Fellowship: Knoebber: Mutational Analysis of Conserved Regions in Paramyxovirus Fusion Proteins

The Paramyxoviridae are a diverse family of enveloped viruses, with homologous members such as simian virus 5 (SV5) and mumps virus and newly emerged and disparate members such as Hendra and Nipah viruses. Paramyxoviruses possess a surface glycoprotein, the fusion (F) protein, which promotes fusion between the viral envelope or virus-infected cell membrane and a target cell membrane. While several domains within the F protein have identified roles in membrane fusion, the majority of the protein has no defined function. Our central hypothesis is that conserved regions among diverse members of the Paramyxoviridae F proteins have important conserved functions, either in the folding of fusion proteins or in aiding F-mediated fusion events. Sequence alignment of F proteins from disparate members of the Paramyxovirus family and analysis by the Block Maker program were used to identify three blocks of conserved amino acid sequence. Two of the identified blocks of conservation (CBF1 and CBF2) are in regions which currently have no known function. The proposed research will examine the function of conserved residues within paramyxovirus fusion proteins utilizing site-directed mutagenesis and transient expression systems, along with metabolic labeling and immunoprecipitation or fusion assays. Our initial studies found that point mutations in CBF1 led to defects in homotrimerization, an event that is essential for the fusogenic activity of the F protein. These results both provide the first identification of a region important for trimer formation of paramyxovirus F proteins, and support our hypothesis that conserved domains playa critical role in folding or function. Specific Aim 1 will analyze the effects of mutations in the second conserved block of unknown function, CBF2. Experiments in Specific Aim 2 will address the function of residues in the fusion peptide that are modified in the newly emerged paramyxovirus Hendra as compared to previously characterized members of the family. Accomplishing these goals will provide new insight into the role of these sequences in the folding of, or fusion promotion by, these critical viral proteins. These studies should also shed light on the folding of glycoproteins in the endoplasmic reticulum and the basic process of membrane fusion. In addition, domains demonstrated to be critical in membrane fusion promotion could serve as targets for antiviral therapy.