ISOLATION AND ANALYSIS OF MURINE LEUKEMIA VIRUS RECEPTOR

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Ecotropic host range in murine type C retroviruses is determined by the expression of a specific virus receptor. The goal of this proposal is to understand the molecular basis for viral receptor function in infection. The specific aims are to: 1. Complete the cloning of an intact ecotropit virus receptor gene. Expression of an intact gene in non-permissive human EJ cells will confer ecotropic host range. 2. Determine if receptor expression is the sole determinant of ecotropic host range by introducing the receptor gene into a broad range of eucaryotic species and testing for infection. 3. Investigate the relationship between the receptor structure and function. a. Determine if the transfected murine gene encodes the receptor directly or by specifying the modification of an endogenous human protein. b. Compare the nucleotide sequence of the coding region to a sequence bank to determine if the receptor gene has been identified as a cellular protein. c. Model receptor structure with respect to the membrane. Identify extracellular domain(s) that may be involved in envelope binding. d. Test this model by preparing antisera against synthetic peptides from the predicted extracellular domains. 1. Test the ability of these antisera to block envelope binding and infection. 2. Demonstrate direct interaction between virus envelope protein (gp70) and receptor by co-immunoprecipitation. 3. Investigate the role of membrane gp70-receptor interaction in interference. e. Identify specific sites of virus envelope-receptor interaction. The approach will be to create null mutations in the binding domain of the receptor and then to select mutant viruses that complement. These studies will help elucidate the molecular determinants of retrovirus host range, the mechanism of retrovirus entry into cells, and the phenomenon of interference. Further, they will identify a cellular protein that is utilized by retroviruses for an essential step in their lifecycle. In human retroviruses these interactions have been shown to play an important role in the pathogenesis of AIDS and retrovirus-induced cancer.