Chemical determination of the m1 Moloney sarcoma virus pP60gag gene order: evidence for unique peptides in the carboxy terminus of the polyprotein.

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Tryptic peptide maps from more than 50 isolates of murine leukemia virus (MuLV) have shown that, in general, the structure of core protein p30 is highly conserved. However, a structurally variable region of p30 has been identified that is functionally associated with Fv-1 tropism. On the basis of this structural variability, MuLV strains can be classified as B-tropic, N-tropic, xenotropic, and/ MATERIALS AND METHODSThe sources of the MuLV strains utilized in this study are listed in Table 1. Viruses were concentrated and purified from tissue culture media obtained from the third and fourth subcultures by differential centrifugation and banding in 15-50% sucrose gradients, as previously described (11, 13). Virus with a buoyant density of 1.16 g/cm3 was collected and the viral proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (24). Tryptic peptide maps were derived as previously described (1, 13). RESULTSWe have made two-dimensional tryptic peptide maps of the p30 from more than 50 strains of ecotropic, amphotropic, and xenotropic MuLVs isolated from numerous strains of mice. As expected from the NH2-terminal amino acid sequence homology (25), all of the MuLV p3Os displayed a remarkable structural similarity. This indicated that these viruses are all members of a single closely related group, despite their rather wide phenotypic diversity. Fig. 1 upper left is a representative tryptic peptide map of an MuLV p30 from a BALB/c mouse. Twelve peptides have been numbered in Fig. 1 upper right to facilitate subsequent discussion. Some of the lighter spots (e.g., 1, 4, 6, and 9) always appear with a darker spot as a doublet. The spots that appear as doublets (1-2, 4-5, 6-7, 9-10, and 12) could represent the same tyrosine-containing peptide, the darker of the two containing monoiodotyrosine and the lighter one diiodotyrosine (26). As long as labeling and trypsin treatment conditions were held constant, identical peptide maps were generated from the same protein time after time. Several spots or "smudges" were seen in the right half of the MuLV p30 maps. These regions were usually not resolved as discrete spots but showed a similar pattern in most of the maps and were not considered in the comparisons described below.Fine Structural Differences in MuLV p3Os Are Excellent Markers for Specific Viral Strains. Although all MuLV p3Os obviously belong to a single structural group, there are minor differences in the peptide profiles indicative of small variances in the amino acid sequence of these molecules. These differences are reproducibly seen and are thus excellent markers for certain specific MULV strains.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Structural markers on core protein p30 of murine leukemia virus: functional correlation with Fv-1 tropism.

Gautsch¹,

Elder²,

Schindler³

et al. 1978

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“…Specifically, we have studied two variants of the defective transforming Moloney murine sarcoma virus (MuSV) called ml (1)(2)(3) and (4). Although these variants were originally derived from a tumor-inducing uncloned MuSV stock (5), they have been maintained separately for more than 10 years in different hosts and cells.…”

Section: Introductionmentioning

confidence: 99%

Cloning of integrated Moloney sarcoma proviral DNA sequences in bacteriophage lambda.

Woude

Oskarsson

Enquist

et al. 1979

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100

We have identified integrated proviral DNA sequences of m1 and HT-1 isolates of Moloney sarcoma virus (MuSV) in EcoRI digests of transformed mink cell genomic DNA and have cloned these fragments in bacteriophage lambda. Both the lambda-HT1 phage recombinant, containing a 12.3-kilobase MuSV pair (kb) fragment, and the lambda-m1 phage recombinant, containing a 7.0-kb fragment, possess full copies of the sarcoma viruses along with 5' and 3' host flanking sequences. The MuSV proviral DNA sequences, 6.7 kb for HT-1 and 5.2 kb for m1, are colinear by heteroduplex microscopy with the 1.5-kb difference in size accounted for by two approximately equal to 0.8-kb deleted regions in m1. Both integrated viral genomes are terminally redundant and have integrated at the same site in the provirus but at different sites on the host chromosome. The host sequence flanking integrated HT-1 MuSV have been identified as a single EcoRI restriction fragment of 5.6 kb in normal mink cells.

“…We (8). Tryptic peptide analysis of the mlMSV p60 indicated the presence of an as yet unidentified peptide (<20,000 Mr) at the COOH terminal of the molecule (9). Antisera to the mlMSV p60 have been raised to protein purified from the mlMSV(FeLV) pseudotype (2).…”

mentioning

confidence: 99%

Thermolabile protein kinase molecules in a temperature-sensitive murine sarcoma virus pseudotype.

Sen¹,

Todaro²,

Blair³

et al. 1979

Murine sarcoma virus-associated protein kinases that bind to actin have been purified by affinity chromatography on actin coupled to Sepharose. Heat inactivation studies showed the presence of thermolabile enzyme activity in pseudotypes containing a temperature-sensitive mutant of murine sarcoma virus (MSV) but not in two independent wildtype MSV pseudotypes. Studies with Sephadex G-75 column fractions showed that a low molecular weight form, ;t15,000, is the major thermolabile kinase in the temperature-sensitive MSV virions. Antibodies raised against the MSV-coded p60 protein, when added to the in vitro reaction mixtures, showed specific phosphorylation of the IgG heavy chain and a simultaneous reduction in the extent of phosvitin phosphorylation catalyzed by the various MSV pseudotype kinases. Thus a transforming retrovirus-coded enzyme activity that interacts directly with a major cytoskeletal protein and whose activity parallels the transforming ability of a conditional MSV mutant has now been identified. We reported that murine sarcoma virus (MSV) pseudotype virions package a low molecular weight (LMW) protein kinase activity that binds to actin. This activity was found in association with the MSV pseudotypes and was absent in nontransforming helper virus particles (1). We now report the presence of a thermolabile actin-binding protein kinase activity packaged in virion particles containing a temperature-sensitive (ts) MSV. Protein kinases, obtained by affinity chromatography on actin-Sepharose conjugate, from ts or wild-type (wt) MSV particles were compared for their thermal stabilities. The ts MSV pseudotype-associated enzyme preparation was inactivated up to -50% at 43°C with a half time of -40 min. A parallel enzyme preparation from wt MSV was not inactivated even after incubation for up to 2 hr at 43°C. Gel filtration of actin-binding kinases showed the LMW form to be the major thermolabile species (>80% inactivation in 1 hr at 43°C). Antibodies raised against the mlMSV-specified p60 protein, purified from a mlMSV-feline leukemia virus (FeLV) pseudotype (2), inhibited both the wt and the ts MSV-associated kinase.MATERIALS AND METHODS Cells and Viruses. The ts 110 MSV (3) was made into a pseudotype with Moloney murine leukemia virus (MuLV) by an acute infection (multiplicity of infection > 10) of the rat nonproducer line (clone 6M2-31) and virus was collected at 320 C. The MSVcl349-MuLV complex (4) and the m3MSV(IC-MuLV) complex (5) were obtained from producer mouse cell lines. The cultures were grown and maintained in Dulbecco's modification of Eagle's medium or minimal Eagle's medium supplemented with glutamine and 10% fetal calf serum (GIBCO). ['y-32P]ATP at ;20 Ci/mmol (New England Nuclear; 1 Ci = 3.7 X 1010 becquerels); phosvitin was added to a final concentration of 250 ,ug/ml. Assays were incubated at the indicated temperatures for 30 min. Radioactive phosphoprotein precipitated by 10% trichloroacetic acid was measured by liquid scintillation spectrometry. When necessary, reaction product...