University of Arizona
Structure, Function, and Evolution of Viral Scaffolding Proteins in a Two-Scaffolding Protein System
Dr. Fane was a former undergraduate student at Brandeis who spent much of his college career working under the direction of Kalpana White. During his introduction he spoke of the formative changes that Kalpana had on him both scientifically and personally. Memorably he spoke of her being the “jackhammer” that hewed chunks off the as-yetunsculpted granite block he was as an undergraduate, thanking Kalpana for playing that role as he became a better scientist and person from his time with her.
Unlike most viral assembly systems, two scaffolding proteins, B and D, mediate bacteriophage phiX174 morphogenesis. The external scaffolding protein D is highly ordered in the atomic structure, and proper function is very sensitive to mutation. In contrast, the internal scaffolding protein B is relatively unordered, and extensive alterations do not eliminate function. Despite this genetic laxity, protein B is absolutely required for virus assembly. To address the biochemical functions of a dual scaffolding protein system and the evolution of complexity, progressive and targeted genetic selections were employed to lessen and finally eliminate B protein-dependence. The biochemical and genetic bases of adaptation were characterized throughout the analysis that led to the sextuple mutant with a B-independent phenotype, as evaluated by plaque formation in wild-type cells. The primary adaptation appears to be the over-expression of a mutant external scaffolding protein. Progeny production was followed in lysisresistant cells. The ability to produce infectious virions does not require all six mutations. However, the lag 24 phase before progeny production is shortened as mutations accumulate. The results suggest that the primary function of the internal scaffolding protein may be to lower the critical concentration of the external scaffolding protein needed to nucleate procapsid formation. Moreover, they demonstrate a novel mechanism by which a stringently required gene product can be bypassed, even in a system encoding only eight strictly essential proteins.