Respiratory syncytial virus (RSV), is the single most important infectious cause of severe respiratory disease in infants. Infection begins when the virion attachment protein binds to a cellular receptor and the virion membrane fuses with the target cell membrane. RSV has three surface glycoproteins in a position to perform the attachment and fusion functions: G (glycoprotein), F (fusion), and the small hydrophobic (SH) protein. Laboratory strains of RSV attach to glycosaminoglycans (GAGs) to initiate infection, but this characteristic might be selected by passage in cultured cells. The first Specific Aim of this study will compare the viral glycoprotein gene sequences derived directly from patient samples to those from cultured virus from the same patient. The second Specific Aim will examine the ability of the RSV F protein to bind to target cells, an idea derived from the surprising fact that both the G and SH genes can be deleted from RSV without loss of infectivity. This characteristic, unique among paramyxoviruses, strongly suggests that the RSV F protein is able to attach to cells, and together with its well-established role in membrane fusion, to initiate infection. F protein attachment to target cells will be explored using radioactively labeled recombinant RSV virions that contain the F protein as their only viral glycoprotein. These viruses also express the green fluorescent protein (GFP), allowing easy assay of infectivity and determination of the efficiency of infection once the virus is bound. The third Specific Aim will identify the region(s) of the F protein involved in cell attachment and fusion, through blocking studies with Fspecific monoclonal antibodies and overlapping peptide libraries, and through targeted mutations in the F protein. These mutations will be incorporated into recombinant virions expressing GFP, and their ability to support infection tested. The overall goal of this project is to understand the entry mechanism of RSV for eventual use in designing antiviral agents.
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