Retroviruses are RNA viruses capable of inducing various diseases including cancers and immunodeficiencies. Many retroviruses cause cellular transformation because they carry oncogenes transduced from cellular genes. Due to their high efficiency in transferring genes, retroviral vectors, ironically, are a preferred means of introducing genes into cells for gene therapy. The long-term goal of this proposal is to understand on the molecular level the mechanism by which Murine Leukemia Viruses (MuLV) choose and enter their host cells. The viral gene products which are known to be required for the entry of the virus into the cells (env gene products) will be studied genetically and biochemically. Identification of the various functional domains of the env gene products provides the basis for future experiments aimed at targeting the entry of the virus to specific cell types and facilitates the use of retroviruses for gene therapy. The env gene products from two MuLV isolates with differing host-range will be analyzed and compared. Linker-insertion mutations will be generated throughout the env gene of the Moloney ecotropic MuLV, which only infects rodent cells, as well as the amphotropic 4070A isolate, which can infect many mammalian species. The effects of the mutations on the viral life-cycle will be determined. Functional domains within the proteins will be localized by the clustering of mutations with identical phenotypes. Regions required for correct processing and transport to the cell surface, association with the viral particle, syncytium formation, and receptor binding may be identified. A second approach to identify regions required for receptor binding and recognition will involve the generation of hybrid amphotropic/ecotropic env gene products. Variations within the env gene alter the host-range of the virus and direct the entry using differing host-cell receptors. Hybrid env molecules in which the majority of the amphotropic env gene has replaced the ecotropic gene has produced virus with the amphotropic host- range. Using cloning techniques, various sections of the amphotropic env gene will be systematically introduced into the ecotropic backbone. The host-range of the hybrid gene product will be examined and regions required for the receptor binding and tropism will be determined. env gene products with altered oligosaccharide moieties will be generated to address the role of the multiple glycosylations. The potential glycosylation sites will be changed using oligonucleotide-directed mutagenesis. The effects of the loss of the individual sites as well as multiple glycosylation sites will be examined. The final processed form of the env gene product contains two proteins, SU and TM, are covalently bound through a disulfide bridge. the cysteine residue involved in this bond in the TM protein will be identified.
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