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

I. Katoh

Y. Yoshinaka

R. B. Luftig^1,

Department of Microbiology and Immunology, Mie University School of Medicine, 174-Edobashi-2-Chome, Tsu, Mie, Japan 514
and¹ Department of Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina 29208, U.S.A.

We have utilized two-dimensional (2D) gel electrophoresis [the first dimension being a linear pH gradient (5 to 8) and the second an 8 to 15% acrylamide gradient] to characterize the virion protein, p30, from several strains of purified murine leukaemia virus (MuLV). In all cases, we found that there was a predominant (70 to 90%) Coomassie Brilliant Blue-staining p30 spot, as well as several other species which differed in pI. The major p30 spot differed in pI among different MuLV strains and the minor spots varied depending on the host cell used to grow the virus. Specifically, (i) Moloney (M)-MuLV/NIH-3T3 showed two spots, a major one at pI 6.3 and a more acidic one, (ii) AKR/NIH-3T3, AKR/mouse embryo, and Gross/NIH-3T3 showed four spots, with the two basic, minor spots of AKR/NIH-3T3 appearing relatively decreased in intensity, and (iii) Rauscher (R)-MuLV/JLS-V9 (BALB/c) showed two spots, a major one with greater than 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 our finding that another technique, chromato-focusing, gave the same results. Specifically, M-MuLV/JLS-V9 p30, when separated on chromatofocusing columns under non-denaturing conditions yielded three peaks, each of which directly corresponded to the three spots (pI:6.1, 6.3, 6.6) observed on 2D gels. Furthermore, 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 five clearly defined differences. These results indicate (i) that the p30s of several N- and NB-tropic viruses are heterogeneous in pI, and (ii) for one 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 p30s in each virus particle and/or a heterogeneity in the virus population.

Keywords: MuLV, p30, 2D gel electrophoresis

Present address: Basic Research Program, NCI, Frederick Cancer Research Facility, Frederick, Maryland 21701, U.S.A.

Present address: Department of Microbiology and Immunology, LSU Medical Center, 1901 Perdido Street, New Orleans, Louisiana 70112-1393, U.S.A.

Received 5 August 1983; accepted 17 January 1984.