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

Abstract

In the X174 procapsid crystal structure, 240 external scaffolding protein D subunits form 60 pairs of asymmetric dimers, D 1 D 2 and D 3 D 4 , in a non-quasi-equivalent structure. To achieve this arrangement, ␣-helix 3 assumes two different conformations: (i) kinked 30°at glycine residue 61 in subunits D 1 and D 3 and (ii) straight in subunits D 2 and D 4 . Substitutions for G61 may inhibit viral assembly by preventing the protein from achieving its fully kinked conformation while still allowing it to interact with other scaffolding and structural proteins. Mutations designed to inhibit conformational switching in ␣-helix 3 were introduced into a cloned gene, and expression was demonstrated to inhibit wild-type morphogenesis. The severity of inhibition appears to be related to the size of the substituted amino acid. For infections in which only the mutant protein is present, morphogenesis does not proceed past the first step that requires the wild-type external scaffolding protein. Thus, mutant subunits alone appear to have little or no morphogenetic function. In contrast, assembly in the presence of wild-type and mutant subunits is blocked prematurely, before D protein is required in a wild-type infection, or channeled into an off-pathway reaction. These data suggest that the wild-type protein transports the inhibitory protein to the pathway. Viruses resistant to the lethal dominant proteins were isolated, and mutations were mapped to the coat and internal scaffolding proteins. The affected amino acids cluster in the atomic structure and may act to exclude mutant subunits from occupying particular positions atop pentamers of the viral coat protein.

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

“…However, in wild-type infections (Fig. 1A), these mutant proteins inhibit morphogenesis by removing 12S* intermediates into the insoluble fraction, thus displaying a dominant lethal phenotype (12). As the wild-type D protein forms dimers and higher-order oligomers in solution (11), these observations suggested that lethal dominant G61X proteins cannot self-oligomerize, a hypothesis directly tested in this study.…”

“…The heterodimers displayed a slight loss of helicity (data not shown). The D D34G protein, which was previously shown to elevate the most evolved strain's fitness, was also analyzed for the ability to form homodimers and heterodimers with the D G61D protein (11,12). It behaved like the wild-type species in these assays (data not shown).…”

“…Earlier results suggested that the inhibitory D G61D protein alone cannot properly dimerize but will form inhibitory oligomers with wild-type subunits (11,12). To investigate this further, purified mutant and wild-type D proteins (0.5 mg/ml) were examined by analytical ultracentrifugation (AUC).…”

“…The first resistant strain isolated contained three mutations in the coat protein F (F D44H , F D205N , and F S227P ), which were identical or similar to resistance mutations isolated in single-step selections. In general, all of these mutations cluster underneath the D 3 subunit in the X-ray structure (12,16). There was also a mutation in the DNA pilot protein H (H D136G ).…”

“…Mutant D genes that encode large side chain substitutions (D G61X , where X is valine, aspartic acid, lysine, or proline) do not complement nullD strains and inhibit wild-type (WT) morphogenesis (12). During nullD infections, D G61X proteins do not interact with assembly intermediates.…”