimera displayed a temperature-sensitive assembly defect that could easily be corrected through single mutations that promote productive assembly. Gene transfer in the other direction was more problematic. The initial φXG4G chimera required an exogenous supply of both the φX174 major spike G and DNA pilot H proteins. Elevated DNA pilot protein levels may be required to compensate for off-pathway reactions that may have become thermodynamically and/or kinetically favored when the foreign spike protein was present. After three targeted genetic selections, the foreign spike protein was productively integrated into the φX174 background. The first adaption involved a global decrease in gene expression. This was followed by modifications affecting key protein-protein interactions that govern assembly. Finally, gene expression was re-elevated. Although the first selection suppresses nonproductive reactions, subsequent selections promote productive assembly and ultimately viability. However, viable chimeric strains exhibited reduced fitness compared with wild-type. This chimera's path to recovery may partially explain how unusual recombinant viruses could persist long enough to naturally emerge.