Functional relationship between the J proteins of bacteriophages phi X174 and G4 during phage morphogenesis

2013

C-terminal, aromatic amino acids in the X174 internal scaffolding protein B mediate conformational switches in the viral coat protein. These switches direct the coat protein through early assembly. In addition to the aromatic amino acids, two acidic residues, D111 and E113, form salt bridges with basic, coat protein side chains. Although salt bridge formation did not appear to be critical for assembly, the substitution of an aromatic amino acid for D111 produced a lethal phenotype. This side chain is uniquely oriented toward the center of the coat-scaffolding binding pocket, which is heavily dominated by aromatic ring-ring interactions. Thus, the D111Y substitution may restructure pocket contacts. Previously characterized B ؊ mutants blocked assembly before procapsid formation. However, the D111Y mutant produced an assembled particle, which contained the structural and external scaffolding proteins but lacked protein B and DNA. A suppressor within the external scaffolding protein, which mediates the later stages of particle morphogenesis, restored viability. The unique formation of a postprocapsid particle and the novel suppressor may be indicative of a novel B protein function. However, genetic data suggest that the particle represents the delayed manifestation of an early assembly error. This seemingly late-acting defect was rescued by previously characterized suppressors of early, preprocapsid, B ؊ assembly mutations, which act on the level of coat protein flexibility. Likewise, the newly isolated suppressor in the external scaffolding protein also exhibited a global suppressing phenotype. Thus, the off-pathway product isolated from infected cells may not accurately reflect the temporal nature of the initial defect.

Section: Methodsmentioning

confidence: 99%

Effects of an Early Conformational Switch Defect during ϕX174 Morphogenesis Are Belatedly Manifested Late in the Assembly Pathway

Gordon

2013

“…Escherichia coli C strain C122 (sup 0 ) is the wild-type host; BAF5 contains a supE mutation (22). BAF30 is a recA-null derivative of C122 (23). Plasmids pXB and pXDJ contain IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible clones of the denoted X174 genes (8,9).…”

Section: Methodsmentioning

confidence: 99%

φX174 Genome-Capsid Interactions Influence the Biophysical Properties of the Virion: Evidence for a Scaffolding-Like Function for the Genome during the Final Stages of Morphogenesis

Hafenstein

2002

During the final stages of X174 morphogenesis, there is an 8.5-Å 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, X174 was packaged with foreign, single-stranded, covalently closed, circular DNA molecules identical in length to the 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-tovirion transition.Unlike many double-stranded DNA (dsDNA) viruses which use one scaffolding protein (14, 34), X174 morphogenesis is dependent on two species (30). Together, these two proteins perform the full spectrum of functions found in one-protein systems. However, after procapsid assembly, the final stages of X174 morphogenesis differ. In dsDNA systems, procapsids expand during packaging (32,37,41), and the genome forms a dense core (18). In contrast, X174 morphogenesis concludes with the collapse of coat proteins around the single-stranded genome, which is associated with the inner surface of the capsid (15,16,31,38,39).As illustrated in Fig. 1, X174 genome replication is coupled to DNA packaging. The preinitiation complex, consisting of the host cell rep and viral A and C proteins, associates with the procapsid forming the 50S complex (21,40). The viral A protein binds the origin of replication in replicative-form DNA. This is both necessary and sufficient for packaging specificity (3,4,26). Upon binding, protein A nicks the origin (47) and forms a covalent ester bond with the DNA (19). After one round of rolling circle synthesis, protein A cuts and ligates the newly generated origin, generating a circular single-stranded molecule (7,20). This packaging mechanism produces precise genomes of identical length (4).The highly basic DNA-binding protein (J) enters the procapsid, along with the single-stranded DNA (ssDNA) genome, (27), associating with the genome via charge-charge interactions (13, 33). Once in the procapsid, the C terminus binds to a coat protein cleft (38,39). This may facilitate further interactions with a small cluster of adjacent basic capsid amino acids. Accordingly, a portion of the packaged DNA (8 to 10%) is icosahedral...

“…Escherichia coli C strains C122 (sup°) and BAF30 (recA) have been previously described (8,9). The host slyD mutation confers resistance to E protein-mediated lysis (17).…”

Section: Methodsmentioning

confidence: 99%

Complete Virion Assembly with Scaffolding Proteins Altered in the Ability To Perform a Critical Conformational Switch

Cherwa

2009

In the X174 procapsid, 240 external scaffolding proteins form a nonquasiequivalent lattice. To achieve this arrangement, the four structurally unique subunits must undergo position-dependent conformational switches. One switch is mediated by glycine residue 61, which allows a 30°kink to form in ␣-helix 3 in two subunits, whereas the helix is straight in the other two subunits. No other amino acid should be able to produce a bend of this magnitude. Accordingly, all substitutions for G61 are nonviable but mutant proteins differ vis-à-vis recessive and dominant phenotypes. As previously reported, amino acid substitutions with side chains larger than valine confer dominant lethal phenotypes. Alone, these mutant proteins appear to have little or no biological activity but rather require the wild-type protein to interact with other structural proteins. Proteins with conservative substitutions for G61, serine and alanine, have now been characterized. Unlike the dominant lethal proteins, these proteins do not require wild-type subunits to interact with other viral proteins and cause assembly defects reminiscent of those conferred by the lethal dominant proteins in concert with wild-type subunits. Although atomic structures suggest that only a glycine residue can provide the proper torsion angle for assembly, mutants that can productively utilize the altered external scaffolding proteins were isolated, and the mutations were mapped to the coat and internal scaffolding proteins. Thus, the ability to isolate strains that could utilize the single mutant D protein species would not have been predicted from past structural analyses.Proper virion assembly requires a series of precise proteinprotein interactions that proceed along an ordered morphogenetic pathway. During Tϭ1 Microvirus assembly (canonical species: X174, G4, and ␣3), early intermediates are directed into larger macromolecular structures by a class of transiently associated proteins called scaffolding proteins. These proteins mediate the conformational switching of structural proteins, assist in lowering the nucleation barrier for assembly, and ensure morphogenetic fidelity (10). While many large DNA viruses rely on a single internal scaffolding protein, the small microviruses and the satellite P4-like viruses require both internal and external scaffolding proteins (7,11,20). The atomic structures of the X174 virion, procapsid, and assembly naïve external scaffolding protein have been determined by crystallography (5, 6, 14-16). Thus, biochemical and genetic data can be interpreted within a defined structural context.The morphogenetic roles of the X174 internal and external scaffolding proteins are illustrated in Fig. 1A. The first identifiable assembly intermediates are pentamers of the viral coat F and major spike G proteins; the respective 9S and 6S particles, which form independently of both scaffolding proteins (21). Five internal scaffolding B proteins bind to the underside of the 9S particle, yielding the 9S* intermediate (3). This interaction also induces...