Differences in the pI heterogeneity of virion and intracellular Moloney murine leukaemia virus p30s.
At least three different p30 forms which vary in isoelectric point (pI) were previously shown by two-dimensional (2D) gel electrophoresis to be present in purified virions obtained from several strains of murine leukaemia virus (MLV). This heterogeneity which had been identified by Coomassie Brilliant Blue staining has been further characterized by immunological techniques. Using as substrates two Moloney (M) MLV chronically infected cell lines (MJD-54 and clone 2 cells), we found that (i) all p30s had the antigenicity of M-MLV p30, when analysed by immunoblotting of virion proteins with anti-p30 sera, and (ii) when cells were labelled with [35S]methionine, a 14C-amino acid mixture, or [14C]serine and lysates of purified virions were immunoprecipitated with goat anti-p30 sera, four p30 spots (pI 6.0, 6.1, 6.3 and 6.6) could be clearly identified. These results strongly support the viral origin of the heterogeneous p30 spots. We next examined infected cell lysates in an attempt to pinpoint the molecular basis of this heterogeneity. When we immunoprecipitated p30s from labelled cell lysates utilizing goat anti-p30 sera it was observed that in contrast to the four virion p30s, there were only three intracellular p30s (pI 6.0, 6.3 and 6.6), there was a threefold greater amount of the intracellular compared to the virion form of p30 with pI 6.0, tryptic peptide maps of both virion and intracellular p30s labelled either with [35S]methionine or 125I showed basically similar patterns with only slight differences in intensity among certain peptides for the p30s with pI 6.1, 6.3 and 6.6, and the intracellular p30 with pI 6.0 had a peptide that was not present in any of the other p30s. These results suggest that due to some as yet uncharacterized modification(s) of p30 and/or some structural differences between different p30s, a heterogeneity in pI exists. This may be important for assembly of the virion capsid. However, it is also possible that the p30 heterogeneity reflects the presence of multiple M-MLV proviruses within each of the infected cell clones.
D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D002460:Cell Line; D015683:Gene Products, gag; D007424:Intracellular Fluid; D007526:Isoelectric Point; D008979:Moloney murine leukemia virus / Q000032:analysis* / Q000276:immunology; D010449:Peptide Mapping; D012191:Retroviridae Proteins / Q000276:immunology / Q000302:isolation & purification*; D014771:Virion / Q000032:analysis*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D015683:Gene Products, gag; D012191:Retroviridae Proteins