Respiratory syncytial virus is the most common cause of lower respiratory tract disease in young children worldwide. The F and G glycoproteins of this virus are known to be targets of a protective immune response; however, following natural infection, immunity is incomplete. Repeated infections with viruses of the homologous or heterologous subtypes occur despite high levels of neutralizing antibody. In addition, antibodies that neutralize extracellular virus may not be able to inhibit fusion, allowing cell-to-cell spread of virus. These findings suggest that B cell responses seen following infection may be deficient in antibodies that prevent virus- receptor interactions. In order to better understand the virus-cell interactions required for infection, studies were initiated to more completely define the receptor (s) used by RSV. The binding of viral proteins to cell surface glycosaminoclycans was evaluated using heparin agarose affinity chromatography. Studies using whole virions,infected cell lysates, purified envelope glycoproteins, and lysates of vaccinia recombinant viruses expressing individual RSV F or G glycoprotein, showed that both F and G bind heparin. Strain RSV/B cp 52, which expresses F glycoprotein but lacks G and SH, was used to show that F-heparin binding was independent of G. In addition, cp52 infection of Vero cells was diminished in the presence of heparin, Bovine lung heparin and de-N sulfated heparin and this inhibition was dose-dependent. These results indicate that RSV-F-heparin sulfate interactions are important in the biology of the virus. We identified linear F sequences responsible for heparin binding using overlapping F peptides. These peptides were evaluated for their ability to bind in heparin agarose affinity chromatography, their ability to bind to Vero, Hep-2 and A549 cells, ability to block virus attachment and capacity to inhibit infection with A and B strains as well as strain cp52. Binding to cells was specifically inhibited by soluble heparin and bovine lung heparin, suggesting that the interaction was specific for cell surface glycosaminoglycans. These studies identified a linear sequence within RSV subgroup F that bound heparin. Work in this laboratory has also shown that RSV F,G and SH are expressed in a complex on the surface of infected cells. Interestingly, the affinity of the F-G-SH complex for heparin is greater than the affinity of F or G glycoproteins alone. These results may explain, in part, the incomplete inhibition observed when heparin and heparin sulfate derivatives are used to inhibit RSV infection. In addition, working with colleagues in OBRR, we have determined that the dominant human neutralizing antibody response is isotype IgG1 and identified linear domains on RSV F and G recognized by this subclass of human antibody.
