Conformational Switch-Defective ϕX174 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.

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

“…1B). Altering these contacts strongly inhibits coat-scaffolding protein interactions (5). Considering the proximity and orientation of the D111 side chain toward the cleft's center, the introduction of an aromatic amino acid at this position may abrogate coat-internal scaffolding protein interactions, which would lead to the intracellular coat protein aggregation (5), or introduce a novel interaction that redirects assembly.…”

“…The host slyD mutation confers resistance to E protein-mediated lysis (31). The construction of the complementing plasmid pXB has been previously reported (5).…”

“…1B). The results of a previous genetic analysis demonstrate that nonaromatic substitutions for residue F120, the last amino acid, or the absence of this amino acid, abolish coat protein binding (5). Substitutions at the other aromatic sites produced defective conformational switching phenotypes.…”

“…Alteration of an amino acid that mediates a protein-protein interaction, either via a salt bridge or via an aromatic ring, is typically regarded as a loss-of-function, or loss-ofcontact, mutation. The previously characterized mutations at aromatic sites most likely fall into this category (5). In contrast, the introduction of aromatic residue into an environment already rich in aromatic content could create a gain-of-contact mutation, which may compete with wild-type interactions.…”

“…Since many assembly systems involve the rapid addition of small elongation units to short-lived nucleation complexes, morphogenesis is typically divided into three stages: coat protein binding, nucleation, and elongation (1)(2)(3). While the absence of coat protein binding can be easily distinguished from other defects (4)(5)(6), the fluid nature of assembly can obscure the differences between defects in the later stages. An altered nucleation complex could result in the formation of an aberrantly shaped assembled particle (4,7).…”