The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus φX174 1 1Edited by I. A. Wilson

Dokland, Terje; Bernal, Ricardo A.; Burch, April D.; Pletnev, Sergei; Fane, Bentley A.; Rossmann, Michael G.

doi:10.1006/jmbi.1999.2699

Cited by 82 publications

(128 citation statements)

References 54 publications

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“…Wild-type homodimers and WT/D G61D heterodimers were analyzed by circular dichroism. In agreement with the X-ray structure (13)(14)(15), the wild-type D protein homodimer was largely helical. The heterodimers displayed a slight loss of helicity (data not shown).…”

Section: Resultssupporting

confidence: 81%

“…The D G61D mutation would inhibit the kinking of ␣-helix 3. Amino acid residues at the end of the helix participate in dimer formation (13)(14)(15). The altered orientation of the helix may inhibit those interactions.…”

Section: Discussionmentioning

confidence: 99%

“…The microvirus internal scaffolding protein shares many properties and functions with the scaffolding proteins found in other viruses (7,9,10,14,15,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). However, it does not physically construct the procapsid or control morphogenetic fidelity.…”

Section: Discussionmentioning

confidence: 99%

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

Cherwa

Tyson

Bedwell

et al. 2017

J Virol

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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).

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Section: Resultssupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

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

Cherwa

Tyson

Bedwell

et al. 2017

J Virol

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“…Ref. 68 ) and the proteins required for release of viruses from the infected cell 14 . The amount of information that is required constrains the length of the genome molecule from below.…”

Section: A Simple Description Of a Virusmentioning

confidence: 99%

Energies and pressures in viruses: contribution of nonspecific electrostatic interactions

Šiber

Božič

Podgornik

2012

Phys. Chem. Chem. Phys.

132

153

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We summarize some aspects of electrostatic interactions in the context of viruses. A simplified but, within well defined limitations, reliable approach is used to derive expressions for electrostatic energies and the corresponding osmotic pressures in single-stranded RNA viruses and double-stranded DNA bacteriophages. The two types of viruses differ crucially in the spatial distribution of their genome charge which leads to essential differences in their free energies, depending on the capsid size and total charge in a quite different fashion. Differences in the free energies are trailed by the corresponding characteristics and variations in the osmotic pressure between the inside of the virus and the external bathing solution.

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“…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).…”

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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

Fane

2002

J Virol

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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...

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The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus φX174 1 1Edited by I. A. Wilson

Cited by 82 publications

References 54 publications

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

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

Energies and pressures in viruses: contribution of nonspecific electrostatic interactions

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

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