Murine Leukaemia Virus p30 Heterogeneity as Revealed by Two-dimensional Gel Electrophoresis Is Not an Artefact of the Technique

Abstract

Two-dimensional (2D) gel electrophoresis [the 1st dimension being a linear pH gradient (5-8) and the 2nd an 8-15% acrylamide gradient] was used to characterize the virion protein, p30, from several strains of purified murine leukemia virus (MuLV). In all cases, there was a predominant (70-90%) Coomassie Brilliant Blue-staining p30 spot, as well as several other species which differed in pI [isoelectric point]. The major p30 spot differed in pI among different MuLV strains and the minor spots varied depending the host cell used to grow the virus. Specifically, Moloney (M)-MuLV/NIH-3T3 showed 2 spots, a major one at pI 6.3 and a more acidic one; AKR/NIH-3T3, AKR/mouse embryo and Gross/NIH-3T3 showed 4 spots, with the 2 basic, minor spots of AKR/NIH-3T3 appearing relatively decreased in intensity; and Rauscher (R)-MuLV/JLS-V9 (BALB/c) showed 2 spots, a major one with > 90% of the estimated Coomassie Brilliant Blue stain at a pI of 6.5 and a minor, acidic one. The major spots of AKR and M-MuLV viruses also differed in pI. The major spot of the AKR and Gross N-tropic viruses had a pI of 6.7 while that of NB-tropic virus M-MuLV had a pI of 6.3. The possibility that the heterogeneity observed in p30 was an artefact of the 2D gel technique had to be considered since urea was used to denature proteins in the first dimension of the gel. This possibility was made unlikely by the finding that another technique, chromatofocusing, gave the same results. Specifically, M-MuLV/JLS-V9p30, when separated on chromatofocusing columns under non-denaturing conditions yielded 3 peaks, each of which directly corresponded to the 3 spots (pI: 6.1, 6.3, 6.6) observed on 2D gels. Tryptic peptide maps of the major (pI 6.3) and one of the minor (pI 6.6) M-MuLV spots, although very similar in peptide composition, showed about 5 clearly defined differences. Evidently, the p30 of several N- and NB-tropic viruses are heterogeneous in pI and for 1 particular MuLV, the p30 heterogeneity can be explained by a difference in amino acid composition. These findings of p30 charge heterogeneity may reflect either the presence of several different p30 in each virus particle and/or a heterogeneity in the virus population.