Receptor utilization for influenza virus entry in vivo

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DESCRIPTION (provided by applicant): The entry of influenza virus into its host cell is a fundamental part of the virus life cycle. Initial binding to cell surface sialic acid moieties and low pH-dependent envelope fusion within the endosome have generally been well characterized for influenza virus; however, the cellular internalization and trafficking events between binding and fusion remain incompletely understood. Our preliminary data show that sialic acid binding per se is not sufficient for functional influenza virus entry in vivo. Using Lec 1 cells (a mutant cell line of CHO that lacks terminal sialic acid on its N-linked glycoproteins), we show that influenza entry and infection is severely inhibited. In contrast, virus binding to the cell surface is normal in Lec 1 cells. We hypothesize that Lec 1 cells lack a functional receptor normally required for influenza virus internalization and/or endocytic sorting. Our goals in this project are to use Lec 1 cells to identify a functional secondary, or "co"-receptor for influenza virus infection in vivo. Specifically, we will; 1) characterize the defect in virus entry for Lec 1 cells using a variety of well-characterized binding, internalization and fusion assays, and 2) identify the missing receptor by isolation and screening of monoclonal antibodies that differentially recognize CHO/Lec 1 cell surface epitopes and that will prevent virus entry. Our preliminary data clearly show a block in virus entry in mutant cells, however the identification of a proposed "co-receptor" and molecular characterization of virus entry in the mutant cells requires some degree of experimentation of an exploratory nature. The characterization of the entry block in Lec 1 cells and the identification of receptor requirements for internalization and/or endocytic trafficking of influenza virus in vivo will be of fundamental importance to our understanding of this highly pathogenic and potentially deadly virus, which is classified as an NIAID category C priority pathogen for biodefense and emerging infectious disease research.