Transmembrane domain interactions in pathogenic viruses

Using a novel assay involving sedimentation equilibrium centrifugation to directly analyze interactions between transmembrane (TM) regions of viral fusion proteins, we found that TM domains of three different paramyxovirus F proteins best fit a monomer-trimer equilibrium, demonstrating for the first time that F protein TM domains self-associate as trimeric complexes in the absence of the rest of the protein. Our results also suggested a role for TM-TM interactions in the stabilization of the pre-fusion form of the paramyxovirus F protein. Given these findings, we hypothesized that TM-TM interactions are broadly important for regulating viral fusion protein function, and that peptides targeting these interactions could represent a new class of antivirals agents. To test this hypothesis, we first examined type I and type Ill fusion proteins from pathogenic viruses by analysis of TM-TM interactions for the influenza HA protein TM, the Ebola GP protein TM, the SARS-CoV S protein TM, and the rabies G TM. TM association was observed for all four glycoprotein TM domains, supporting our hypothesis, but interesting differences were found. For influenza HA and rabies G protein TM domains, a monomer-trimer best fit was found at both pH 7 and at pH 5, suggesting that pH-induced alterations to TM interactions are not a driving force for the conformational changes that promote membrane fusion. In contrast, analysis of TM domain interactions of Ebola GP and SARS CoV S at pH 7 indicated that higher order species than trimer were present, with a best fit monomer-trimerhexamer model, suggesting potential interaction between trimeric TM components. Interestingly, for both of these proteins, analysis at pH 5 indicated a best fit monomer-trimer model, and addition of a hexamer component did not improve the curve fit, indicating that interactions to form higher order oligomers than trimers were significantly reduced at lower pH. These results suggest a preliminary model where modulation of interactions between Ebola GP and SARS CoV S TM regions of differing trimers may play a role in fusion at endosomal pH. We will continue analysis of these type I and type Ill fusion protein interactions in the coming year by focusing on known mutations in the TM domain which alter folding or fusion when present in the whole protein. We are also analyzing TM interactions in the Dengue virus E type II fusion protein, which interestingly has two TM domains. We have successfully made chimeric proteins containing the TM1 and TM2 domains, or the separate TM domains, and analysis of the interaction of these domains will be performed in the coming year. Finally, we are working to assess the ability of TM or HRB-TM peptides to inhibit paramyxovirus entry. Our initial studies with peptides corresponding to the full length TM domain indicated an effect of viral entry, but additional issues with cell integrity in the presence of the peptide complicated analysis. We will focus on shorter peptides containing a portion of the TM domain, with or without the HRB region, in the coming year. We have also completed synthesis of Hendra F protein mutants which lack the ectodomain, to determine if expression of just the TM domain (with or without flanking regions) inhibits F protein folding or function, and this analysis will be performed during the next year of the grant. The work to date therefore breaks new ground on our understanding of TM association in viral glycoproteins, showing for the first time that TM domains from influenza HA, Ebola GP, SARS-CoV S and rabies G associate in monomer-trimer or monomer-trimer-hexamer equilibria in the absence of the rest of the protein.