Molecular cloning of proviral DNA and structural analysis of the transduced myc oncogene of avian oncovirus CMII.

Molecularly cloned proviral DNA of avian oncogenic retrovirus CMII was isolated by screening a genomic library of a CMII-transformed quail cell line with a myc-specific probe. On a 10.4-kilobase EcoRI fragment, the cloned DNA contained 4.4 kilobases of CMII proviral sequences extending from the 5' long terminal repeat to the EcoRI site within the partial (delta) complement of the env gene. The gene order of CMII proviral DNA is 5'-delta gag-v-myc-delta pol-delta env-3'. All three structural genes are partially deleted: the gag gene at the 3' end, the env gene at the 5' end, and the pol gene at both ends. The delta gag (0.83 kilobases)-v-myc (1.50 kilobases) sequences encode the p90gag-myc transforming protein of CMII. In comparison with the p110gag-myc protein of acute leukemia virus MC29, p90gag-myc lacks amino acids corresponding to additional 516 bases of gag sequences and 12 bases of 5' v-myc sequences present in the MC29 genome. Nucleotide sequence analysis of CMII proviral DNA at the delta gag-v-myc and the v-myc-delta pol junctions revealed significant homologies between avian retroviral structural genes and the cellular oncogene c-myc precisely at the positions corresponding to the gene junctions in CMII. Furthermore, the delta gag-v-myc junction in CMII corresponds to sequence elements in gag and C-myc that are possible splicing signals. The data suggest that transduction of cellular oncogenes may involve RNA splicing and recombination with homologous sequences on retroviral vectors. Different sequence elements of both the retroviral vectors and the c-myc gene recombined during genesis of highly oncogenic retroviruses CMII, MC29, or MH2.     

Two unrelated cell-derived sequences in the genome of avian leukemia and carcinoma inducing retrovirus MH2.

Molecularly cloned proviral DNA of avian replication-defective retrovirus Mill Hill No. 2 (MH2) was analyzed. The MH2 provirus measures 5.5 kb including two long terminal repeats (LTR), and contains a partial complement of the structural gene gag, 1.5 kb in size, near the 5' terminus, and a 1.3-kb segment of the v-myc transforming gene near the 3' terminus. These v-myc sequences are closely related to the v-myc transforming gene of avian acute leukemia virus MC29, and to the cellular chicken gene c-myc. The gag and myc domains on the MH2 provirus are separated by unique sequences, 1.3 kb in size and termed v-mil, which are unrelated to v-myc, or to other oncogenes or structural genes of the avian leukemia-sarcoma group of retroviruses. Normal chicken DNA contains sequences closely related to v-mil, termed c-mil. Analyses of chicken c-mil clones isolated from a recombinant DNA library of the chicken genome reveal that c-mil is a single genetic locus with a complex split gene structure. In the MH2 genome, v-mil is expressed via genome-sized mRNA as a gag-related hybrid protein, p100gag-mil, while v-myc is apparently expressed via subgenomic mRNA independently from major coding regions of structural genes. The presence in the MH2 genome of two unrelated cell-derived sequences and their independent expression may be significant for the oncogenic specificities of this virus.     

Avian oncovirus MH2: molecular cloning of proviral DNA and structural analysis of viral RNA and protein

Viral RNA, molecularly cloned proviral DNA, and virus-specific protein of avian retrovirus MH2 were analyzed. The complexity and sequence conservation of the transformation-specific v-myc sequences of MH2 RNA were compared with those of the other members of the MC29 subgroup of acute leukemia viruses, MC29, CMII, and OK10, and with chicken cellular c-myc sequences. All T1 oligonucleotides mapping within the 1.3-kilobase coding region of MC29 v-myc have homologous counterparts in the RNAs of all MC29 subgroup viruses and in c-myc. These counterparts are either identical in composition or altered by single point mutations. Hence, the 47,000-dalton carboxy-terminal sequences of the transforming proteins of these viruses and of the cellular gene product are probably highly conserved but may contain single amino acid substitutions. T1 oligonucleotide mapping of MH2 RNA indicated that the MH2 v-myc sequences map close to the 3' end of viral RNA. A genomic library of an MH2-transformed quail cell line was prepared by using the Charon 4A vector system. By screening with an myc-specific probe, a clone containing the entire MH2 provirus (lambda MH2-1) was isolated. Digestion of cloned DNA with KpnI yielded a 5.1-kilobase fragment hybridizing to both gag- and myc-specific probes. Further restriction mapping of lambda MH2-1 DNA showed that about 1.6 kilobases of the gag gene are present near the 5' end of proviral DNA, and the conserved part of v-myc, i.e., 1.3 kilobases, is present near the 3' end of proviral DNA. These two domains are separated by a segment of at least 1 kilobase of different genetic origin, including additional unique sequences unrelated to virion genes. Tryptic peptide analysis of the gag-related protein of MH2, p100, revealed gag-specific peptides and several unique methionine-containing peptides. One of the latter is possibly shared with the polymerase precursor protein Pr180gag-pol, but no myc-specific peptides, defined for the MC29 protein p110gag-myc, appear to be present in MH2 p100. The data on viral RNA, proviral DNA, and protein of MH2 reveal a unique genetic structure for this virus of the MC29 subgroup and suggest that its v-myc gene is not expressed as a gag-related protein.     

Nucleotide sequence of a transduced myc gene from a defective feline leukemia provirus.

The nucleotide sequence of a feline v-myc gene and feline leukemia virus (FeLV) flanking regions was determined. Both the nucleotide and predicted amino acid sequences are very similar to the murine and human c-myc genes (ca. 90% identity). The entire c-myc coding sequence is represented in feline v-myc and replaces portions of the gag and env genes and the entire pol gene. The coding sequence is in phase with the gag gene reading frame; v-myc, therefore, appears to be expressed as a gag-myc fusion protein. Viral sequences at the 3' myc-FeLV junction begin with the hexanucleotide CTCCTC, which is also found at the 3' fes-FeLV junction of both Gardner-Arnstein and Snyder-Theilen feline sarcoma viruses. These similarities suggest that some sequence specificity may exist for the transduction of cellular genes by FeLV. Feline v-myc lacks a potential phosphorylation site at amino acid 343 in the putative DNA-binding domain, whereas both human and murine c-myc have such sites. Avian v-myc has lost a potential phosphorylation site which is present in avian c-myc five amino acids from the potential mammalian site. If these sites are actually phosphorylated in normal c-myc proteins, their loss may alter the DNA-binding affinity of v-myc proteins.     

Oligoribonucleotide map and protein of CMII: detection of conserved and nonconserved genetic elements in avian acute leukemia viruses CMII, MC29, and MH2.

RNA and protein of the defective avian acute leukemia virus CMII, which causes myelocytomas in chickens, and of CMII-associated helper virus (CMIIAV) were investigated. The RNA of CMII measured 6 kilobases (kb) and that of CMIIAV measured 8.5 kb. By comparing more than 20 mapped oligonucleotides of CMII RNA with mapped and nonmapped oligonucleotides of acute leukemia viruses MC29 and MH2 and with mapped oligonucleotides of CMIIAV and other nondefective avian tumor viruses, three segments were distinguished in the oligonucleotide map of CMII RNA: (i) a 5' group-specific segment of 1.5 kb which was conserved among CMII, MC29, and MH2 and also homologous with gag-related oligonucleotides of CMIIAV and other helper viruses (hence, group specific); (ii) an internal segment of 2 kb which was conserved specifically among CMII, MC29, and MH2 and whose presence in CMII lends new support to the view that this class of genetic elements is essential for oncogenicity, because it was absent from an otherwise isogenic, nontransforming helper, CMIIAV; and (iii) a 3' group-specific segment of 2.5 kb which shared 13 of 14 oligonucleotides with CMIIAV and included env oligonucleotides of other nondefective viruses of the avian tumor virus group (hence, group specific). This segment and analogous map segments of MC29 and MH2 were not conserved at the level of shared oligonucleotides. CMII-transformed cells contained a nonstructural, gag gene-related protein of 90,000 daltons, distinguished by its size from 110,000-daltom MC29 and 100,000-dalton MH2 counterparts. The gag relatedness and similarity to the 110,000-dalton MC29 counterpart indicated that the 90,000-dalton CMII protein is translated from the 5' and internal segments of CMII RNA. The existence of conserved 5' and internal RNA segments and conserved nonstructural protein products in CMII, MC29, and MH2 indicates that these viruses belong to a related group, termed here the MC29 group. Viruses of the MC29 group differ from one another mainly in their 3' RNA segments and in minor variations of their conserved RNA segments as well as by strain-specific size markers of their gag-related proteins. Because (i) the conserved 5' gag-related and internal RNA segments and their gag-related, nonvirion protein products correlate with the conserved oncogenic spectra of the MC29 group of viruses and because (ii) the internal RNA sequences and nonvirion proteins are not found in nondefective viruses, we propose that the conserved RNA and protein elements are necessary for oncogenicity and probably are the onc gene products of the MC29 group of viruses.     

The second oncogene mil of avian retrovirus MH2 is related to the src gene family.

The nucleotide sequence of a PstI fragment prepared from a cloned MH2 virus genome, pMH2-Hd, has been deduced using chemical and enzymatic methods. This fragment, 1862 nucleotides in length, starts with the gag gene, encodes the v-mil sequence and stops within the v-myc gene. This sequence shows that the v-mil gene is fused to the gag gene giving rise to a fused polyprotein of 98 000 daltons: 515 amino acids at the amino terminus would correspond to p10, p19, p27 and part of p12 determinants, 347 amino acids at the carboxy terminus correspond to the v-mil specific sequence. The mil protein shares homology with a number of onc proteins such as src, fes, fms, mos, yes, fps and erbB, as well as with the catalytic chain of the cAMP-dependent protein kinase. This PstI fragment also encodes the beginning of the myc gene which was integrated in MH2 along with the 3' end of the preceding intron placing an acceptor splice site in front of the used open reading frame. As deduced from the sequence, the MH2 myc protein is not identical to the MC29 myc protein. It differs at its amino terminus, which contains little or no gag determinants, depending on the ATG used to initiate translation.     

RNA and protein encoded by MH2 virus: evidence for subgenomic expression of v-myc.

MH2 and MC29 are highly related myc-containing avian retroviruses. We found that MH2, unlike MC29, synthesizes a 2.6-kilobase subgenomic mRNA containing myc sequences as well as sequences from the 5' end of the genome. A 57-kilodalton protein containing myc, but not gag, sequences (p57myc) was detected by hybrid selection and in vitro translation of RNA from MH2-transformed cells. Gradient separation of MH2 intracellular RNAs indicated that p57myc is encoded by the subgenomic RNA. A highly oncogenic MH2 virus variant (MH2YS3) (M. Linial, Virology 119:382-391, 1982) was shown to encode only p57myc and not P100, the previously described MH2-encoded polyprotein (Hu et al., Virology, 89:162-178, 1978). Cells transformed by subclones of this virus synthesized predominantly the 2.6-kilobase RNA rather than genomic 5.4-kilobase RNA. These results suggest that only p57myc is required for maintenance of the transformed state after MH2 infection.     

gag as well as myc sequences contribute to the transforming phenotype of the avian retrovirus FH3.

The avian retrovirus FH3, like MC29 and CMII, encodes a Gag-Myc fusion protein. However, the FH3-encoded protein is larger, about 145 kDa, and contains almost the entire retroviral gag gene. In contrast to the other gag-myc avian retroviruses, FH3 fails to transform fibroblasts in vitro, although macrophages are transformed both in vitro and in vivo (C. Chen, B. J. Biegalke, R. N. Eisenman, and M. L. Linial, J. Virol. 63:5092-5100, 1989). We have used the polymerase chain reaction technique to obtain a molecular clone of FH3. Sequence analysis of the FH3 myc oncogene revealed a single proline----histidine change (position 223) relative to c-myc. However, substitution of the FH3 myc sequence with the chicken c-myc sequence did not alter the transformation potential of the virus. Hence, overexpression of the proto-oncogene as a Gag-Myc retroviral protein is sufficient for macrophage, but not fibroblast, transformation. After passage of FH3 in fibroblast cultures, a virus (FH3L) that is capable of rapidly transforming fibroblasts appears. The Gag-Myc protein encoded by FH3L is smaller (ca. 130 kDa) than that encoded by the original viral stock (FH3E). Sequencing of an FH3L molecular clone revealed a 212-amino-acid deletion within the Gag portion. Using FH3E/FH3L recombinants, we have demonstrated that the ability of encoded viruses to transform fibroblasts directly correlates with the presence of this deletion. Moreover, the addition of the Gag sequence deleted from FH3L to the MC29 oncoprotein significantly reduces its transforming activity as measured by focus assay. These data suggest that the C-terminal segment of Gag attenuates the oncogenic potential of Gag-Myc fusion proteins.     

Quail embryo fibroblasts transformed by four v-myc-containing virus isolates show enhanced proliferation but are non tumorigenic.

Quail embryo fibroblasts infected with any of the four natural avian myc gene-containing virus strains (MC29, CMII, OK10 and MH2) or with the myb, ets-containing E26 acute leukemia virus, were examined for their expression of several transformation-associated parameters. All myc-containing viruses, but not E26 or Rous sarcoma virus (used as a control) induced a dramatic stimulation of cell proliferation. In addition, the myc virus-transformed cells exhibited prominent nucleoli, possibly as a consequence of their increased proliferation. Cells transformed by MC29, OK10, MH2 and E26 were capable of growing in semi-solid medium and showed a loss of actin cables and, in most cases, of an ordered fibronectin distribution. All of the myc virus-transformed fibroblasts, as well as the E26-transformed cells, were unable to form tumors in nude mice, indicating that the myc gene (and the myb/ets genes) are not sufficient for the induction of a fully malignant phenotype in avian fibroblasts.     

The transforming protein of the MC29-related virus CMII is a nuclear DNA-binding protein whereas MH2 codes for a cytoplasmic RNA-DNA binding polyprotein.

The acute avian leukemia viruses MH2 and CMII belong to the group of avian myelocytomatosis viruses, the prototype virus of which is MC29. This group of viruses is characterized by myc-specific oncogenes which are presumably expressed as gag-myc polyproteins. These polyproteins are synthesized in non-producer cells transformed by MH2 and CMII and have mol. wts. of 100 000 (p100) and 90 000 (p90), respectively. Monoclonal antibodies against the N terminus of gag, p19, were used to localize the protein in MH2- and CMII-transformed non-producer fibroblasts. Immunofluorescence and cell fractionation indicated that greater than 90% of p100 from MH2 was located in the cytoplasm, whereas greater than 70% of p90 from CMII resided in the nucleus. Isolation of p100 and p90 by immunoaffinity chromatography resulted in an approximately 2000-fold purification of the two polyproteins. Both of them, as well as p110 of MC29, bound to double-stranded DNA of chick fibroblasts in vitro. However, only the MH2-specific polyprotein p100 bound to RNA in vitro. Such a binding was not observed for p90 or p110, or for the purified gag precursor Pr76. Another polyprotein, gag-erbA, from avian erythroblastosis virus, which is also located in the cytoplasm, did not bind to RNA. Our results indicate that the CMII-specific polyprotein p90 behaved indistinguishably from the p110 of MC29. However, the MH2-specific polyprotein p100 exhibited unique and novel properties which were distinct from a gag-myc-type protein.     

Myc oncogene activation in B and T lymphoid tumours

The chromosome translocations characteristic of certain B lymphoid tumours associate the myc oncogene and immunoglobulin loci. The typical t(12;15) in murine plasmacytomas and analogous t(14;8) in Burkitt lymphomas couple the myc coding region to one of the switch recombination regions within the immunoglobulin heavy (H) chain locus; hence the switch machinery may promote some translocations. Significantly, translocation induces constitutive myc expression, the untranslocated myc allele remaining silent. The predilection for breakpoints near the 5' end of the c-myc gene may reflect selection for altered myc regulation. In most tumours, the stimulatory effect of the H locus context is not understood, but an H locus enhancer participates in some tumours, including one displaying a novel transposition. The variant (6;15) translocations found in about 15% of plasmacytomas involve the myc band and the region of chromosome 6 where the kappa locus lies. The t(6;15) is shown here to represent an exchange between C kappa and a chromosome 15 locus (designated pvt-1) which lies unexpectedly far from c-myc. The association of myc expression with pvt-1 alterations suggest that myc can be activated at a distance. Myc has also been implicated in some T lymphomas by detection of proviral inserts near myc and also, surprisingly, within the pvt-1 locus. Inserts near myc appear to activate its expression via the retroviral enhancer.     

A 3' UTR sequence stabilizes termination codons in the unspliced RNA of Rous sarcoma virus

Eukaryotic cells target mRNAs to the nonsense-mediated mRNA decay (NMD) pathway when translation terminates within the coding region. In mammalian cells, this is presumably due to a downstream signal deposited during pre-mRNA splicing. In contrast, unspliced retroviral RNA undergoes NMD in chicken cells when premature termination codons (PTCs) are present in the gag gene. Surprisingly, deletion of a 401-nt 3' UTR sequence immediately downstream of the normal gag termination codon caused this termination event to be recognized as premature. We termed this 3' UTR region the Rous sarcoma virus (RSV) stability element (RSE). The RSE also stabilized the viral RNA when placed immediately downstream of a PTC in the gag gene. Deletion analysis of the RSE indicated a smaller functional element. We conclude that this 3' UTR sequence stabilizes termination codons in the RSV RNA, and termination codons not associated with such an RSE sequence undergo NMD.     

Activation of the c-H-ras proto-oncogene by retrovirus insertion and chromosomal rearrangement in a Moloney leukemia virus-induced T-cell leukemia.

A rearrangement of the c-H-ras locus was detected in a T-cell line (DA-2) established from a Moloney leukemia virus-induced tumor. This rearrangement was associated with the high-level expression of H-ras RNA and the H-ras gene product, p21. DNA from DA-2 cells transformed fibroblasts in DNA transfection experiments, and the transformed fibroblasts contained the rearranged H-ras locus. The rearrangement involved one allele and was present in tissue from the primary tumor from which the cell line was isolated. Cloning and sequencing of the rearranged allele and comparison with the normal allele demonstrated that the rearrangement was complex and probably resulted from the integration of a retrovirus in the H-ras locus between a 5' noncoding exon and the first coding exon and a subsequent homologous recombination between this provirus and another newly acquired provirus also located on chromosome 7. These events resulted in the translocation of the coding exons of the H-ras locus away from the 5' noncoding exon region to a new genomic site on chromosome 7. Sequencing of the coding regions of the gene failed to detect mutations in the 12th, 13th, 59th, or 61st codons. The possible reasons for the complexity of the rearrangement and the significance of the activation of the H-ras locus to T-cell transformation are discussed.     

The effect of alterations in myc gene expression on B cell development in the bursa of Fabricius

Infection of 18-day embryonic bursal lymphocytes with a v-myc-containing retrovirus leads directly to a polyclonal proliferation of surface immunoglobulin-positive (slg+) cells in the bursa of Fabricius detected four weeks after hatching. These v-myc-expressing bursal cells repopulate the follicles of chemically ablated bursae more efficiently than total normal 18-day embryonic bursal cells. In contrast, comparable normal bursal cells lose the ability to repopulate follicles by four weeks. Bursal lymphocytes expressing either a retroviral v-myc or a c-myc gene deregulated by adjacent retroviral integration retain the ability of embryonic bursal lymphocytes to diversify their immunoglobulin light chain genes. These results suggest that retroviral deregulation of myc expression during avian B cell development induces outgrowth of a population of cells with the cardinal phenotypic characteristics of bursal stem cells.     

3''-Terminal region of avian carcinoma virus MH2 shares sequence elements with avian sarcoma viruses Y73 and SR-A.

We determined the nucleotide sequence of the acute transforming avian retrovirus MH2 from an HgiAI site within the coding region of its oncogene, v-myc, to the KpnI site within the long terminal repeat. Comparison with published sequences from other retroviruses allowed us to identify all sequence elements in this region. We conclude that MH2 contains a unique assembly of 3'-terminal sequences, which includes part of the helper virus-derived SPC region of avian sarcoma virus Y73 and the complete F3 and F1 segments of Rous sarcoma virus strain SR-A.     

Provirus of M7 baboon endogenous virus: nucleotide sequence of the gag-pol region

A 3,023-base nucleotide sequence of the M7 baboon endogenous virus genome, spanning the 5' noncoding region as well as the entire gag gene and part of the pol gene, is reported. Within the 562-base 5' noncoding region, a 21-base sequence complementary to the OH terminus of tRNApro is located immediately downstream from the long terminal repeat. Amino acid sequences were deduced from the 1,596 nucleotides comprising the gag gene, and the four structural gag polypeptides, p12, p15, p30, and p10, appeared to be coded contiguously. Only one termination codon interrupted the M7 gag and pol genes. The data suggest that 55 additional amino acids may be attached to the NH2 terminus of the gag precursor protein. However, such a sequence was not detected in virions or in virus-infected cells. With the exception of the p15 region, nucleotide and amino acid sequences of the gag and pol regions of M7 virus exhibited strong homologies to those of Moloney leukemia virus.