Secretion, glycosylation, and phosphorylation of rat-specific, transformation-associated proteins in Moloney murine sarcoma virus-transformed rat cells.

Previously, we detected, by monoclonal antibody, three Moloney murine sarcoma virus (Mo-MSV)-activated intracellular transformation-associated proteins (TAP), (P66, P63, and P60) in several Mo-MSV-transformed rat cell lines (Chan et al., Biochem. Biophys. Res. Commun., 134: 1223-1230, 1986) and found the release of TAP into the extracellular medium with a change from three intracellular polypeptides to two extracellular polypeptides (P68 and P64) (Li et al., Virology, 156: 91-100, 1987). Since then, we have further analyzed TAP in terms of their secretion, glycosylation, and phosphorylation in a temperature-sensitive Mo-MSV-transformed normal rat kidney (NRK) cell line, the 6M2 line, and found these results. Extracellular TAP were detected by immunoblotting and immunoprecipitation techniques as two polypeptides (P68 and P64). The secretion of TAP was rapid, with a 50% secretion rate of 78 min. Both intracellular (except for P63) and extracellular TAP were glycosylated. As a result of inhibition of glycosylation by the antibiotic tunicamycin, a fourth intracellular TAP (P58) and a third extracellular TAP (P60) were found. The new results suggest that the intracellular TAP were probably changed from four polypeptides (P66, P63, P60, and P58) to three (P66, P63, and P60) during the glycosylation process. Likewise, the three extracellular TAP were changed to two (P68 and P64) as a result of further glycosylation and subsequent secretion into the extracellular medium. Inhibition of glycosylation by tunicamycin (0.5 microgram/ml) reduced the TAP secretion rate by about 39%. Extracellular TAP (P68 and P64) as well as P85gag-mos and P58gag were found to be phosphoproteins.

Detection of the myristylated gag-raf transforming protein with raf-specific antipeptide sera.

The post-translational modifications of the gag-raf fusion proteins of the 3611 murine sarcoma virus (MSV) have been examined by inhibiting glycosylation with tunicamycin and by in vivo labeling with [3H]myristic acid. The results show that P75gag-raf is myristylated but not glycosylated and that P90gag-raf is glycosylated but not myristylated (and is now termed gP90gag-raf). gP90gag-raf expression appeared to become lost during passage of the transformed cells, and consequently does not appear to be necessary for the maintenance of transformation. raf-specific sera for detecting gag-raf fusion proteins have been obtained from synthetic peptides made from different regions of the predicted v-raf sequence. Immunoprecipitation of P75gag-raf with raf-specific sera directly confirmed the deduced v-raf sequence. The fact that P75gag-raf is both myristylated and precipitated by antiserum to a predicted carboxyl-terminal peptide of the v-raf gene established that the mature protein represents the entire coding region. The gP90gag-raf thus appears to be a glycosylated form of P75gag-raf specified by the gag sequences of the fusion protein, in analogy with Pr65gag and gPr80gag of murine leukemia viruses. Antiserum to the carboxyl-terminal P75gag-raf peptide was the most efficient in immunoprecipitation, and will be useful for detecting the product of the c-raf gene.

Recognition of mos-related proteins with an antiserum to a peptide of the v-mos gene product.

A mos-specific antiserum was generated by injection of rabbits with a peptide predicted from the sequence of the v-mos gene of Moloney murine sarcoma virus (MuSV) strain 124. The peptide is composed of amino acids 37-55 (cyclized at the cysteine residues) conjugated to keyhole limpet haemocyanin. This serum [anti-mos(37-55c)] specifically recognized p37mos in MuSV-124 acutely infected NIH-3T3 cells, P85gag-mos in 6m2 cells, an NRK clone infected with the temperature-sensitive mutant (ts110) of Moloney MuSV, and P100gag-mos in 54-5A4 cells, an NRK clone infected with a spontaneous revertant of ts110. An additional protein of Mr 55000 from uninfected cells was recognized by this serum. Reactivity of the serum toward the v-mos-containing proteins and the 55K protein was completely inhibited by prior incubation with free peptide. The 55K protein was not recognized by antisera made from synthetic peptides prepared from the C-terminal eight or 12 amino acids of v-mos.

In vitro cleavage of Pr65gag by the Moloney murine leukaemia virus proteolytic activity yields p30 whose NH2-terminal sequence is identical to virion p30.

In vitro cleavage of Gazdar murine sarcoma virus Pr65gag, which has all of the antigenic determinants of Moloney murine leukaemia virus Pr65gag, i.e. p15, p12, p30 and p10, by the Moloney murine leukaemia virus proteolytic activity yielded a p30 whose partial NH2-terminal sequence was identical to Moloney murine leukaemia virus. Both [3H]leucine-labelled and unlabelled Pr65gag were used to generate the cleaved p30.

Further characterization of the in vitro products generated by proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

In vitro proteolytic cleavage of the Gazdar murine sarcoma virus (Gz-MuSV) p65gag polypeptide (Gz-p65gag) was facilitated by detergent-disrupted Moloney murine leukaemia virus (Mo-MuLV). Incubation of radioactively labelled Gz-p65gag in the presence of unlabelled Mo-MuLV under optimal conditions resulted in the cleavage of Gz-p65gag to proteins of 40000 (P40) and 25000 (P25) Mr. P40 and P25 appeared to be similar in both mobility and antigenicity to Mo-MuLV intermediates, Pr40gag and Pr25gag, previously found in infected cells. Additional proteins of 30000 (Gz-p30), 15000 (Gz-p12), 12000 (Gz-p15) and 10000 (Gzp10) Mr were also generated upon cleavage of Gz-p65gag and contained antigenic determinants of Mo-MuLV structural proteins p30, pp12, p15 and p10, respectively. Both detergent-disrupted Mo-MuLV and Rauscher murine leukaemia virus produced similar cleavage profiles. Trypsin and detergent-disrupted mouse mammary tumour virus generated cleavage patterns very different from that produced by Mo-MuLV. Both visual and quantitative time studies of the reaction indicated that P40 gave rise to Gz-p30 and Gz-p10. Tryptic peptide mapping of Gz-p65gag and its cleavage products supported the results obtained from both immunoprecipitation studies with anti-gag sera and the kinetics of cleavage of Gz-p65gag. Both Mo-MuLV Pr65gag and Gz-p65gag were found to be very similar in primary sequence as judged by peptide mapping. P40 produced tryptic peptides that co-migrated with Mo-MuLV p30 peptides; P25 contained tryptic peptides that were also found in Mo-MuLV p15. Gz-p30 and Gz-p15 contained the tryptic peptides of Mo-MuLV p30 and p15, respectively, that were found in P40 and P25. The Gz-p10 fraction contained a tryptic peptide that was also found in P40, but not p30. These results provide good evidence that the protease packaged within Mo-MuLV can cleave, in vitro, the gag-related polyprotein of Gz-MuSV in a manner very similar to the processing of Mo-MuLV Pr65gag in infected cell culture systems.

Murine retrovirus Pr65gag forms a 130K dimer in the absence of disulfide reducing agents.

Gazdar-murine sarcoma virus (Gz-MSV) particles, obtained from tissue culture fluids of chronically infected HTG-2 hamster cells are immature in morphology and contain uncleaved Pr65gag as the predominant protein (greater than 95% Coomassie blue stain) (A. Pinter and E. deHarven, 1979, Virology 99, 103-110; Y. Yoshinaka and R. B. Luftig, 1982, Virology 118, 380-388). When Gz-MSV particles are disrupted in 1% sodium dodecyl sulfate (SDS) and then analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) in the absence of reducing agents, such as beta-mercaptoethanol (beta-MSH) almost half of the Pr65gag Coomassie blue-stained band is detected as a band at a Mr of 130K. Electrophoretic blotting studies with monospecific antisera against MuLV p30, p15, p12, and p10 showed that the 130K band cross-reacted with all four antigens suggesting that it was a dimer of Pr65gag. Two-dimensional (2D) SDS-PAGE where the first dimension was run under nonreducing conditions and the second with beta-MSH, supported the contention that the 130K band was a dimeric complex of Pr65gag. One also saw minor amounts of a 260K and higher polymeric forms of Pr65gag on the SDS gels, suggesting that polymeric forms may exist as well. When 32P-labeled Gz-MSV particles obtained by in vivo labeling of infected HTG-2 cells with [32P]PPi were electrophoresed on SDS-PAGE, only 10% of the 32P label was detected at the 130K position. In contrast, 30% of the Coomassie blue-stained Pr65gag material was found at 130K on the 2D gels. This suggests that unphosphorylated Pr65gag is more likely to participate in dimer formation than phosphorylated Pr65gag. Pr65gag of Moloney murine leukemia virus (M-MuLV), which is present as a minor (5% of stain) protein band on SDS-PAGE also showed 130K dimers. Further, in beta-MSH-deficient SDS preparations of Gz-MSV, electrophoresed after trypsin treatment, a 32K band that stained with p15, but not p10, p12, nor p30, antisera was observed. If beta-MSH was added, this band was no longer present. Thus Pr65gag dimerization in immature MuLV particles appears to at least involve the p15 region of the polyprotein. Since p15 is an extremely hydrophobic protein, formation of Pr65gag dimers may occur when virion precursor proteins are brought to the cell membrane during virus assembly.

Characterization of the FBR-murine osteosarcoma virus complex: FBR-MuSV encodes a FOS-derived oncogene.

The FBR murine osteosarcoma virus complex, isolated from a radiation-induced osteosarcoma of an X/Gf mouse causes the rapid appearance of osteosarcomas in newborn mice and transforms fibroblasts in vitro. The two components of the FBR-viral complex have been isolated separately in tissue culture: FBR-MuLV by end-point dilution and FBR-MuSV by the establishment of mouse [FBR-NP 117 (NIH 3T3)] and rat non-producer cell lines [FBR-NP415 (REF)]. The host range and RNase Tl fingerprint analysis of FBR-MuLV demonstrated a pattern closely related to, but distinguishable from, Akv-MuLV. Transformed cells from both mice and rats contain a rescuable FBR-MuSV genome. These pseudotypes produce foci in tissue culture and induce osteosarcomas in susceptible mouse strains. An FBR-MuSV (FBR-MuLV) cDNA probe detects a 5.2 kb HindIII and a 9.5 kb EcoRI FBR-MuSV-specific fragment in FBR-MuSV-transformed non-producer rat cells. The same fragments hybridized with a fos specific probe, demonstrating that FBR-provirus contains a c-fos-derived onc-gene.

Detection of an 85,000-dalton phosphoprotein in ts110 murine sarcoma virus-infected cells with antiserum against a v-mos peptide.

We have used an antiserum directed against a synthetic v-mos peptide (anti-C3 serum) to screen ts110 murine sarcoma virus (MuSV)-infected cells for the presence of v-mos-encoded proteins. Anti-C3 serum specifically recognized an 85,000-dalton protein doublet (P85) from [35S]methionine-labeled ts110 MuSV-infected producer cells grown at 32 degrees C, the permissive temperature for transformation. The P85 doublet was also recognized by an antiserum directed against the viral gag protein p15. P85 was present but at 2- to 10-fold-lower levels in ts110 MuSV-infected producer cells grown at 39 degrees C, the restrictive temperature for transformation. The P85gag-mos fusion product was the only v-mos protein reproducibly detected in this ts110 MuSV-transformed cell line. Immunoprecipitation of 32P-labeled cells with anti-C3 serum revealed that the upper band of the P85 doublet is phosphorylated, containing mostly phosphoserine and some phosphothreonine. Cells acutely infected with ts110 MuSV contained slightly higher levels of P85 than did the ts110 MuSV-infected producer cell line. Anti-C3 serum specifically recognized a 33,000-dalton protein (p33) in the acutely infected cells labeled with [35S]methionine. p33 was present in trace amounts and may represent a previously unidentified ts110 MuSV-encoded v-mos protein.

Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family.

We have isolated eight rat lymphocyte-myeloma hybrid cell lines producing monoclonal antibodies that react with the 21,000-dalton transforming protein (p21) encoded by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV). These antibodies specifically immunoprecipitate both phosphorylated and non-phosphorylated forms of p21 from lysates of cells transformed by Ha-MuSV. All eight react with the products of closely related ras genes expressed in cells transformed by two additional sarcoma viruses (rat sarcoma virus and BALB sarcoma virus) or by a cellular Harvey-ras gene placed under the control of a viral promoter. Three of the antibodies also react strongly with the p21 encoded by the v-ras gene of Kirsten MuSV. These same three antibodies immunoprecipitate the predominant p21 species synthesized normally in a variety of rodent cell lines, including the p21 produced at high levels in 416B murine hemopoietic cells. This suggests that an endogenous gene closely related to Kirsten-ras is expressed in these cells. The monoclonal antibodies have been used to confirm two properties associated with p21; localization at the inner surface of the membrane of Ha-MuSV-transformed cells, assayed by immunofluorescence microscopy, and binding of guanine nucleotides.

In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution containing dithiothreitol in addition to 0.1% Nonidet P-40 allowed the efficient processing of P40 to p30 and a band migrating with p10. Immunoprecipitation with monospecific sera indicated that P40 contained p30 and p10, whereas P25 contained p15 and pp12 determinants. P40 and P25 are similar in size and antigenic properties to Pr40gag and Pr25gag observed in infected cells (Naso et al, J. Virol. 32:187-198, 1979).

Biological and molecular biological characterization of the virus progeny from transformed clones MuSV-124 and MuSV-349: evidence for MuLV-specific nucleotide sequences in the MoMuSV size class of RNA from MoMuSV-124.

The genetic information of MoMuSV-349 and MoMuSV-124, two clones of productively transformed TB cells, was distributed between two size classes of RNA (mol. wt. 2.9 x 10(6)) in the proportions of 5:1. Some preparations of MoMuSV-124 lacked the large RNA. The virions produced by both clones also contained all the nucleotide sequences of Moloney leukaemia virus and the ratio of MuSV:MuLV produced by the two clones differed markedly. The distribution of the sequences specific for Moloney murine leukaemia virus (MoMuLV) between the two size classes of RNA was studied using molecular hybridization to DNA probes complementary to and representative of: (i) the Moloney murine sarcoma virus (MoMuSV) RNA genome (mol. wt. 1.9 x 10(6)); (ii) those nucleotide sequences shared by MoMuSV and MoMuLV; (iii) nucleotide sequences specific for MoMuSV; (iv) nucleotide sequences specific for MoMuLV. The only detectable Moloney leukaemia virus-specific nucleotide sequences present in MoMuSV-124 virions were in the RNA of mol. wt. 1.9 x 10(6), whereas these sequences were detected in the RNA of mol. wt. 2.9 x 10(6) isolated from MoMuSV-349 virions. The biological properties of the replicating information in MoMuSV-124 suggest that, consistent with the small size of RNA, it is defective. whereas MoMuSV-349 produces virions containing an intact MoMuLV genome, competent for replication.

Structure of murine sarcoma virus DNA replicative intermediates synthesized in vitro.

Moloney murine sarcoma virions synthesize discrete DNA products in vitro which closely resemble those found in vivo shortly after infection. These in vitro products have been isolated by electrophoresis and mapped with restriction endonucleases. In addition to the full-genome-length 6-kilobase pair linear DNA, a 5.4-kilobase pair circular DNA molecule, an incomplete linear DNA molecule, and a 600-base pair molecule were detected. The 6-kilobase pair DNA contained a 600-base pair direct terminal repeat which was missing from the circular form and was partially represented on the incomplete linear DNA molecule. The 600-base pair DNA contained sequences which were present in the 600-base pair direct repeat on the 6-kilobase pair DNA. The order of synthesis and the structure of these molecules detected in the in vitro reaction suggest that they are crucial intermediates in the formation of the final product of in vitro reverse transcription. A model which accounts for the synthesis of all of these molecules during the initial stages of viral replication is suggested.

Comparative study of different isolates of murine sarcoma virus.

The RNA genomes of a variety of murine sarcoma viruses (MSV) were compared by heteroduplex analysis. These viruses included the Moloney-derived isolates 124-MSV, m1-MSV, m3-MSV, HT1-MSV, and NP-MSV and also two independent isolates, Gazdar MSV and 1712-MSV. All of these viral genomes exhibited the acquired cellular sequences previously identified in 3124-MSV and thought to be responsible for transformation and sarcomagenesis. The location of the acquired cellular sequences within the envelope gene was variable in different MSV isolates, suggesting that the cellular sequences can be expressed in different positions relative to murine leukemia virus-derived information present in MSV. Deletions in the gag coding region of the different MSVs were consistent with their known gag-related gene products. Based on several features of the hetero-duplex analysis and the known genealogical relationships of the different MSVs, various possible mechanisms for the formation of MSV are considered.

Heteroduplex analysis of the RNA of clone 3 Moloney murine sarcoma virus.

Heteroduplex analysis of the RNA isolated from purified virions of clone 3 Moloney murine sarcoma virus (M-MSV) hybridized to cDNA's from Moloney murine leukemia virus (M-MLV) and clone 124 M-MSV shows that the main physical component of clone 3 RNA is missing all or most of the 1.5-kilobase (kb) clone 124 M-MSV specific sequence denoted beta s (S. Hu et al. Cell 10:469--477, 1977). This sequence is either deleted in clone 3 RNA or substituted by a very short (0.3-kilobase) sequence. In other respects, clone 3 and clone 124 RNAs show the same heteroduplex structure relative to M-MLV. Since beta s is believed to contain the src gene(s) of clone 124 RNA, this result leaves as an unresolved question the nature of the src gene(s) of the clone 3 M-MSV RNA complex.

Physical map of biologically active Harvey sarcoma virus unintegrated linear DNA.

BALB/c JLS V9 cells recently infected with Harvey sarcoma virus-murine leukemia virus (HSV-MuLV) complex contained unintegrated HSV linear DNA of 6.0-kilobase pair mass. The cells also contained two HSV closed circular DNA species along with MuLV-encoded linear and closed circular DNA species. HSV 6.0-kilobase pair linear DNA induced focal transformation upon transfection of NIH 3T3 mouse fibroblasts, and the biological activity of HSV DNA did not require helper MuLV functions. A physical map of restriction endonuclease cleavage sites along HSV 6.0-kilobase pair linear DNA was derived. Comparison of this map with one for Moloney MuLV DNA showed that the HSV and Moloney MuLV genomes are identical near their viral RNA 3' ends.

Heteroduplex analysis of the sequence relationships between the genomes of Kirsten and Harvey sarcoma viruses, their respective parental murine leukemia viruses, and the rat endogenous 30S RNA.

The sequence relations between Kirsten murine sarcoma virus (Ki-SV), Harvey murine sarcoma virus (Ha-SV), and a rat endogenous 30S RNA were studied by electron microscope heteroduplex analysis. The sequence relationships between the sarcoma viruses and their respective parental murine leukemia viruses (Kirsten and Moloney murine leukemia viruses), as well as between the two murine leukemia viruses, were also studied. The only observed nonhomology feature of the Kirsten murine leukemia virus/Moloney murine leukemia virus heteroduplexes was a substitution loop with two arms of equal length extending from 1.80 +/- 0.18 kilobases (kb) to 2.65 +/- 0.27 kb from the 3' end of the RNA. It is believed that this feature lies in the env gene region of the viral genomes. The Ha-SV and Moloney murine leukemia virus genomes (respective lengths, 6.0 and 9.0 kb) were homologous in a 1.0 +/- 0.05-kb region at the 3' end and possibly over a 200-nucleotide region at the 5' ends; otherwise, they were nonhomologous. Ha-SV and Ki-SV (length, 7.5 kb) were homologous in the first 4.36 +/- 0.37-kb region from the 3' end and in a 0.70 +/- 0.15-kb region at the 5' end. In between, there was a nonhomology region, possibly containing a short (0.23-kb) region of partial or total homology. The heteroduplex analysis between rat endogenous 30S RNA and Ki-SV shows that there are mixed regions of sequence homology and nonhomology at both the 5' and 3' ends. However, there is a large (4-kb) region of homology between Ki-SV and the rat 30S RNA in the center of the genomes, with only a small nonhomology hairpin feature. These studies help to define the regions of homology between the Ha-SV and Ki-SV genomes with each other and with the rat endogenous 30S RNA. These regions may be related to the sarcoma genicity of the viruses. In particular, the 0.7-kb region of homology of Ha-SV with Ki-SV at the 5' ends may be related to the formation of a 21,000-dalton phosphoprotein in cells transformed by either virus.

Molecular cloning of the Harvey sarcoma virus closed circular DNA intermediates: initial structural and biological characterization.

Supercoiled Harvey sarcoma virus (Ha-SV) DNA was extracted from newly infected cells by the Hirt procedure, enriched by preparative agarose gel electrophoresis, and digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized Ha-SV DNA was then inserted into lambda gtWESlambda B at the EcoRI site and cloned in an approved EK2 host. Ha-SV DNA inserts from six independently derived recombinant clones have been analyzed by restriction endonuclease digestion, molecular hybridization, electron microscopy, and infectivity. Four of the Ha-SV DNA inserts were identical, contained about 6.0 kilobase pairs (kbp), and comigrated in agarose gels with the infectious, unintegrated, linear Ha-SV DNA. One insert was approximately 0.65 kbp smaller (5.35 kbp) and one was approximately 0.65 kpb larger (6.65 kpb) than the 6.0 kpb inserts. R-looping with Ha-SV RNA revealed that the small (5.35 kbp) insert contained one copy of the Ha-SV RNA. Preliminary restriction endonuclease digestion of the recombinant DNAs suggested that the middle-size inserts contained a 0.65-kbp tandem duplication of sequences present only one in the small-size insert; this duplication corresponded to the 0.65-kpb terminal duplication of the unintegrated linear Ha-SV DNA. The large-size insert apparently contained a tandem triplication of these terminally located sequences. DNA of all three sized inserts induced foci in NIH 3T3 cells, and focus-forming activity could be rescued from the transformed cells by superinfection with helper virus. Infectivity followed single-hit kinetics, suggesting that the foci were induced by a single molecule.