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Journal of Virology, June 2005, p. 6751-6756, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6751-6756.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of an Interacting Coat-External Scaffolding Protein Domain Required for both the Initiation of {phi}X174 Procapsid Morphogenesis and the Completion of DNA Packaging

Asako Uchiyama and Bentley A. Fane*

Department of Veterinary Science and Microbiology, University of Arizona, Tucson, Arizona

Received 2 December 2004/ Accepted 4 February 2005

The {phi}X174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven {alpha}-helices. The protein's core, {alpha}-helices 2 to 6, mediates the vast majority of intra- and interdimer contacts and is strongly conserved in all Microviridae (canonical members are {phi}X174, G4, and {alpha}3) external scaffolding proteins. On the other hand, the primary sequences of the first {alpha}-helices have diverged. The results of previous studies with {alpha}3/{phi}X174 chimeric external scaffolding proteins suggest that {alpha}-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and {phi}X174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and DNA packaging. {phi}X174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external 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.

* Corresponding author. Mailing address: Department of Veterinary Sciences and Microbiology, Building 90, University of Arizona, Tucson AZ 85721-0090. Phone: (520) 626-6634. Fax: (520) 621-6366. E-mail: bfane{at}u.arizona.edu.

Journal of Virology, June 2005, p. 6751-6756, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6751-6756.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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