Fellowship for Rachel Schowalter: Host Cell Surface Components Required for HMPV Fusion Protein Binding and Fusion

Human metapneumovirus (HMPV) was identified in 2001 in samples from infants and young children experiencing acute respiratory tract disease. HMPV is associated with severe respiratory disease, particularly in infants, the elderly or immunocompromised patients, and evidence suggests that HMPV is the second most common cause of childhood bronchiolitis. Underlying cardiopulminary disease is a major risk factor for severe HMPV disease in children and adults. Sequence analysis clearly places HMPV within the paramyxovirus family, and, like other paramyxoviruses, HMPV contains genes encoding for glycoproteins that are likely function in attachment and entry into host cells. The entry of paramyxoviruses is primarily mediated by the F (fusion) protein, which promotes fusion of viral and cellular membranes. Attachment of the virus to the cell is usually mediated by a second glycoprotein. However, HMPV and several closely related viruses have been shown to enter cells in the absence of the attachment protein, suggesting the F protein can bind to cell surface receptors and trigger fusion alone. We have recently established the first efficient assays for the study of membrane fusion promoted by the HMPV F protein. Interestingly, HMPV F protein-mediated cell-cell fusion only occurs when transfected cells are exposed to low pH. As previously studied paramyxovirus F proteins promote fusion at neutral pH at the plasma membrane, the triggering of HMPV F protein fusion activity by low pH suggests that this protein functions in a manner unique among the paramyxoviruses. We hypothesize that HMPV F interacts with one or more defined cellular receptors, and that this interaction is necessary to prime the F protein for low-pH induced promotion of membrane fusion. To address this hypothesis we will first systematically define the target cell components required for HMPV F protein-promoted membrane fusion by utilizing varying cell lines and by treatment of target cells with enzymes or reagents that specifically modify cell surface proteins or proteoglycans. In addition, we will create a soluble form of the HMPV F protein and utilize it to analyze the conditions necessary for binding of the F protein to cell surfaces. The soluble F protein will then also be used to identify a specific cellular receptor required for HMPV entry. Accomplishing these goals will set the stage for future analysis of inhibitors of this important viral protein.