Structural analysis of Rauscher virus Gp70 using monoclonal antibodies: Sites of antigenicity and p15(E) linkage

A linear map of 19 monoclonal antibody-binding domains on Rauscher retroviral gp70 was generated using a technique we have designated PEC-MAP (partial enzymatic cleavage-monoclonal antibody precipitation). We used eight proteolytic enzyme preparations in limited digests to produce 39 gp70 fragments. Immune precipitation of these fragments by monoclonal antibodies from 51 cell lines allowed us to define 19 binding sites by virtue of overlapping fragments and differential precipitation patterns. The sites most accessible to proteolytic attack and antibody binding were then mapped by analyzing the apparent molecular weights of the various gp70 fragments. This analysis revealed three hyperreactive regions in gp70 located approximately within the first 2000 daltons of the amino end of gp70 as well as 18,000 and 38,000 daltons from the amino end of the M r 47,000 deglycosylated gp70 molecule. In addition, we used monoclonal antibodies directed against the disulfide-linked p15(E) molecule to localize its linkage site to Domain XVII, estimated to be between 34,000 and 38,000 daltons from the amino end of deglycosylated gp70. These data place certain constraints on the tertiary structure of gp70 and suggest a mechanism for the generation of leukemogenic MCF recombinants.