The Functions of the N Terminus of the φX174 Internal Scaffolding Protein, a Protein Encoded in an Overlapping Reading Frame in a Two Scaffolding Protein System

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

“…70S particles usually represent degraded procapsids, which were never filled and which lost the scaffolding proteins (2,18). To determine whether the 70S particles observed were degraded procapsids, spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed.…”

Section: Vol 79 2005 X174 Coat-external Scaffolding Protein Interacmentioning

confidence: 99%

“…The first detectable intermediates are the 9S and 6S particles, respective pentamers of the major coat F and spike G proteins (22). Five internal scaffolding B proteins bind to the underside of the 9S particles, inducing a conformational change that enables interactions with major spike G, minor spike H, and external scaffolding D proteins (5,6,18,21,22). While the identity of the next intermediate remains obscure, structural data suggest that it contains the F, G, H, B, and D proteins (5,6).…”

mentioning

confidence: 99%

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

Uchiyama

2005

The X174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven ␣-helices. The protein's core, ␣-helices 2 to 6, mediates the vast majority of intra-and interdimer contacts and is strongly conserved in all Microviridae (canonical members are X174, G4, and ␣3) external scaffolding proteins. On the other hand, the primary sequences of the first ␣-helices have diverged. The results of previous studies with ␣3/X174 chimeric external scaffolding proteins suggest that ␣-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and X174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and DNA packaging. X174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.

“…The N termini, which can tolerate large deletions, exhibit a high degree of variation (4,21). Accordingly, the N terminus produces diffuse density in the X174 procapsid crystal structure, whereas the C terminus is highly ordered (8,9).…”

mentioning

confidence: 99%

Conformational Switch-Defective ϕX174 Internal Scaffolding Proteins Kinetically Trap Assembly Intermediates before Procapsid Formation

Gordon

Knuff

2012

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.