Molecular dissection of Rauscher virus gp70 by using monoclonal antibodies: localization of acquired sequences of related envelope gene recombinants.

Abstract

Using hybridoma-specific immune precipitations of fragments derived from Rauscher virus [glycoprotein] gp70, coupled with peptide patterns (fingerprinting) and partial amino acid sequence analyses, a linear map of Rauscher gp70 was generated. A panel of 56 hybridomas derived from the fusion of the drug-selected SP-2 myeloma line with spleen cells from either a 129 GIX+ or a GIX- mouse immunized with purified Rauscher virus gp70. The results showed that by natural breakdown, gp70 splits into predominant fragments with MW 45,000 (P45), 34,000 (P34) and 32,000 (P32). Peptide fingerprinting of these as well as overlapping fragments coupled with partial amino acid sequence analyses allowed us to align the fragments into the linear arrangement NH2-P45-P32-COOH, with P34 being an NH2-terminal degradation product of P45. Of the 56 hybridomas, 20 immunoprecipitated both P45 and P34; 18 immunoprecipitated only P45; and 18 immunoprecipitated only P32. The hybridomas thus define 3 domains of the molecule as NH2-P45/P34, P45 only, and P32-COOH. Allowing these hybridomas to react with 2 Rauscher-derived envelope gene recombinant viruses yielded the following results: all 20 P45/34 reactors bound to the 2 Rauscher recombinants; of 18 P45-only hybridomas, 10 reacted; and only 1 of 18 P32 reactors bound to the Rauscher recombinants. This last hybridoma reacted with various murine retroviruses, indicating that it was directed at conserved determinants of gp70. Peptide fingerprinting of R-gp70, the recombinant gp70, and their respective breakdown products confirmed the homologies and nonhomologies defined by the hybridomas. Peptide patterns showed that these acquired sequences on the COOH-terminal portion of the recombinant gp70 are related to xenotropic virus gp70.