Identification of an Interacting Coat-External Scaffolding Protein Domain Required for both the Initiation of φX174 Procapsid Morphogenesis and the Completion of DNA Packaging

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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: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Gordon

Fane

2013

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“…To distinguish between these two alternatives, the large particles produced in wild-type-infected cells expressing the G61S, G61A, and G61T proteins were characterized. The concentration of the isopropyl-␤-D-thiogalactopyranoside (IPTG) inducer used in these experiments yields a wild-type-to-mutant D protein ratio of approximately 1:1 (22). After the removal of unabsorbed virions, incubation, and chemical lysis, soluble material was subjected to rate-zonal sedimentation.…”

Section: Resultsmentioning

confidence: 99%

“…Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) of gradient fractions was used to detect particles with S values of less than 50. The protocols used to examine the kinetics of virion production have been previously described (22).…”

Section: Methodsmentioning

confidence: 99%

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

Cherwa

Fane

2009

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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...

“…Thus, procapsids accumulate in infected cells (57). Particles were isolated and purified as previously described (58).…”

Section: Methodsmentioning

confidence: 99%

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro

Cherwa

Tyson

Bedwell

et al. 2017

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During X174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro. In previous studies, X174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. IMPORTANCE Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo. We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During X174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.KEYWORDS bacteriophage X174, Microviridae, microvirus, scaffolding protein, virus assembly D ue to their rapid replication cycle, detailed structural information, and well developed genetic and biochemical assays, the microviruses have become an ideal model system for experimental evolution (1-4). The morphogenetic pathway can be characterized under restrictive conditions and adaptive mutations can be mapped onto X-ray structures, providing insights into evolutionary mechanisms and protein structure-function relationships (2-8).