Transmembrane domain interactions in the pathogenic viruses

We have recently established a technique to directly analyze interactions between transmembrane (TM) regions of viral fusion proteins using sedimentation equilibrium centrifugation. Applying this assay to the TM domains of three paramyxovirus F proteins, we found a monomer-trimer equilibrium best fit for all three tested TM domains. These results demonstrate for the first time that F protein TM domains self-associate as trimeric complexes in the absence of the rest of the protein. Our data also suggests a role for TM-TM interactions in the stabilization of the pre-fusion form of the paramyxovirus F protein. Given our findings with paramyxovirus fusion proteins, 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, and we are testing our hypotheses in this developmental project. To extend our studies to other type I fusion proteins, we created expression constructs for analysis of TM-TM interactions for the influenza HA protein TM, the Ebola Gp protein TM, and the SARS-CoV S protein TM. High level expression following IPTG induction resulted in chimeric proteins which contain the staphylococcal nuclease (SN) protein linked to the TM segment of interest which we are currently analyzing by sedimentation equilibrium analytical ultracentrifugation. In addition to analysis of wild-type TM interactions, we will study the effect of mutations known to effect membrane fusion on any TM-TM interactions observed. Second, to examine type III fusion proteins from pathogenic viruses, we have created an SN- rabies G TM chimeric protein, and the TM-TM interactions of this protein, and the effect of low pH known to trigger fusion, will be assessed. Next, we will establish a system to examine the intramolecular and intermolecular TM interactions for the Dengue E protein, which contains two TM regions. Using our system, we will determine whether the two TM regions from the Dengue virus E protein display stable interactions with each other or with themselves, providing the first direct analysis of TM-TM interactions in a type II viral fusion protein, and providing insight into control of the E protein fusion mechanism. Finally, we are working to assess the ability of TM or HRB-TM peptides to inhibit paramyxovirus entry. First, we are currently creating 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. We will be examining the effect of a peptide containing portions of the Hendra TM domain on cell-cell fusion or pseudotype viral entry. We will also examine whether addition or TM residues to heptad repeat B peptides, already known to function as antivirals, increases efficacy. These experiments will therefore test TM peptides as novel antivirals. PHS The primary goal of the Southeast Regional Center of Excellence for Emerging Infections and Biodefense (SERCEB) is to assist the nation in developing and deploying effective and rapid responses to emerging infectious diseases and biothreats. Through our research programs in host-pathogen interactions and interventions, we aim to discover new therapies and principles by which effective vaccines and other intervention measures can be developed for diseases and pathogens that threaten the health of our citizens. Our Developmental Projects Program, described here, offers investigators across our region up to 2 years of funding for novel and innovative research projects that may lead to new discoveries in the area of emerging infectious diseases and biodefense (EID/BD).