Project 4: Molecular Mechanisms of RSV F Activation and Inhibition

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Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections that result in substantial morbidity and mortality in infants and the elderly. RSV entry into the host cell is facilitated by a fusion (F) glycoprotein that in its active form adopts a metastable prefusion conformation. After attachment of F to host-cell factors, it is hypothesized that one or more of these factors trigger the conformational rearrangement that results in fusion of the viral and cellular membranes. Since F is essential for RSV infection, humans elicit neutralizing antibodies that target it, with the most potent recognizing the prefusion conformation. Thus, this conformation of F is considered to be the ideal vaccine antigen, and antibodies and small molecules that disrupt its structure and function are rapidly being pursued. Development of effective therapeutics will be greatly enhanced by a molecular understanding of how the F glycoprotein interacts with host-cell factors that promote entry, and how neutralizing antibodies are able to inhibit one or more steps in the entry process. Through a collaborative effort we have determined X-ray crystal structures of the pre- and postfusion conformations of RSV F, stabilized a soluble form of the prefusion conformation, and identified a new class of potent neutralizing antibodies. This proposal builds upon these results by leveraging our prefusion-stabilized F glycoprotein and panel of antibodies to test the hypothesis that host-cell factors trigger RSV F rearrangement and that the most potent neutralizing antibodies target receptor binding sites and block conformational changes. In Aim 1, we propose to characterize specific interactions between prefusion F and host-cell factors, and determine which factors trigger F. In Aim 2, we plan to structurally determine the epitopes of F-directed neutralizing antibodies, and determine which steps in the viral entry process these antibodies inhibit. These studies will be greatly facilitated by the interactions and resources provided by the iTarget COBRE. Our mass spectrometry-based identification of RSV F receptors will benefit from interactions with Dr. Kettenbach, and our development of structure-based antivirals and their clinical testing will be shaped through interactions with Drs. Grigoryan and Ashare, a physician scientist. The Molecular Tools Core will provide support for the expression and purification of F glycoproteins, receptors, and antibodies. The functional and biophysical characterization of these proteins and their interactions will be facilitated by the Visualizing Molecular Interactions Core through access to SPR, ITC, FP, X-ray and flow cytometry equipment. The proposed project will advance our molecular understanding of the activation and inhibition of viral fusion glycoproteins by discovering and defining interactions of host factors with RSV F. Through involvement with the iTarget COBRE, this knowledge will be translated into the screening and development of RSV entry inhibitors, vaccine antigens and prophylactic antibodies. The COBRE will also provide an intellectual environment and mentoring support that will allow my research, and my lab, to expand and flourish.