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Conformational switch-defective X174 internal scaffolding proteins kinetically trap assembly intermediates before procapsid formation.

ABSTRACT: Conformational switching is an overarching paradigm in which to describe scaffolding protein-mediated virus assembly. However, rapid morphogenesis with small assembly subunits hinders the isolation of early morphogenetic intermediates in most model systems. Consequently, conformational switches are often defined by comparing the structures of virions, procapsids and aberrantly assembled particles. In contrast, X174 morphogenesis proceeds through at least three preprocapsid intermediates, which can be biochemically isolated. This affords a detailed analysis of early morphogenesis and internal scaffolding protein function. Amino acid substitutions were generated for the six C-terminal, aromatic amino acids that mediate most coat-internal scaffolding protein contacts. The biochemical characterization of mutant assembly pathways revealed two classes of molecular defects, protein binding and conformational switching, a novel phenotype. The conformational switch mutations kinetically trapped assembly intermediates before procapsid formation. Although mutations trapped different particles, they shared common second-site suppressors located in the viral coat protein. This suggests a fluid assembly pathway, one in which the scaffolding protein induces a single, coat protein conformational switch and not a series of sequential reactions. In this model, an incomplete or improper switch would kinetically trap intermediates.

SUBMITTER: Gordon EB

PROVIDER: S-EPMC3446603 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Conformational switch-defective X174 internal scaffolding proteins kinetically trap assembly intermediates before procapsid formation.

Gordon Emile B EB Knuff Christopher J CJ Fane Bentley A BA

Journal of virology 20120703 18

Conformational switching is an overarching paradigm in which to describe scaffolding protein-mediated virus assembly. However, rapid morphogenesis with small assembly subunits hinders the isolation of early morphogenetic intermediates in most model systems. Consequently, conformational switches are often defined by comparing the structures of virions, procapsids and aberrantly assembled particles. In contrast, X174 morphogenesis proceeds through at least three preprocapsid intermediates, which c ...[more]

PMID: 22761377

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Project description:Like many viruses, bacteriophage phi X174 packages its DNA genome into a procapsid that is assembled from structural intermediates and scaffolding proteins. The procapsid contains the structural proteins F, G and H, as well as the scaffolding proteins B and D. Provirions are formed by packaging of DNA together with the small internal J proteins, while losing at least some of the B scaffolding proteins. Eventually, loss of the D scaffolding proteins and the remaining B proteins leads to the formation of mature virions.phi X174 108S 'procapsids' have been purified in milligram quantities by removing 114S (mature virion) and 70S (abortive capsid) particles from crude lysates by differential precipitation with polyethylene glycol. 132S 'provirions' were purified on sucrose gradients in the presence of EDTA. Cryo-electron microscopy (cryo-EM) was used to obtain reconstructions of procapsids and provirions. Although these are very similar to each other, their structures differ greatly from that of the virion. The F and G proteins, whose atomic structures in virions were previously determined from X-ray crystallography, were fitted into the cryo-EM reconstructions. This showed that the pentamer of G proteins on each five-fold vertex changes its conformation only slightly during DNA packaging and maturation, whereas major tertiary and quaternary structural changes occur in the F protein. The procapsids and provirions were found to contain 120 copies of the D protein arranged as tetramers on the two-fold axes. DNA might enter procapsids through one of the 30 A diameter holes on the icosahedral three-fold axes.Combining cryo-EM image reconstruction and X-ray crystallography has revealed the major conformational changes that can occur in viral assembly. The function of the scaffolding proteins may be, in part, to support weak interactions between the structural proteins in the procapsids and to cover surfaces that are subsequently required for subunit-subunit interaction in the virion. The structures presented here are, therefore, analogous to chaperone proteins complexed with folding intermediates of a substrate.

Project description:In the present work, the conformational dynamics and folding pathways of i-motif DNA were studied in solution and in the gas-phase as a function of the solution pH conditions using circular dichroism (CD), photoacoustic calorimetry analysis (PAC), trapped ion mobility spectrometry-mass spectrometry (TIMS-MS), and molecular dynamics (MD). Solution studies showed at thermodynamic equilibrium the existence of a two-state folding mechanism, whereas during the pH = 7.0 → 4.5 transition a fast and slow phase (ΔHfast + ΔHslow = 43 ± 7 kcal mol-1) with a volume change associated with the formation of hemiprotonated cytosine base pairs and concomitant collapse of the i-motif oligonucleotide into a compact conformation were observed. TIMS-MS experiments showed that gas-phase, kinetically trapped i-motif DNA intermediates produced by nanoESI are preserved, with relative abundances depending on the solution pH conditions. In particular, a folded i-motif DNA structure was observed in nanoESI-TIMS-MS for low charge states in both positive and negative ion mode (e.g., z = ±3 to ±5) at low pH conditions. As solution pH increases, the cytosine neutralization leads to the loss of cytosine-cytosine+ (C·CH+) base pairing in the CCC strands and in those conditions we observe partially unfolded i-motif DNA conformations in nanoESI-TIMS-MS for higher charge states (e.g., z = -6 to -9). Collisional induced activation prior to TIMS-MS showed the existence of multiple local free energy minima, associated with the i-motif DNA unfolding at z = -6 charge state. For the first time, candidate gas-phase structures are proposed based on mobility measurements of the i-motif DNA unfolding pathway. Moreover, the inspection of partially unfolded i-motif DNA structures (z = -7 and z = -8 charge states) showed that the presence of inner cations may or may not induce conformational changes in the gas-phase. For example, incorporation of ammonium adducts does not lead to major conformational changes while sodium adducts may lead to the formation of sodium mediated bonds between two negatively charged sides inducing the stabilization towards more compact structures in new local, free energy minima in the gas-phase.

Project description:During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro In previous studies, ϕX174 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. 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 ϕX174 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.

Dataset Information

Conformational switch-defective X174 internal scaffolding proteins kinetically trap assembly intermediates before procapsid formation.

Publications

Conformational switch-defective X174 internal scaffolding proteins kinetically trap assembly intermediates before procapsid formation.

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