Foreign and Chimeric External Scaffolding Proteins as Inhibitors of Microviridae Morphogenesis

Viral assembly is an ideal system in which to investigate the transient recognition and interplay between proteins. During morphogenesis, scaffolding proteins temporarily associate with structural proteins, stimulating conformational changes that promote assembly and inhibit off-pathway reactions. Microviridae morphogenesis is dependent on two scaffolding proteins, an internal and an external species. The external scaffolding protein is the most conserved protein within the Microviridae, whose canonical members are fX174, G4, and a3. However, despite 70% homology on the amino acid level, overexpression of a foreign Microviridae external scaffolding protein is a potent cross-species inhibitor of morphogenesis. Mutants that are resistant to the expression of a foreign scaffolding protein cannot be obtained via one mutational step. To define the requirements for and constraints on scaffolding protein interactions, chimeric external scaffolding proteins have been constructed and analyzed for effects on in vivo assembly. The results of these experiments suggest that at least two cross-species inhibitory domains exist within these proteins; one domain most likely blocks procapsid formation, and the other allows procapsid assembly but blocks DNA packaging. A mutation conferring resistance to the expression of a chimeric protein (chiD r ) that inhibits DNA packaging was isolated. The mutation maps to gene A, which encodes a protein essential for packaging. The chiD r mutation confers resistance only to a chimeric D protein; the mutant is still inhibited by the expression of foreign D proteins. The results presented here demonstrate how closely related proteins could be developed into antiviral agents that specifically target virion morphogenesis. The proper assembly of viral proteins and nucleic acids into a biologically active virion involves numerous and diverse macromolecular interactions. While structural proteins must correctly interact with other structural proteins, proper morphogenesis is equally dependent on interactions between structural and scaffolding proteins. Viral scaffolding proteins are transiently associated with morphogenetic intermediates but are not found in the mature viral particle (10, 12, 17, 22). These proteins promote the efficiency and fidelity of particle formation by ensuring proper interactions between viral proteins, promoting the nucleation of assembly, and aiding in the formation of a precisely sized capsid (13, 16, 19). In the Microviridae, assembly is dependent on two scaffolding proteins, an internal and an external species. The assembly pathway has been extensively characterized (8), and the atomic structures of both the fX174 virion and a morphogenetic intermediate containing a full complement of scaffolding proteins have been solved (2, 3, 14, 15). Therefore, the results of genetic and biochemical analyses can be interpreted within a structural context. The Microviridae assembly pathway is illustrated in Fig. 1. The first detectable morphogenetic intermediates are the 9S and 6S particles, respective pentamers of the viral coat and spike proteins. In a reaction mediated by the internal scaffolding (or B) protein, these intermediates associate, forming the 12S particle. Twelve 12S particles are then organized into the procapsid by 240 copies of the external scaffolding (or D) protein. The preinitiation complex, consisting of one copy of viral proteins A and C, replicative-form (RF) DNA, and the host cell Rep protein, associates with the procapsid, probably along a twofold axis of symmetry (5), forming the 50S complex. Single-stranded genomic DNA is then concurrently synthesized and packaged. The internal scaffolding protein is extruded during this reaction, yielding the infectious provirion, which may represent the end of the intracellular assembly pathway. The external scaffolding protein may dissociate upon cell lysis. External scaffolding proteins are not unique to the Microviridae. The coliphage P4 Sid protein and the triplex proteins of the Herpesviridae perform analogous functions (13, 23). Although the Microviridae morphogenetic pathway has been well characterized, the mechanism in which viral scaffolding proteins enable nascent intermediates to reach a biologically active form remains somewhat obscure. Here we report that the expression of closely related external scaffolding proteins inhibits morphogenesis. Using chimeric proteins, we have defined the existence of two inhibitory domains. These results have implications for the use of closely related proteins in the development of antiviral analogs specific to viral morphogenesis.