Expression and characterization of respiratory syncytial virus fusion protein for evaluation of lipid raft association and early human immune responses

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory illness in both infant and elderly populations. Repeat RSV infections are common throughout life and sequelae from infection can include long-term asthma-like symptoms. Currently, there is no safe and efficacious vaccine or specific anti-viral therapy. The RSV fusion (F) protein resides on the surface of both the virus and the infected cell. F protein is important in vaccine and anti-viral therapy development since, it is necessary for entry into permissive cells. The purpose of this dissertation is to describe methods to study the F protein independent of other RSV proteins as a means to investigate the cellular biology of RSV F protein and the early human immune responses. Previous studies focusing on F protein achieved expression through use of vaccinia vectors. To overcome disadvantages introduced by use of vaccinia, we generated a synthetic gene construct of F that retained the wild-type amino acid sequence while optimizing the DNA sequence for mammalian cell expression. The F protein was expressed in several cell types, and was properly processed and expressed at the cell surface. We further analyzed specific domains of the fusion protein through molecular mutagenesis. We observed some differences in cell surface expression and ability to form syncytia, or fused cells, when portions of the cytoplasmic tail domain of F were truncated. We also found that the F protein was able to target specialized regions of the plasma membrane, termed lipid rafts, independent of other RSV proteins. The raft-association region of F protein was mapped to the extracellular domain. We also present data demonstrating reduced immediate-early T cell responses-to RSV that may account for the ability of RSV to cause reinfection. We speculate that this reduced T cell response is mediated by the F protein and discuss how our synthetic F gene construct can be used to evaluate this response. Collectively, the results in this dissertation further the understanding of RSV F protein cellular biology and provide potential methods to elucidate the described immune suppression.