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.
PMID: 7498760;PMCID: PMC1206684;Abstract:
The morphogenetic pathway of bacteriophage φX 174 was investigated in rep mutant hosts that specifically block stage III single-stranded DNA synthesis. The defects conferred by the mutant rep protein most likely affect the formation or stabilization of the 50S complex, a single-stranded DNA synthesizing intermediate, which consists of a viral prohead and a DNA replicating intermediate (preinitiation complex). φX 174 mutants, ogr (rep), which restore the ability to propagate in the mutant rep hosts, were isolated. The ogr(rep) mutations confer amino acid substitutions in the viral coat protein, a constituent of the prohead, and the viral A protein, a constituent of the preinitiation complex. Four of the six coat protein substitutions are localized on or near the twofold axis of symmetry in the atomic structure of the mature virion.
Unlike tailed bacteriophages, which use a preformed tail for transporting their genomes into a host bacterium, the ssDNA bacteriophage ΦX174 is tailless. Using cryo-electron microscopy and time-resolved small-angle X-ray scattering, we show that lipopolysaccharides (LPS) form bilayers that interact with ΦX174 at an icosahedral fivefold vertex and induce single-stranded (ss) DNA genome ejection. The structures of ΦX174 complexed with LPS have been determined for the pre- and post-ssDNA ejection states. The ejection is initiated by the loss of the G protein spike that encounters the LPS, followed by conformational changes of two polypeptide loops on the major capsid F proteins. One of these loops mediates viral attachment, and the other participates in making the fivefold channel at the vertex contacting the LPS.
The three-dimensional structure of bacteriophage φX174 external scaffolding protein D, prior to its interaction with other structural proteins, has been determined to 3.3 Å by X-ray crystallography. The crystals belong to space group P41212 with a dimer in the asymmetric unit that closely resembles asymmetric dimers observed in the φX174 procapsid structure. Furthermore, application of the crystallographic 41 symmetry operation to one of these dimers generates a tetramer similar to the tetramer in the icosahedral asymmetric unit of the procapsid. These data suggest that both dimers and tetramers of the D protein are true morphogenetic intermediates and can form independently of other proteins involved in procapsid morphogenesis. The crystal structure of the D scaffolding protein thus represents the state of the polypeptide prior to procapsid assembly. Hence, comparison with the procapsid structure provides a rare opportunity to follow the conformational switching events necessary for the construction of complex macromolecular assemblies.
The phiX174 DNA pilot protein H contains four predicted C-terminal coiled-coil domains. The region of the gene encoding these structures was cloned, expressed in vivo, and found to strongly inhibit wild-type replication. DNA and protein synthesis was investigated in the absence of de novo H protein synthesis and in wild-type-infected cells expressing the inhibitory proteins (DeltaH). The expression of the DeltaH proteins interfered with early stages of DNA replication, which did not require de novo H protein synthesis, suggesting that the inhibitory proteins interfere with the wild-type H protein that enters the cell with the penetrating DNA. As transcription and protein synthesis are dependent on DNA replication in positive single-stranded DNA life cycles, viral protein synthesis was also reduced. However, unlike DNA synthesis, efficient viral protein synthesis required de novo H protein synthesis, a novel function for this protein. A single amino acid change in the C terminus of protein H was both necessary and sufficient to confer resistance to the inhibitory DeltaH proteins, restoring both DNA and protein synthesis to wild-type levels. DeltaH proteins derived from the resistant mutant did not inhibit wild-type or resistant mutant replication. The inhibitory effects of the DeltaH proteins were lessened by the coexpression of the internal scaffolding protein, which may suppress H-H protein interactions. While coexpression relieved the block in DNA biosynthesis, viral protein synthesis remained suppressed. These data indicate that protein H's role in DNA replication and stimulating viral protein synthesis can be uncoupled.