Human metapneumovirus (HMPV) is a recently identified paramyxovirus associated with severe respiratory disease, particularly in infants, with symptoms that closely resemble those seen with respiratory syncytial virus. We have established efficient assays for the study of membrane fusion promoted by the HMPV fusion (F) glycoprotein. Interestingly, HMPV F protein-mediated cell-cell fusion only occurs when transfected cells are both treated with trypsin (to proteolytically process the HMPV F protein) and 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. Our results also demonstrate that HMPV F is competent to promote fusion in the absence of the attachment (G) protein, consistent with reports that recombinant HMPV viruses lacking the HMPV G protein replicate efficiently both in cell culture and in hamster and non-human primate animal model. Our long-term goal is to understand the attachment and entry of paramyxoviruses. Our specific hypothesis is 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 pursue three research aims. First, we will utilize our recently developed, efficient reporter gene assay to systematically define the target cell components required for HMPV F protein-promoted membrane fusion. Second, we will analyze the effect of treatments to the HMPV F protein on promotion of membrane fusion, including effects of prior low pH treatment, proteolytic processing of the F protein or addition of soluble glycosaminoglycans. Finally, we will utilize a soluble form of the HMPV F protein to analyze binding and identify the specific cellular receptor(s) for HMPV F. Accomplishing these goals will provide crucial information on the cellular components that interact with the HMPV F protein, and may well lead to identification of the first receptor for a paramyxovirus fusion protein. In addition, these studies will further clarify the mechanism by which the HMPV F protein promotes critical early steps in infection, and set the stage for future analysis of inhibitors of this important viral protein. Human metapneumovirus (HMPV) is a recently identified human pathogen responsible for significant levels of severe respiratory disease, particularly in infants. This research will address significant questions concerning the cellular factors which interact with the HMPV fusion protein to facilitate attachment of the virus and entry into cells. [unreadable] [unreadable] [unreadable]