Grants and Contracts Details
Recent studies have altered the perception of transmembrane domains (TMDs) from hydrophobic anchors to important factors in protein function, including protein oligomerization, cell signaling, and regulation of channel function (cite). Despite these advancements, the role of TMDs in viral glycoprotein stability and function remains poorly understood. Previous studies from our lab utilized sedimentation equilibrium (SE) ultracentrifugation analysis to demonstrate that the isolated TMD of several viral glycoproteins associate in a monomer-trimer equilibrium. Functional analysis of mutations in the TMD of the full length Hendra virus F protein also revealed altered expression levels and fusion activity. These studies implicate TM-TM interactions in regulation of F protein folding and function. Preliminary data from our lab identified the presence of an L/I zipper within the TMD of HeV F that continues in frame with the L/I zipper of the upstream heptad repeat domain B (HRB). Using site directed mutagenesis, I demonstrated that by replacing the four L/I residues that continue into the TMD with alanine, TM-TM association is dramatically reduced as determined by analytical ultracentrifugation. Introducing these mutations into the full length F protein, I also found that F protein mediated membrane fusion was dramatically reduced. Further characterization of the TMD, including features of the environment important for association, will provide critical information regarding F protein function, and therefore provide insights into viral membrane fusion. The overall hypothesis of this proposal is that TM-TM association and fusion protein stability are affected by the lipid environment of the viral particle and also specific residues within the TMD, therefore making TMDs a potential target for antiviral therapeutics. To test this important hypothesis, we will define the role of key residues in HeV F protein TM-TM interactions and pre-fusion stability by determining the effect of TMD mutations on TM-TM association in an E. coli reporter system, GALLEX, and also assess the effect the mutations have on the full length fusion protein in eukaryotic cells. Also, I will determine whether the L/I zipper is important for TM-TM association in other viral fusion proteins by analyzing the L/I zipper present in the TMD of Ebola GP and SARS CoV S, two other type I fusion proteins. Finally, since TM-TM interactions take place in the complex environment of lipid bilayers, I will also dissect the role of the lipid environment in pre-fusion stability and TM-TM association. These experiments will utilize virus like particles, which will enable for modulation of lipid content. Overall, these important experiments will provide new insights into the important field of TMDS association, and enhance our understanding of F protein stability.
|Effective start/end date||5/15/15 → 5/14/17|
- National Institute of Allergy and Infectious Diseases: $34,931.00
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