In vitro translation of Harvey murine sarcoma virus RNA.

The viral RNA of the Harvey strain of murine sarcoma virus (Ha-SV), which does not encode for any known viral structural polypeptides, has been translated in a nuclease-digested, cell-free system. The major protein product of the in vitro translation reaction has a molecular weight of 21,000 and is initiated faithfully with [35S]formylmethionine from formyl-[35S]methionyl-tRNAFMET. This polypeptide is clearly distinct from the RNA of the Moloney strain of type C helper virus used to pseudotype the Ha-SV. The intensity of the 21,000-dalton polypeptide on gels correlates well to the concentration of Ha-SV RNA in different viral RNA preparations. These experiments indicate that a polypeptide marker for Ha-SV is now available for the first time. The possibility that this protein is the product of the rat portion of the Ha-SV genome is discussed.     

Dual evolutionary origin for the rat genetic sequences of Harvey murine sarcoma virus.

Detailed restriction endonuclease maps were developed for Harvey murine sarcoma virus (Ha-MuSV) DNA (clone H-1), molecularly closed at its unique EcoRI site in pBR322, for three nonoverlapping subgenomic HindIII clones which together span the entire H-1 clone and for a molecularly cloned DNA copy of a portion of rat 30S RNA (which represents the majority of the rat genetic sequences in Ha-MuSV). Molecular hybridization of the 30S clone to small restriction fragments of clone H-1 revealed a 0.9-to-1.0-kilobase pair region in the 5' half of the Ha-MuSV genome not homologous to the 30S clone, although the 30S clone did contain related sequences in Ha-MuSV on both sides of this nonhomologous region. By using cloned sequences from a segment of the Ha-MuSV nonhomology region as a probe for hybridization to Southern blots of DNA from rat, mouse, bat, and chicken cells, one to three bands were detected in DNA of each species. By contrast, the 30S clone DNA was highly related to many sequences in rat DNA, partially related to fewer mouse DNA sequences, and homologous only to one to three bands in bat and chicken DNA. Earlier work had shown that the 5' half of the Ha-MuSV genome coded for transformation and for the viral p21 protein (Chang et al., J. Virol. 35: 76--92, 1980; Wei et al., Proc. Natl. Acad. Sci. U.S.A., in press). We used two subgenomic HindIII clones whose shared HindIII site mapped within the 5' region of clone H-1 nonhomologous to the 30S clone to test whether the nonhomologous segment might encode the transforming and p21 functions. Although neither of the subgenomic HindIII fragments by themselves induced transformation, ligation of these two nontransforming DNAs to each other did restore p21-mediated transformation. A conclusion consistent with these results is that a region in the 5' half of the Ha-MuSV genome evolutionarily distinct from and not present in rat 30S RNA is essential for transformation and for p21 encoding.     

Helper-independent transformation by unintegrated Harvey sarcoma virus DNA.

We have studied the unintegrated infectious DNA of Harvey sarcoma virus (Ha-SV) and Moloney leukemia virus (Mo-MuLV). The source of infectious viral DNA was the Hirt supernatant fraction from cells acutely infected with Ha-SV and Mo-MuLV. To obtain a direct quantitative assay for infectious viral DNA, recipient mouse cells were first exposed to calcium phosphate-precipitated viral DNA and then treated with dimethyl sulfoxide. Infectivity was monitored by focus formation for Ha-SV and XC plaque formation for Mo-MuLV. The viral DNA titration pattern followed single-hit kinetics for both foci and plaques, indicating that a single molecule carried information for each function. Focus-forming and plaque-forming activity were present in different molecules, since these two biological activities could be separated from each other by agarose gel electrophoresis. The focus-forming molecule was linear DNA with a molecular weight of about 4 x 10(6) daltons. The focus-forming activity of the viral DNA was sensitive to EcoRI and resistant to XhoI restriction endonucleases, whereas the plaque-forming activity was resistant to EcoRI and sensitive to XhoI. The generation of helper-independent foci indicates that Ha-SV DNA can transform mouse cells in the absence of helper virus or its proteins.     

Functional organization of the Harvey murine sarcoma virus genome.

The comparative infectivity of Harvey murine sarcoma virus (Ha-MuSV) DNA for NIH 3T3 cells was determined for supercoiled Ha-MuSV DNA molecularly cloned in lambda phage and pBR322 at its unique EcoRI site (which is located near the middle of the 6-kilobase pair [kbp] unintegrated linear viral DNA) and for two cloned subgenomic fragments: one was 3.8 kbp and lacked about 1 kbp from each side of the EcoRI site, and the second did not contain the 3 kbp of the unintegrated linear viral DNA located on the 3' side of the EcoRI site. Each subgenomic DNA induced foci of transformed cells, but with a lower relative efficiency then genomic DNA. Transfection with intact vector Ha-MuSV DNA yielded results similar to those obtained after separation of Ha-MuSV DNA from vector DNA. Cells lines were then derived from individual foci transformed with each type of viral DNA. Focus-forming virus was recovered from transformed cells after superinfection with a helper-independent virus, but the efficiency varied by several orders of magnitude. For several transformed lines, the efficiency of recovery of focus-forming virus was correlated with the structure of the Ha-MuSV DNA in the cells before superinfection. When 32P-labeled Ha-MuSV DNA probes specific for sequences on either the 3' or 5' side of the EcoRI site were used to analyze the viral RNA in the transformed cell lines, all lines were found to hybridize with the 5' probe, but some lines did not hybridize with the 3' probe. The transformed lines contained high levels of the Ha-MuSV-coded p21 or its associated GDP-binding activity. We conclude that the transforming region and the sequences that code for the viral p21 protein are both located within the 2 kilobases closest to the 5' end of the Ha-MuSV genome.     

Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat.

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.     

Polyomavirus tumor induction in mice: influences of viral coding and noncoding sequences on tumor profiles.

We determined the DNA sequences of the noncoding regions of two polyomavirus strains that differ profoundly in their abilities to induce tumors in mice. Differences between strains were found, both on the late side of the replication origin in the region containing known enhancer elements and on the early side of the origin, affecting the number and location of large-T-antigen-binding sites. By constructing and analyzing recombinant viruses between these high- and low-tumor strains, we attempted to localize determinants which affect the frequency and histotype of tumors. Seven recombinants were constructed and propagated in vitro, and the tumor profile of each was established by inoculation into newborn C3H mice. Recombinants containing noncoding sequences from the high-tumor strain and coding sequences from the low-tumor strain behaved like the latter, inducing tumors at a low frequency and strictly of mesenchymal origin. Reciprocal recombinants with noncoding sequences of the low-tumor strain linked to structural determinants from the high-tumor strain induced several types of epithelial tumors typical of the high-tumor strain but at reduced frequency, in addition to mesenchymal tumors. A high frequency and full diversity of epithelial tumors required, in addition to structural regions from the high-tumor strain, noncoding sequences on the early side of the origin also present in this strain. A high-tumor profile thus resulted from the combined effects of structural and regulatory determinants in the high-tumor strain, with the former affecting primarily the tissue tropism and the latter affecting the frequency of tumors. No differential effects of the enhancer regions from the late side of the origin in the two virus strains were seen in this study.     

Generation of a recombinant Moloney murine leukemia virus carrying the v-src gene of avian sarcoma virus: transformation in vitro and pathogenesis in vivo.

A Moloney murine leukemia virus (M-MuLV) recombinant carrying the v-src gene of avian sarcoma virus was generated by the introduction of a cloned portion of v-src from Schmidt-Ruppin A avian sarcoma virus into a molecular clone of M-MuLV provirus at the recombinant DNA level. The v-src sequences (lacking a portion of the 5' end of v-src) were inserted into the p30 region of the M-MulV gag gene so that M-MuLV gag and v-src were in the same reading frame. Transfection of this chimeric clone, pMLV(src), into NIH 3T3 cells which were constitutively producing M-MuLV gag and pol protein resulted in the formation of foci of transformed cells. Infectious and transforming virus could be recovered from the transformed cells. This virus was designated M-MuLV(src). M-MuLV(src)-transformed cells contained two novel proteins of 78 and 90 kilodaltons. The 78-kilodalton protein, p78gag-src, contained both gag and src determinants, exhibited kinase activity in an immune kinase assay, and is probably a fusion of Pr65gag and src. The 90-kilodalton protein, which is of the appropriate size to be the gPr80gag fused to src, contained gag determinants as well as a V8 protease cleavage fragment typical of the carboxy terminus of avian sarcoma virus pp60src. However, it could not be immunoprecipitated with an anti-v-src serum. M-MuLV(src)-transformed cells showed elevated levels of intracellular phosphotyrosine in proteins, although the elevation was intermediate compared with cells transformed with wild-type v-src. M-MuLV and amphotropic murine leukemia virus pseudotypes of M-MuLV(src) were inoculated into newborn NIH Swiss mice. Inoculated mice developed solid tumors at the site of inoculation after 3 to 6 weeks, with most animals dying by 14 weeks. Histopathological analysis indicated that the solid tumors were mesenchymally derived fibrosarcomas that were both invasive and metastatic.     

Further genetic localization of the transforming sequences of the p21 v-ras gene of Harvey murine sarcoma virus.

The sequences encoding the 21-kilodalton transforming protein (p21 ras) of Harvey murine sarcoma virus have previously been localized genetically to a 1.3-kilobase segment of the viral DNA (E. H. Chang, R. W. Ellis, E. M. Scolnick, and D. R. Lowy, Science 210:1249-1251, 1980). Within this segment, DNA sequence analysis has found a single open reading frame large enough to encode the viral p21 (R. Dhar, R. W. Ellis, T. Y. Shih, S. Oroszlan, B. Shapiro, J. Maizel, D. Lowy, and E. M. Scolnick, Science 217:934-937, 1982). There are three potential in-frame ATG initiation codons at the 5' end of this open reading frame. By constructing a mutant of Harvey murine sarcoma virus DNA from which the first two ATG codons of this open reading frame have been deleted, we now show by transfection of the mutant viral DNA into NIH 3T3 cells that only the third ATG codon is necessary and sufficient for synthesis of the viral p21 and for cellular transformation.     

The malignant histiocytosis sarcoma virus, a recombinant of Harvey murine sarcoma virus and Friend mink cell focus-forming virus, has acquired myeloid transformation specificity by alterations in the long terminal repeat.

The malignant histiocytosis sarcoma virus (MHSV), in contrast to other viruses with the ras oncogene, induces acute histiocytosis in newborn and adult mice. Molecular structure and function studies were initiated to determine the basis of its unique macrophage-transforming potential. Characterization of the genomic structure showed that the virus evolved by recombination of the Harvey murine sarcoma virus (Ha-MuSV) and a virus of the Friend-mink cell focus-forming virus family. Structural analysis of MHSV showed two regions of the genome that are basically different from the Ha-MuSV: (i) the ras gene, which is altered by a point mutation in codon 181 leading to a Cys----Ser substitution of the p21 protein, and (ii) the U3 region of the long terminal repeat, which is largely derived from F-MCFV and contains a deletion of one direct repeat as well as a duplication of an altered enhancer-like region. Biological studies of Ha-MuSV, MHSV, and recombinants between the two viruses show that the U3 region of the MHSV long terminal repeat is essential for the malignancy and specificity of the disease. A contributing role of the ras point mutation in determining macrophage specificity, however, cannot be excluded.     

Rat sequences of the Kirsten and Harvey murine sarcoma virus genomes: nature, origin, and expression in rat tumor RNA.

Two murine sarcoma viruses, the Kirsten and the Harvey, were isolated by passage of mouse type C leukemia viruses through rats. These sarcoma viruses have genomes containing portions of their parental type C mouse leukemia virus genomes, in stable association with specific rat cellular sequences that we find to be quite likely not those of a rat type C leukemia virus. To determine if these murine sarcoma viruses provide a model relevant to the events occurring in spontaneous tumors, we have hybridized DNA and RNA prepared from rat tumors and normal rat tissues to [3H]DNA prepared from the Kirsten murine sarcoma virus. We have also hybridized these rat tissue nucleic acids to [3H]DNA prepared from a respresentative endogenous rat type C leukemia virus, the WFU (Wistar-Furth). Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected in the DNA of both tumor and normal tissues, with no evidence of either gene amplification or additional sequences being present in tumor DNA. Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected at elevated concentrations in the RNA of many rat tumors and in specific groups of normal tissues.     

Leukemogenicity and cell transformation mechanisms in vitro by Gross murine leukemia virus: analysis of virus subpopulations.

The leukemogenic activity of Gross murine leukemia virus adapted to rats was tested in W/Fu rats and NIH/Swiss mice. All animals infected with this virus developed thymic and nonthymic T-cell leukemia with a short latency period. It was observed that cell-free extracts from thymic lymphoma tissue of mice and rats, induced by either Gross murine leukemia virus or Gross murine leukemia virus adapted to rats, consisted of both small-plaque-forming and large-plaque-forming viruses, as determined by the XC plaque test. MCF-type virus was found in these virus complexes. Transformed cell foci were induced in SC-1 cell layers by double infection of the cloned MCF-type virus and an ecotropic virus. SC-1 cells containing transformed cell foci were shown to be tumorigenic upon inoculation into nude mice. The formation of transformed cell foci in mink lung cells was also observed after double infection with the cloned MCF-type virus and a xenotropic virus. The possible mechanism of leukemogenesis by endogenous viruses is discussed.     

Myeloid cell transformation by ras-containing murine sarcoma viruses.

BALB and Harvey murine sarcoma viruses contain ras transforming genes capable of altering the proliferation and differentiation of cells within the erythroid and lymphoid lineages (W. D. Hankins and E. M. Scolnick, Cell 26:91-97, 1981; J. H. Pierce and S. A. Aaronson, J. Exp. Med. 156:873-887, 1982; E. M. Scolnick et al., Mol. Cell. Biol. 1:68-74). The present studies demonstrate hematopoietic targets of ras-containing viruses within the myeloid lineage. Diffuse colonies were induced by BALB or Harvey marine sarcoma virus infection of murine bone marrow cells. Generally, these colonies were made up of relatively mature macrophages which exhibited increased self-renewal capacity but eventually underwent terminal differentiation in culture. Cells from one BALB murine sarcoma virus-induced colony displayed phenotypic markers of more immature myelomonocytic cells. This colony, designated BAMC1, readily established as a continuous cell line and was highly malignant in vivo. Exposure of these cells to 12-O-tetradecanoylphorbol-13-acetate led to the induction of a more mature myeloid phenotype, which was associated with decreased growth potential in vitro and in vivo. The effects of the inducing agent were not mediated by an alteration in the level of expression of the ras-coded p21 transforming protein. Our present findings extend the spectrum of targets whose growth is altered by ras-containing retroviruses to cells at several stages of differentiation within each of the major hematopoietic lineages.     

An altered DNA sequence encompassing the ras gene of Harvey murine sarcoma virus.

The DNA fragment encompassing the ras gene of Harvey murine sarcoma virus was sequenced and assigned the coding region of a transforming protein, p21, to the sequence. Examination of nucleotide sequence, taken together with the result of analysis of the ras mRNAs (1), has revealed that p21 is encoded from a continuous coding region starting with the 5' proximal initiation codon but not a processed protein. However, there were found several differences between the sequence published by Dhar et. al. (2) and ours, including 9 deletions, 7 substitutions and 2 insertions of nucleotides in the published sequence of 997 nucleotides in length. Among these, one of the substitutions occurring in the coding region resulted in amino acid replacement of glycine by alanine at position 122 of p21. The evidences are presented with some of actual gel autoradiographs.     

In vitro transformation of murine pre-B lymphoid cells by Snyder-Theilen feline sarcoma virus

Snyder-Theilen feline sarcoma virus (ST-FeSV) codes for a protein kinase with specificity for tyrosine residues (Barbacid et al., Proc. Natl. Acad. Sci. U.S.A. 77:5158-5163, 1980), properties analogous to those of the transforming gene product of Abelson murine leukemia virus (Witte et al., Nature (London) 283:826-831, 1980). In the present report, ST-FeSV was demonstrated to transform murine hematopoietic cells under in vitro assay conditions which detect lymphoid cell transformation by Abelson murine leukemia virus. Bone marrow colony formation was shown to require ST-FeSV, follow single-hit kinetics, and require the presence of mercaptoethanol in the agar medium. ST-FeSV-induced colonies could be established in culture as continuous cell lines that demonstrated unrestricted self-renewal capacity and leukemogenicity in vivo. The hematopoietic blast cells transformed by ST-FeSV in culture appeared to be at an early stage of B cell differentiation. They possessed Lyb 2 surface antigens, were dependent on mercaptoethanol for growth, and contained only low levels of terminal deoxynucleotidyl transferase. Moreover, a large fraction of the lines synthesized immunoglobulin mu chain in the absence of light chains. Thus, the phenotype of ST-FeSV hematopoietic transformants was indistinguishable from that of the pre-B lymphoblast transformants induced by Abelson murine leukemia virus. These findings indicate that the in vitro functional similarities in the onc gene products of ST-FeSV and Abelson murine leukemia virus may reflect a common pathway by which they exert their oncogenic potential.     

Homologies between a brain-specific identifier (ID) sequence and regions of Harvey murine sarcoma virus and Rous sarcoma virus genomes. Putative role of identifier sequences in the tissue specificity of malignant transformation by RNA tumor viruses

As a step toward understanding of the tissue specificity of cellular transformation by RNA tumor viruses were looked for the presence of a putative brain specific regulatory (identifier) sequence (C82B) in the genome of various oncornaviruses. The genomes of Harvey murine sarcoma virus and Rous sarcoma virus contain sequences flanking the viral oncogenes with greater than 80% and greater than 60% homology to C82B, respectively. We suggest that identifier sequences acquired by oncoviruses may determine the potential target cells of malignant transformation after virus penetration.     

Clustering of identical oligomers in coding and noncoding DNA sequences.

We develop a quantitative method for analyzing repetitions of identical short oligomers in coding and noncoding DNA sequences. We analyze sequences presently available in the GenBank separately for primate, mammal, vertebrate, rodent, invertebrate and plant taxonomic partitions. We find that some oligomers "cluster" more than they would if randomly distributed, while other oligomers "repel" each other. To quantify this degree of clustering, we define clustering measures. We find that (i) clustering significantly differs in coding and noncoding DNA; (ii) in most cases, monomers, dimers and tetramers cluster in noncoding DNA but appear to repel each other in coding DNA. (iii) The degree of clustering for different sources (primates, invertebrates, and plants) is more conserved among these sources in the case of coding DNA than in the case of noncoding DNA. (iv) In contrast to other oligomers, we find that trimers always prefer to cluster. (v) Clustering of each particular oligomer is conserved within the same organism.     

Entropies of coding and noncoding sequences of DNA and proteins.

The entropies of protein coding genes from Escherichia coli were calculated according to Boltzmann's formula. Entropies of the coding regions were compared to the entropies of noncoding or miscoding ones. With nucleotides as code units, the entropies of the coding regions, when compared to the entropies of complete sequences (leader and coding region as well as trailer), were seen to be lower but with a marginal statistical significance. With triplets of nucleotides as code units, the entropies of correct reading frames were significantly lower than the entropies of frameshifts +1 and -1. With amino acids as code units, the results were opposite: Biologically functional proteins had significantly higher entropies than proteins translated from the frameshifted sequences. We attempt to explain this paradox with the hypothesis that the genetic code may have the ability of lowering information content (increasing entropy) of proteins while translating them from DNA. This ability might be beneficial to bacteria because it would make the functional proteins more probable (having a higher entropy) than nonfunctional proteins translated from frameshifted sequences.