The murine leukemia virus (MuLV) env gene is an important determinant of viral infectivity, and the products of this gene provide useful models for studying the biosynthetic pathways of membrane proteins. The spleen focus-forming virus (SFFV), a defective MuLV variant, is a potent pathogenic agent, causing extensive proliferation of spleen cells shortly after infection and leading to the rapid development of erythroleukemia. Although SFFV contains a defective env gene which is not involved in viral replication, genetic evidence strongly implicates the SFFV env gene as the major pathogenic determinant of these viruses. We have recently found that several SFFV glycoproteins, including the mature env gene product, gp65, are efficiently secreted from cells, and that partially purified preparations of gp65 stimulate the proliferation of erythroid progenitor cells in vitro. These results suggest that gp65 or a related component may be the molecule responsible for the pathogenicity of these viruses. We now propose to extend our studies of both the MuLV and SFFV env components, to further elucidate the structure and function of these components and to clarify their roles in the leukemogenic process. The functional roles of oligosaccharide substituents of gp70 in the transport and secretion of the MuLV env proteins will be determined by treatment with a number of drugs which interfere with the normal processing of N-linked sugars. The structures of the oligosaccharides on molecules secreted in the presence of these drugs will be determined, and the assembly and infectivity of the resulting virions analyzed. The sites of O-glycosylation on gp70 will be identified, and the nature of the O-linked sugars determined. Extracellular SFFV products will be purified to homogeneity and the proliferative activities of the purified material for hematopoietic cells will be assayed. Further biochemical studies of SFFV glycoproteins will be preformed, including amino- and carboxy-terminal sequencing and the complete characterization of the post-translational modifications of these molecules. Site-specific mutations will be introduced into molecularly cloned genomes of SFFV and MuLVs by oligonucleotide- directed mutagenesis, in order to determine the functional roles of O-linked glycosylation and the structural features specific for the SFFV env components in the biological activities of these proteins and in the pathogenicity of these viruses. Monoclonal antibodies and site-specific antisera will be prepared against different domains of gp65 and the active site of gp65 identified by examining the effects of such antibodies on the in vitro and in vivo biological activities of this molecule. The effect of the regulatory Fv-2 gene on the response of hematopoietic cells to gp65 will be examined as an additional test of the biological specificity of the proliferation activity of this molecule. Finally, binding assays will be developed for both gp70 and gp65, and cell surface receptors for these molecules isolated and characterized in order to study the role of receptor interactions in viral leukemogenesis.
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