EXCEED THE SPACE PROVIDED. The goal of this project is to elucidate the molecular events of ecotropic murine leukemia virus (MLV) entry. Infection by these retroviruses shows several characteristic anomalies, including entry by an atypical endocytic pathway and a much slower rate of membrane fusion, suggesting that the mechanisms of MLV entry might be fundamentally different from those of other enveloped viruses.
Specific Aim 1 proposes to study the influence of receptor membrane localization on infection. Depletion of cholesterol reduces infection of NIH 3T3 cells, suggesting parallels with other viruses that require lipid raft components on the host cell for membrane fusion. The proposed studies will determine if infection is also reduced by depletion of glycosphingolipids, and will identify the step in membrane fusion that is blocked by perturbing host cell membrane rafts using cell-cell and virus-cell fusion assays developed in influenza virus studies, and a new virus-cell fusion assay we developed in preliminary studies. Three candidates for sequences on the receptor that influence its membrane localization will also be examined.
Aims 2 and 3 will focus on the molecular mechanisms of envelope protein (Env) function in membrane fusion.
In Aim 2, the step in membrane fusion that is blocked by Env fusion mutants will be identified using the virus- and cell-cell fusion assays and a detailed functional map of the Env will be built by assigning mutants to genetic complementation groups using a subunit trans-complementation assay.
Aim 3 will address basic questions about the conformation of the Env. The usefulness of structural data on the receptor binding domain (RBD) and heptad repeat domains of Env in elucidating molecular mechanisms of Env function has been limited by lack of information on these aspects. One question will be addressed by mapping the epitopes recognized by existing monoclonal antibodies (mAb). Others will be addressed using the epitope-mapped mAb as probes of Env conformation. PERFORMANCE SITE ========================================Section End===========================================
|Bolin, Lisa L; Chandhasin, Chandtip; Lobelle-Rich, Patricia A et al. (2011) Distinctive receptor binding properties of the surface glycoprotein of a natural feline leukemia virus isolate with unusual disease spectrum. Retrovirology 8:35|