Further characterization of the in vitro products generated by proteolytic cleavage of Gazdar murine sarcoma virus p65gag

Abstract

In vitro proteolytic cleavage of the Gazdar murine sarcoma virus (Gz-MuSV) p65gag polypeptide (Gz-p65gag) was facilitated by detergent-disrupted Moloney murine leukaemia virus (Mo-MuLV). Incubation of radioactively labelled Gz-p65gag in the presence of unlabelled Mo-MuLV under optimal conditions resulted in the cleavage of Gz-p65gag to proteins of 40000 (P40) and 25000 (P25) Mr. P40 and P25 appeared to be similar in both mobility and antigenicity to Mo-MuLV intermediates, Pr40gag and Pr25gag, previously found in infected cells. Additional proteins of 30000 (Gz-p30), 15000 (Gz-p12), 12000 (Gz-p15) and 10000 (Gzp10) Mr were also generated upon cleavage of Gz-p65gag and contained antigenic determinants of Mo-MuLV structural proteins p30, pp12, p15 and p10, respectively. Both detergent-disrupted Mo-MuLV and Rauscher murine leukaemia virus produced similar cleavage profiles. Trypsin and detergent-disrupted mouse mammary tumour virus generated cleavage patterns very different from that produced by Mo-MuLV. Both visual and quantitative time studies of the reaction indicated that P40 gave rise to Gz-p30 and Gz-p10. Tryptic peptide mapping of Gz-p65gag and its cleavage products supported the results obtained from both immunoprecipitation studies with anti-gag sera and the kinetics of cleavage of Gz-p65gag. Both Mo-MuLV Pr65gag and Gz-p65gag were found to be very similar in primary sequence as judged by peptide mapping. P40 produced tryptic peptides that co-migrated with Mo-MuLV p30 peptides; P25 contained tryptic peptides that were also found in Mo-MuLV p15. Gz-p30 and Gz-p15 contained the tryptic peptides of Mo-MuLV p30 and p15, respectively, that were found in P40 and P25. The Gz-p10 fraction contained a tryptic peptide that was also found in P40, but not p30. These results provide good evidence that the protease packaged within Mo-MuLV can cleave, in vitro, the gag-related polyprotein of Gz-MuSV in a manner very similar to the processing of Mo-MuLV Pr65gag in infected cell culture systems.