Genetic, biochemical, and structural analyses of the Microviridae scaffolding proteins

Abstract

Scaffolding proteins are transiently associated with morphogenetic intermediates but are not found in the mature viral particle. 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 determining appropriate capsid size. The goal of the proposed research is to understand how scaffolding proteins recognize and interact with viral precursors thus enabling them to obtain an assembly active form and defining the requirements for, and constrains on, these interactions. Microviridae morphogenesis is dependent upon two scaffolding proteins, an internal and external species. The genes encoding three Microviridae (ØX174, G4 and α3) internal scaffolding proteins (B proteins) have been cloned, expressed in vivo and assayed for the ability to complement null mutations of different Microviridae species. Despite divergence as great as 70% in amino acid sequence over the aligned length, cross-complementation was observed, indicating that these proteins are capable of directing the assembly of foreign structural proteins into infectious particles. These results suggest that the Microviridae internal scaffolding proteins may be inherently flexible. There was one condition in which a B protein could not cross-function. Substitutions conferring utilization map to the viral coat. The more efficient substitution is located in a region where coat-scaffolding interactions have been observed in the atomic structure and may emphasize the importance of interactions in this region. This is supported by chimeric analyses where efficient complementation was observed only when the viral coat protein and COOH-terminus of internal scaffolding were of the same origin. Despite 70% homology on the amino acid level, over-expression of a foreign Microviridae external scaffolding protein is a potent cross-species inhibitor of morphogenesis. 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 domain allows procapsid assembly but blocks DNA packaging. These results demonstrate how closely-related proteins could be developed into antiviral agents that specifically target virion morphogenesis.