Structure-function relationships in Microviridae external scaffolding proteins

Microviruses (canonical members: oX174, G4, and alpha3) are T=1 icosahedral virions with a two scaffolding protein-mediated assembly pathway. The external scaffolding protein D mainly mediates the assembly of coat protein pentamers into procapsids. The results of previous genetic studies suggest that alpha-helix 1 of D protein may act as a substrate specificity domain, mediating the initial coat-scaffolding protein recognition in a species-specific manner. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were initially constructed in a plasmid, which over-expresses the protein, between the closely related phages G4 and oX174. The results of biochemical and genetic analyses identify coat-scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.; However, protein concentrations higher than those found in typical infections could drive reactions that may not occur under physiological conditions. In order to elucidate a more detailed mechanistic model, the same chimeric external scaffolding gene was placed directly in the oX174 genome, and the chimeric virus was characterized. The results of the genetic and biochemical analyses indicate that alpha-helix 1 most likely mediates the nucleation reaction for the formation of the first assembly intermediate containing the external scaffolding protein. Mutants that can more efficiently use the chimeric scaffolding protein were isolated. These second-site mutations appear to act on a kinetic level, shortening the lag phase before virion production.; Finally, by using improved protocols, two novel early assembly intermediates, the 9S* and 12S* particles, have been isolated and characterized. The 9S* particle consists of a coat protein pentamer associated with the internal scaffolding protein. The 12S* intermediate is a complex of a 9S* particle with the major spike protein, and the DNA pilot protein. The existence of internal scaffolding and DNA pilot proteins that were absent in previously characterized intermediates suggest that 9S* and 12S* particles are biologically active intermediates. Moreover, preliminary in vitro assembly experiments performed with the 12S* particle and exogenous external scaffolding protein yield empty capsids-like particle, strongly suggesting that these are the physiologically relevant intermediates.