We will study the processing and assembly of the vesicular stomatitis virus (VSV) envelope glycoprotein using biochemical, immunochemical, and radiochemical techniques. Preparative scale fractionation of the VSV G protein will be carried out and selected, purified peptides will be characterized to determine their sequence, location within the linear G structure, and immunogenicity and immunochemical reactivity of the peptide fragments. Polyclonal antisera will be prepared against selected purified fragments. Polyclonal antisera against intact VSV, native (non-denatured) purified G protein, denatured G. trypsin digested G, and purified G fragments will be characterized immunochemically and correlated with reactivity to specific sequences of the G polypeptide. Murine hybridomas and monoclonal antibodies will be prepared and those already isolated will be characterized for reactivity with specific domains of the VSV G polypeptide and these domains defined in terms of the linear G. polypeptide sequence. A novel system will be utilized to study the molecular aspects of protein-protein and protein-carbo-hydrate interactions involved in the intracellular transport, glycosylation, and processing of the VSV G glycoprotein. Effects of domain-specific antibodies against the VSV G polypeptide on the glycosylation and processing of the G protein will be analyzed using both in vivo and in vitro systems. Perturbation of the G polypeptide structure during processing will be correlated with alteration of oligosaccharide synthesis using methods developed previously for analyzing the oligosaccharide synthesis. Characterization of intermediates in the synthesis of the oligosaccharide branch structures which occurs in the Golgi will be carried out using both in vivo and in vitro systems. The long term objective of these studies is to define in molecular terms and concepts the processes by which vival polypeptides are synthesized, glycosylated, transported to specific domains of the host cell's membranes, and incorporate into maturing virus progeny. The proposed characterization of polychonal and monoclonal antibodies in terms of their reactivity with G polypeptide sequences will aid in unerstanding and provide insight into the immunochemical mechanisms of antibody interaction with viral surface antigens.
| Etchison, D; Etchison, J R (1987) Monoclonal antibody-aided characterization of cellular p220 in uninfected and poliovirus-infected HeLa cells: subcellular distribution and identification of conformers. J Virol 61:2702-10 |
