The five-year research career development program for Dr. Matthew Buccellato, DVM is divided into two phases. Phase I, which includes Specific Aims 1 and 2, will allow Dr. Buccellato to devote full time to research training that will culminate in the awarding of a Ph.D. in molecular virology. Phase II will allow Dr. Buccellato to further develop his skills as an independent researcher in the post-doctoral period in preparation for a career in biomedical research. The goal of the research proposal is to characterize the regulation of binding interactions between morbilliviral nucleocapsid protein (N) and the major inducible 70kDa heat shock protein (hsp72), establishing a link between modification of this interaction and alterations in viral infection phenotype within the central nervous system. Morbilliviruses and other closely related members of the Paramyxoviridae family, such as the recently emerged Hendra and Nipah viruses, cause significant systemic disease, often involving the central nervous system. Recent work suggests that hsp72, whose expression is the hallmark of the cellular stress response, exerts control over morbilliviral gene expression by associating with the viral nucleocapsid protein, but that this association might be altered by other virus/host determinants. The hypothesis to be tested in this application is that phosphorylation of the highly-conserved tyrosine residue within the hsp72-binding motif on the C-terminus of the morbilliviral N protein reduces hsp72 binding capacity, resulting in a loss of viral responsiveness to hsp72 that is necessary for the establishment of persistent infection of neurons and induction of neurologic disease. This hypothesis will be tested using experimental systems based on both measles and canine distemper as models of morbilliviral infection. Studies undertaken in Specific Aim 1 will identify the role of this tyrosine residue in determining the function of the hsp72-binding motif of morbilliviral nucleocapsid protein using minireplicon systems and biomoloecular interaction analysis.
In Specific Aim 2, a morbillivirus rescue system will be used to produce infectious viral variants incorporating mutations of the tyrosine residue and examine how these changes affect viral phenotype in transgenic murine neuroblastoma cell lines.
Specific Aim 3 is designed to investigate the affects of measles virus nucleocapsid mutations on neurovirulence in hsp72 overexpressing transgenic mice.
