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

Abstract

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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