Scaffolding Proteins Altered in the Ability To Perform a Conformational Switch Confer Dominant Lethal Assembly Defects

Proper virion assembly requires numerous macromolecular interactions proceeding along an ordered morphogenetic pathway. While structural proteins are incorporated into the final product, morphogenesis is equally dependent upon scaffolding proteins, which are not included in the mature virion. During morphogenesis, these proteins mediate conformational switches to ensure the efficiency and fidelity of assembly (13). Although many large DNA viruses rely on a single internal scaffolding protein, the small microviruses (canonical species φX174, G4, and α3) and the satellite P4-like viruses require both internal and external scaffolding proteins (10, 14, 21). Although the internal scaffolding proteins in these dual systems are absolutely essential, several lines of evidence suggest that the external scaffolding proteins may be more critical. Successive targeted genetic selections have led to the isolation of a φX174 multiple mutant that can assemble without its internal scaffolding protein in vivo (5). Moreover, P4 procapsid-like particles can be assembled in vitro using only the capsid and external scaffolding proteins (26). The morphogenetic roles of the φX174 internal and external scaffolding proteins are illustrated in Fig. ​Fig.1A.1A. The first identifiable assembly intermediates are pentamers of the viral coat F and major spike G proteins, the 9S and 6S particles, respectively, which can form in the absence of both scaffolding proteins (23). Five internal scaffolding B proteins bind to the underside of the 9S particle, yielding the 9S* intermediate. This interaction induces a conformational change that allows 9S*-6S particle associations, forming the 12S* intermediate. This particle also includes the DNA pilot protein H (10, 16). Twenty external scaffolding D proteins, most likely in the form of five tetramers or ten asymmetric dimers (18), interact with the 12S* particle to form the short-lived 18S intermediate (4). D-D contacts mediate the construction of the procapsid (108S), an immature virus particle, presumably by allowing 18S pentamers to interact across twofold axes of symmetry (6, 8, 18). FIG. 1. (A) The φX174 morphogenetic pathway. (B) The four external scaffolding subunits (D1 [purple], D2 [blue], D3 [brown], and D4 [green]), coat protein (gray), and internal scaffolding protein (peach) in the asymmetric unit. (C) The four external scaffolding ... In the atomic structure of the viral procapsid (6, 8), there are four structurally distinct external scaffolding proteins (D1 to D4), arranged as dimers of dimers (D1D2, D3D4), per viral coat protein (Fig. 1B and C). To achieve this unique arrangement, one monomer in each asymmetric dimer, D1 and D3, must be bent 30°; this occurs at glycine residue 61 in α-helix 3 (Fig. ​(Fig.1C).1C). This kink is also present in the crystal structure of the assembly naive dimer DADB (18). A mutation at this site may allow the protein to fold into only one of these two conformations. Although the mutant protein would retain the ability to interact with other D and structural proteins, the product of these interactions may inhibit lattice formation and subsequent virus assembly. The putative dominant lethal phenotypes of some glycine 61 substitutions have been reported (3). However, the molecular mechanism of inhibition could not be characterized due to difficulties associated with propagating the mutant strains. To determine the mechanism of inhibition and circumvent the problems associated with propagating dominant lethal viral mutants, genetically engineered D genes were constructed, and the proteins were expressed in wild-type φX174-infected cells. This alternate approach also allowed mutants resistant to the expression of the inhibitory proteins to be isolated, elucidating possible resistance mechanisms that may be anticipated for antiviral therapies that target virus-specific assembly processes. The results of this analysis suggest that the presence of the wild-type external scaffolding protein is required for the mutant inhibitory D protein to enter the morphogenetic pathway. This may lead to premature D protein interactions with structural proteins or off-pathway reactions involving later intermediates. The location of the resistance mutations within the procapsid crystal structure suggest a mechanism in which inhibitory D protein subunits are excluded from occupying particular positions atop 12S* pentamers.