An essential pathway in the assembly of enveloped viruses is the formation of mature, surface glycoproteins. The long-term goal of this project is to understand the maturation of paramyxovirus glycoproteins using Newcastle disease virus (NDV) as a model system. Paramyxoviruses encode two glycoproteins which form the spike structures on the surfaces of virions, the hemagglutinin-neuraminidase (HN) protein and the fusion (F) protein. in the past four years intermediates in the assembly of the HN glycoprotein have been detected using conformationally sensitive monoclonal antibodies and site-directed mutagenesis of cysteine residues. The formation of conformationally sensitive antigenic sites occurs in distinct steps that can be differentiated kinetically. In addition, mutations in different cysteine residues block the maturation of the protein at specific points in the pathway defined by the antibodies. Characterization of the HN protein within the context of this pathway has led to results with significant implications for intramolecular disulfide bond formation, glycosylation site usage, role of carbohydrate side chains on the structure of the protein, and oligomer formation. Exploration of the hypotheses developed from these results forms the basis for this applic ion. The experiments proposed will make use of infected cells, cells transfected with DNA encoding the viral genes, and cell-free systems in which both synthesis and folding occur. Specific Aims: 1. The intramolecular disulfide bonds within the mature HN protein will be defined, and the order in which they form will be determined. 2. Peptide antibodies will be used to detect early steps in the maturation pathway of the HN protein and to define the pathway for the fusion protein maturation. 3. The hypothesis that glycosylation site usage depends upon localized folding events in the vicinity of the site that take place on the nascent chain as it emerges into the lumen of the endoplasmic reticulum will be tested. 4. The role of individual carbohydrate side chains on the maturation and activities of the HN protein will be determined. 5. The hypothesis that HN protein oligomer formation begins on nascent chains and proceeds in several steps will be tested.