Murine leukaemia virus p30 heterogeneity as revealed by two-dimensional gel electrophoresis is not an artefact of the technique.

We have utilized two-dimensional (2D) gel electrophoresis [the first dimension being a linear pH gradient (5 to 8) and the second and 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, chromatofocusing, 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.

Comparison of structural domains of gp70s of ecotropic Akv and dualtropic MCF-247 MuLVs.

Controlled proteolysis of MuLV gp70s results in the generation of several fragments which correspond to distinct structural domains of the molecules. The orientation of these regions in gp70 was determined by analysis of the immunoreactivities of proteolytic products generated from the MuLV PrENV polyprotein toward monoclonal alpha p15(E) and alpha gp70 antibodies, and by fragmentation analysis of gp70s specifically labeled with [35S]cysteine and [35S]methionine. These studies confirmed our previous assignment of a p15(E)-disulfide-linked 33K fragment to the carboxy terminus of Akv gp70 (Pinter, Honnen, Tung, O'Donnell, and Hammerling, Virology 116, 345-351, 1982). Using similar fragmentation procedures, the sizes and structural features of gp70 domains of Akv and MCF 247 MuLV gp70s were compared. Trypsinization of MCF-247 gp70 resulted in the production of a carboxy terminal fragment which resembled that of the ecotropic gp70 in that (1) it was disulfide linked to p15(E) but not to the amino terminal fragments, (2) reacted with monoclonal antibody 35/56, (3) contained cysteines but no methionines, and (4) carried only endo H-resistant oligosaccharide chains. Amino terminal MCF gp70 fragments were obtained with apparent molecular weights of 42K and 30K, considerably smaller than the corresponding Akv fragments of 49K and 35K. These MCF fragments were much more stable to degradation by trypsin than the Akv amino terminal components, indicating the loss or inaccessibility of several trypsin sites in the MCF amino terminal domain. These results demonstrated the Akv and MCF 247 gp70s contained highly conserved carboxy terminal domains but unique amino terminal sequences. Common features for both gp70s were the presence of an endo H-sensitive oligosaccharide chain near the amino terminus, and the presence of internal disulfide bonds in the amino terminal domains which resulted in an increased mobility for these fragments when analyzed under nonreducing conditions. Thus, while the amino terminal domains of the two gp70s are structurally different, certain aspects of glycosylation specificity and secondary conformation are conserved, suggesting that these structural features may be important for common biological properties of these molecules.

Unusual properties of AKR MCF-247 murine leukemia virus unintegrated proviral DNA.

Analyses of Hirt extracts from endogeneous murine leukemia virus (MuLV)-infected cells revealed the presence of 9-kbp linear DNA and two superhelical forms with one or two tandem copies of the long terminal repeat (LTR). In contrast, cells that were infected with AKR MCF247 MuLV yielded two major linear forms of 9.0 and 8.4 kbp and one discrete superhelical DNA. In addition, there was a heterogeneous population of superhelical DNAs that were larger and smaller than the major superhelical DNA species. Restriction endonuclease treatment of purified linear and superhelical DNAs have revealed that MCF247 MuLV unintegrated viral DNA is very heterogeneous. Evidence is presented that there are at least two linear DNAs; one is 9-kbp full-length linear DNA, whereas the other major form contains a 0.6 to 0.7-kbp deletion in the envelope gene adjacent to the right LTR. In addition, there are two size classes of the LTR in at least the full-length linear DNA. The major superhelical DNA species is a 8.4-kbp form which contains one copy of the LTR. Other heterogeneous superhelical DNAs appear to contain env-gene deletions or partially deleted copies of a tandem LTR region.

Identification of ecotropic proviral sequences in high- and low-ecotropic-virus-producing mouse strains.

The arrangement of endogenous ecotropic retroviruses in selected high- and low-ecotropic-virus-producing mouse strains was examined by Southern blot hybridization analysis, using an ecotropic retrovirus-specific DNA probe. High-ecotropic-virus-producing mouse strains of the AKR family displayed heterogeneity with respect to the number of copies and the sites of insertion of endogenous ecotropic specific DNA. This diversity was seen even among individuals of the same AKR subline. Contrastingly, individuals within the same low-ecotropic-retrovirus-producing mouse strain showed no evidence of variability in their endogenous ecotropic proviral sequences. These results favored the hypothesis that germ line proviral reinsertion was responsible for the proviral sequence heterogeneity observed in high-ecotropic-virus-producing mouse strains.

Env gene products of AKR dual-tropic viruses: examination of peptide maps and cell surface expression.

The env gene products of nine AKR dual-tropic murine leukemia viruses were compared by peptide mapping and were assayed for expression on the cell surface of infected fibroblasts. Seven virus isolates expressed the env gene polyprotein on the cell surface. The env gene products of six of the seven viruses had identical peptide maps. The analysis of structure and expression of env gene products carried out in this study characterizes a subset of dual-tropic murine leukemia viruses shown by others to be thymotropic.

Glycoproteins of murine leukemia viruses. III. Glycosylation of env precursor glycoproteins.

We have compared the glycopeptides obtained after extensive pronase digestion of the env precursors (PrENV proteins) of ecotropic, xenotropic, and dual-tropic murine leukemia viruses. Two glycopeptide size classes, having molecular weights of approximately 2,200 and 1,500, were shown to be associated with the PrENV proteins of all murine leukemia viruses studied. Glycopeptides associated with the env precursors were totally susceptible to endo-beta-N-acetyglucosaminidase H. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partial endo-beta-N-acetylglucosaminidase H digestion products of the env precursor of dual-tropic mink cell focus-forming virus (MCF 247) revealed the presence of seven bands, suggesting that six glycosylation sites were present on the precursor molecule. The MCF 247 PrENV protein had been previously shown to be accessible to lactoperoxidase-catalyzed radioiodination on the surface of infected cells. The cell surface PrENV molecules had the same electrophoretic mobility as pulse-labeled PrENV protein, and after endo-beta-N-acetylglucosaminidase H treatment a similar shift in electrophoretic mobility was observed for the cell surface PrENV protein and the pulse-labeled precursors, a finding which indicated that the PrENV protein located on the cell surface also possessed only mannose-rich oligosaccharides. These results indicated that the env precursor glycoproteins of dual-tropic viruses had the unusual property of migrating to the cell surface without undergoing the normal oligosaccharide processing and proteolytic cleavage events that had been observed for ecotropic and xenotropic murine leukemia virus glycoproteins.

Primary structure of the low molecular weight nucleic acid-binding proteins of murine leukemia viruses.

Murine leukemia viruses contain a low molecular weight basic protein, designated p10, which binds to single-stranded nucleic acids. The complete amino acid sequence of p10 from the Rauscher strain of virus has been determined. The partial amino acid sequences of p10s from Moloney, Friend, AKR, Gross, radiation leukemia, and BALB/2 viral strains have also been determined using microsequencing techniques. Rauscher p10 is composed of 56 amino acid residues; the other p10s are similar in size but differ from Rauscher by a few conservative amino acid substitutions. The structure of Rauscher p10 was compared to the structure of a functionally homologous protein from Rous avian sarcoma virus. The comparison revealed regions of amino acid sequence homologies which indicate a phylogenetic relationship between the murine and avian viral strains. The analyses revealed a periodic placement of three Cys residues and a Gly-His sequence. A structure involving these residues is found once in the murine protein and twice in the avian protein. A similar structure is seen in the single stranded nucleic acid binding protein of bacteriophage T4. However, in the latter case, the order of amino acid residues is inverted.

Close similarity between endogenous ecotropic virus of Mus musculus molossinus and AKR virus.

By using seven different restriction endonucleases, the cleavage patterns of the unintegrated provioral DNA from an ecotropic murine leukemia virus isolated from Mus musculus molossinus were found to be identical to those of AKR virus. An AKR [3H]DNA probe can be completely saturated with M. musculus molossinus and M. musculus castaneus DNAs, although the arrangement of viral sequences in M. musculus molossinus DNA differed from that of AKR virus. These studies indicate that an AKR-type ecotropic virus is present in some wild Asiatic mice.

Analysis of the genome of an endogenous, ecotropic retrovirus of the AKR strain of mice: micromethod for detailed characterization of high-molecular-weight RNA.

A detailed characterization of the genome of an endogenous, ecotropic type C virus, the Akv virus, is presented. Approximately 100 RNase T1-resistant oligonucleotides characteristic of the Akv genome were identified by two-dimensional gel electrophoresis, and the complete nucleotide sequence is presented for 75 of these oligonucleotides. A correspondence between the sequence of some of these oligonucleotides and the amino acid sequence of some virus-coded gag gene proteins is reported. For this study we developed methods suitable for the analysis of high-molecular-weight RNA species in nanogram quantities. The in vitro labeling procedures used here led to uniform labeling of the unique oligonucleotides.

Glycopeptides of murine leukemia viruses. I. Comparison of two ecotropic viruses.

The glycopeptides obtained by pronase digestion of two ecotropic strains of murine leukemia virus (MuLV) were compared by gel filtration. Four different glycopeptide size classes, designated G(1), G(2), G(3), and G(4), with molecular weights of approximately 5,100, 2,900, 2,200, and 1,500, respectively, were shown to be associated with Rauscher MuLV virions grown in JLS-V9 cells. Various sugar precursors, including glucosamine, galactose, fucose, and mannose were incorporated into G(1) and G(2), suggesting that these are complex (type I) glycopeptides. The two smaller glycopeptide size classes, G(3) and G(4), were shown to be mannoserich (type II) glycopeptides. G(4) was more sensitive to digestion with endo-beta-N-acetylglucosaminidase H than G(3), suggesting that the core of G(3) may contain fewer mannose residues. Glycopeptides of the same size class as G(1) and G(2) were associated with both Rauscher MuLV and AKR-MuLV grown in III6A (mouse embryo) cells. Previous studies have shown that gp52, a proteolytic cleavage product of gp70, possessed primarily G(1) glycopeptides and that gp52 was more highly sulfated than gp70. We observed that G(1) is approximately twofold more highly sulfated than G(2), explaining the observed difference in sulfation of gp52. The unusually large size of G(1) suggested that infection with MuLV may alter the host cell glycosylation pattern. To test this possibility, glycopeptides from Sindbis virions grown in uninfected and Rauscher MuLV-infected JLS-V9 cells were compared, and no differences were observed. G(1) was not detected in Sindbis virions, indicating that acquisition of G(1) depends on properties of the virus-coded polypeptide backbone of the gp70 molecule.

Analysis of proteins of mouse sarcoma pseudotype viruses: type-specific radioimmunoassay for ecotropic virus p30's.

Murine sarcoma virus pseudotypes were prepared by infection of nonproducer cells (A1-2), which were transformed by the Gazdar strain of mouse sarcoma virus, with Gross (N-tropic), WN1802B (B-tropic), or Moloney (NB-tropic) viruses. The respective host range pseudotype sarcoma viruses were defined by the titration characteristics on cells with the appropriate Fv-1 genotype. Proteins from virus progeny were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bands present in both the 65,000- and the 10,000- to 20,000- molecular-weight regions of the gel distinguished the pseudotype viruses from their respective helpers. Furthermore, two protein bands were noted in the p30 region of murine sarcoma virus (Gross), one corresponding to Gross virus p30, and another of slightly slower mobility. However, since the mobility of the putative sarcoma p30 is nearly indentical to that of WN1802B, its presence could not be established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Type-specific radioimmunoassays for Gross virus p30 and for WN1802B p30 were applied for analysis of pseudotype preparations, and among several ecotropic viruses tested, only the homologous virus scored in the respective assay. By use of these assays, pseudotype viruses were found to contain only 8 to 48% helper-specific p30's; the remainder is presumably derived from the sarcoma virus.

Heteroduplex analysis of the sequence relations between the RNAs of mink cell focus-inducing and murine leukemia viruses.

The sequence relationships betwen AKR ecotropic virus and an AKR-derived "mink cell focus-inducing" (MCF) isolate (AKR MCF 247), between Moloney murine leukemia virus (M-MLV) and an M-MLV MCF isolate (M-MLV83), and between AKR and M-MLV were studied by electron microscopic heteroduplex analysis. The MCF-specific sequences were found to map from 1.95 kilobases (kb) to 2.75 kb (+/- 0.15 kb) from the 3' end of the RNAs for both MCF isolates. The major sequence nonhomology regions between AKR and M-MLV lie between 0.9 and 3.5 kb from the 3' end. However, the AKR and M-MLV sequences immediately adjacent to the 1.95- and 2.75-kb junctions with MCF-specific sequences are relatively similar in AKR and M-MLV. Our results suggest that the env gene of MLVs maps from 1 kb to 3 kb from the 3' end of the genomic RNA and that the carboxyl end of the glycoprotein of each MCF strain is similar (or identical) to that of its ecotropic parent.

High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures.

Several 50 to 70S tumor viral RNAs have previously been shown by electron microscopy to be dimers, with the two monomer subunits joined near their 5' ends. Five additional naturally occurring type C RNA tumor viruses have now been examined: AKR, and endogenous murine ecotropic virus; NZB, an endogenous murine xenotropic virus; and ecotropic and an amphotropic virus isolated from a wild mouse; and the avian reticuloendotheliosis virus (REV). All five 50 to 70S RNAs have similar 5'-to-5' dimer structures. Therefore, the observations support the hypothesis that the dimer linkage is a structural feature common to all type C mammalian viruses. REV is the first example of an avian virus with a clear 5'-to 5' dimer linkage. All of the mammalian viral RNAs, but not REV, showed symmetrically placed loops in each subunit of the dimer. Possible molecular structures and biological functions of the dimer linkages and loops are discussed.

Retrovirus purification: method that conserves envelope glycoprotein and maximizes infectivity.

A Sepharose 4B chromatographic method for purification of retroviruses is described which was less time consuming, increased purified virus yields, conserved viral glycoprotein, and increased recovery of biological infectivity in comparison with conventional sucrose gradient ultracentrifugation techniques.

Viral proteins expressed on the surface of murine leukemia cells.

Leukemic cells of AKR mice contain as constituents of their membranes the murine leukemia virus envelope protein gp70 and the precursor polyprotein of the viral internal (core) structural proteins. Both gp70 and the core polyprotein are represented on the cell surface as glycoproteins, as evidenced by incorporation of [3H]glucosamine into their structure and the binding of these proteins to lectins. The glycosylated core polyprotein exists in at least two serologically distinguishable forms: the 95,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30, p12, and p10, whereas the 85,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30 and p12, but not p10. Additional heterogeneity in these cell surface polyproteins has been observed wtih leukemias induced by exogenous leukemia viruses. Spontaneous leukemia cells of AKR mice invariably express gp70 and the core polyprotein on their cell surface; normal thymocytes of young AKR mice express gp70, but not the core polyprotein on their surface.