Icosahedral Virion Scaffolding Proteins

University Of Arizona, Tucson AZ

Investigators

Abstract

The assembly of proteins and nucleic acids into viruses involves numerous molecular interactions. Often assembly is dependent on scaffolding proteins. Analogous to scaffoldings used in building construction, these proteins assist assembly, but are not found in the final product. The broad objective of this research is to elucidate the molecular mechanisms of scaffolding-mediated viral morphogenesis, using the Microviridae, a family of small single-stranded DNA viruses. Microviruses are somewhat unique: assembly is dependent on two different scaffolding proteins, an internal and an external species. Alterations (mutations) in the external scaffolding protein often result in dominant lethal effects. When the mutant protein is present in infected cells, it is able to interact with other viral proteins and unaltered (wild-type) external scaffolding proteins. These interactions block viral replication in a manner similar to anti-viral chemicals. By analyzing the specific stages of viral assembly affected by the mutant proteins, the protein's functions and the molecular details of virus assembly can be elucidated. In addition, the virus accrues additional mutations that overcome the lethal effects of the originally altered protein, providing insights into viral resistance mechanisms. In contrast to the external scaffolding protein, alterations to the internal scaffolding protein rarely have detrimental consequences to assembly. Consequently, standard genetic analyses have provided limited insights. Therefore, an evolutionary-based paradigm was developed to study this protein. A sextuple mutant that no longer requires the internal scaffolding protein has been isolated by five progressive and targeted genetic selections. By examining virus adaptation, internal scaffolding protein functions and alternate modes of assembly are being elucidated. Although this protein participates in several reactions, one of its prime functions appears to be catalytic, decreasing the time required to build new viral particles in the bacterial cell. Broader Impacts The project's broader impact goes beyond the standard education of graduate and undergraduate students within the investigator's program. The impact reaches the students enrolled in the courses taught by the investigator. Incorporating research into curricula is essential at larger universities, where undergraduates who desire research opportunities far outnumber the available positions. Part of the proposed research will be conducted during a virology/genetics/evolutionary biology module in the laboratory section of an 80-student course. Students will conduct hypothesis-driven experiments that may uncover novel mutations and assembly mechanisms.