Rous sarcoma virus encodes two surface glycoproteins that play a critical role in the replication cycle of the virus. The larger of these two molecules (gp85) defines the host range of the virus by binding to a host cell-encoded receptor, while the smaller protein (gp37) anchors gp85 in the viral membrane and functions in the process of virus-cell membrane fusion. The envelope glycoprotein (env) gene product is synthesized as a glycosylated polyprotein precursor that, during transport to the plasma membrane, is cleaved in the Golgi complex of the host cell to the two structural proteins. At the cell surface the glycoprotein complex must associate with the capsids of assembling virions, since viruses lacking the glycoprotein complex are non-infectious. Thus, the major goals of this proposal are to understand at the molecular level the structural features of the Rous Sarcoma virus envelope glycoprotein complex that are involved in plasma membrane association, assembly into budding virus, receptor binding, and cleavage/activation. In the latter regard, a major goal is to identify and characterize the host-cell encoded protease that is responsible for processing the glycoprotein precursor - an event we have shown to be essential for glycoprotein function. Specifically we will engineer mutations into the carboxyterminal region of the RSV env gene to identify sequences required for recognition and incorporation of the viral glycoproteins into virions. Hybrid viruses containing glycoproteins of viral and cell origin will be constructed to investigate the specificity of incorporation. We will investigate structural features of the env product that influence stability of the protein in the plasma membrane and determine whether a secreted form of the viral glycoprotein can bind the cell receptor in a competitive manner. Experiments are described to identify the nature of the cell receptor. Finally we will identify and characterize the host cell encoded protease that is responsible for cleavage/activation of the glycoprotein precursor. Antibodies made against this protein will be used in cell localization and cDNA library screening experiments. These studies will provide important information on those molecular events encoded by the virus and the host cell that are critical for retroviral replication.
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