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.     

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.     

Phosphorylation of the nonstructural proteins encoded by three avian acute leukemia viruses and by avian fujinami sarcoma virus.

The gag gene-related, nonstructural proteins of three avian acute leukemia viruses (namely, myelocytomatosis viruses MC29 and CMII and avian erythroblastosis virus) and of avian Fujinami sarcoma virus (FSV) isolated by immunoprecipitation from cellular lysates with anti-gag serum were shown to be phosphoproteins in vivo. The specific 32P radioactivity of the nonstructural proteins of MC29, CMII, and FSV was significantly higher than that of helper viral, intracellular gag proteins. Two of these proteins, i.e., the 140,000-dalton FSV and the 110,000-dalton MC29 proteins, were also phosphorylated in vitro by a kinase activity associated with immunocomplexes. This kinase activity is either separated from these proteins or inactivated by incubation of cellular lysates with normal serum followed by adsorption to staphylococcal protein A or sedimentation at 100,000 x g or both. It remains to be resolved whether the 110,000-dalton MC29 and 140,000-dalton FV proteins, in addition to being substrates for phosphorylation, also have intrinsic kinase activity.     

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 acute leukemia virus OK10 has an 8.2-kilobase genome and modified glycoprotein gp 78

We have analyzed the structure of OK10-BM virus, an avian acute leukemia virus produced by a bone marrow-derived cell line of macrophage origin, and compared it with that of OK10 AV, an associated virus originally present in the OK10 virus stock. The RNAs of OK10-BM virus and OK10 AV had the same mobility in agarose gels, corresponding to 8.0 to 8.5 kilobases, a size considerably larger than that of the transforming component (5 to 6 kb) of most other avian acute leukemia viruses. Fingerprint analysis showed a close relationship between OK10-BM virus and OK10 AV RNAs. The polypeptide compositions of OK10-BM and OK10 AV viruses were similar except for the envelope glycoproteins. In analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the large envelope glycoprotein of OK10-BM virus migrated at M_r = 78,000 (gp78), whereas OK10 AV had the characteristic 85,000-dalton glycoprotein (gp85) of nondefective avian leukemia viruses. gp78 was weakly labeled with methionine, glycine, proline, or mannose, suggesting that purified OK10-BM virus had reduced amounts of the modified envelope glycoprotein. In cell-free rabbit reticulocyte lysates, OK10-BM virion RNA directed the synthesis of a 200,000-dalton polypeptide (p200), a 180,000-dalton polypeptide (pr180), and a 76,000-dalton polypeptide (pr76), whereas OK10 AV RNA gave rise only to pr180 and pr76, suggesting that p200 may represent an OK10-BM-encoded transforming protein. No biochemical evidence for the presence of an associated helper virus was found in the OK10-BM virus population produced by the macrophage cell line. However, when OK10-BM virus was serially passaged in chicken embryo fibroblasts, a virus having structural properties similar to those of OK10 AV (OK10 AV-specific oligonucleotides and gp85) appeared after three passages. Moreover, nonproducer clones of transformed cells could be readily obtained in OK10-BM virus-infected quail cell cultures. It is thus likely that the bone marrow-derived macrophage cell line produces a transforming virus defective in its env gene and low amounts of an associated helper virus, which upon transfer to fibroblasts is preferentially replicated.     

Avian erythroblastosis virus produces two mRNA''s.

We analyzed the viral mRNA's present in fibroblast nonproducer clones transformed by avian erythroblastosis virus. Two size classes of mRNA (28 to 30S and 22 to 24S) were identified by solution hybridization with both complementary DNA strong stop and complementary DNA made against the unique sequences of avian erythroblastosis virus. Based upon the kinetics of hybridization with complementary DNA made against the unique sequences of avian erythroblastosis virus, we estimated that there were 400 to 500 copies of the 28 to 30S RNA per cell and 200 to 250 copies of the 22 to 24S RNA per cell. Both RNA species were packaged in the virion. In vitro translation of the 28 to 30S virion RNA yielded a 75,000-dalton protein which was the 75,000-dalton gag-related polyprotein found in avian erythroblastosis virus-transformed cells. In vitro translation of the 22 to 24S virion RNA yielded two proteins (46,000 and 48,000 daltons). This indicates that there may be two genes in avian erythroblastosis virus, one coding for the 75,000-dalton gag-related polyprotein and the second coding for the 46,000- or 48,000-dalton protein or both.     

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.     

Deletions Within the Transformation-Specific RNA Sequences of Acute Leukemia Virus MC29 Give Rise to Partially Transformation-Defective Mutants

The viral RNAs of three nonconditional mutants of avian myelocytomatosis virus MC29 were analyzed. These mutants, which were originally isolated from the quail producer line Q10 and were designated 10A, 10C, and 10H, have lost most of the ability to transform hematopoietic cells in vitro and to induce tumors in vivo, but they still transform cultured fibroblasts with the same efficiency as wild-type (wt) MC29. Electrophoretic analyses showed that the mutant genomic RNAs were smaller than the 5.7-kilobase genome of wt MC29; the genomes of mutants 10A, 10C, and 10H were about 5.5, 5.3, and 5.1 kilobases long, respectively. Analyses of the transformation-specific sequences of these mutant RNAs by a combination of T(1) oligonucleotide fingerprinting and hybridization with cDNA from the transformation-specific sequences myc of wt MC29 or competition hybridization including wt MC29 RNA revealed that deletions of myc-specific sequences had occurred. The deletions in all three mutants overlapped, since they all had lost one particular myc-specific oligonucleotide. In agreement with the size of the genomic RNAs, mutants 10C and 10H had lost two additional myc oligonucleotides, and mutant 10A contained a modified myc oligonucleotide. The locations of the deletions were deduced from comparisons with previously established oligonucleotide maps of several members of the MC29 subgroup of acute leukemia viruses and by hybridization of wt and mutant RNAs to molecularly cloned subgenomic fragments of wt MC29 proviral DNA, representing the 5' and 3' domains of the myc sequence. We found that the deleted sequences represented overlapping internal segments of the myc sequence and that the borders of myc with the partial complements of the virion genes gag and env appeared to be conserved in mutant and wt MC29 RNAs. The correlation between the altered transforming potential for hematopoietic cells and the partial deletion of myc in the mutant RNAs provided direct genetic evidence for the involvement of myc in oncogenesis. However, the unaffected efficiency of these mutants in fibroblast transformation suggested that the deleted sequences are not essential for the fibroblast-transforming potential of the onc gene of MC29.     

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.     

Comparison of the genomic organization of Kirsten and Harvey sarcoma viruses.

Current studies were undertaken to compare the genomes of Kirsten murine sarcoma virus (Ki-MuSV), Harvey murine sarcoma virus (Ha-MuSV), and the replication-defective endogenous rat virus to understand the function of these viral RNAs. Genome organization and sequence homology were studied by fingerprinting large RNase T1-resistant oligonucleotides and by cross-protecting homologous oligonucleotides against RNase A and T1 digestion with complementary DNA prepared from each of the other viral RNA. Ki-MuSV and Ha-MuSV were found to share an extensive series of rat-derived oligonucleotides begining ca. 1 kilobase (kb) from the 3' end and extending to within 1.5 kb of the 5'end of Ki-MuSV RNA. The total map distance covered in ca. 5.5 kb. The eight oligonucleotides covering the 1.5 kb at the 5' end of Ki-MuSV RNA were not found in Ha-MuSV RNA. Five out of these eight oligonucleotides, however, could be designated with certainty to be of rat virus origin. Since Ha-MuSV is 6.5 kb in size and Ki-MuSV is 8 kb in size, the major difference between them is the 1.5 kb from the replication-defective endogenous rat virus sequences at the 5' end of Ki-MuSV not present in Ha-MuSV. Consistent with the difference in the genome structure, these two sarcoma viral RNA'S yielded distinct major translation products in cell-free systems, I.E., A 50,000-dalton polypeptide (P50) from Ki-MuSV and a 22,000-dalton polypeptide (p22) from Ha-MuSV. These polypeptides may provide the necessary protein makers for identifying in vivo virus-coded proteins.     

Cell-free translation of avian erythroblastosis virus RNA

Avian erythroblastosis virus (AEV) RNA rescued from nonproducer cells by superinfection with a helper virus is translated into three polypeptides in the messenger-dependent rabbit reticulocyte lysate. A 75,000 molecular weight polypeptide (P75AEV) is synthesized from 28S RNA and is encoded by the 5' section of the AEV RNA, including gag-related and AEV-specific sequences. The P75AEV synthesized in infected cells and the P75AEV synthesized in the cell-free system are electrophoretically identical. A 44,000 molecular weight polypeptide (P44AEV) is synthesized from 20-24S RNA, apparently from the 3' section of the AEV-specific RNA sequence. A minor 37,000 molecular weight polypeptide (P37AEV) is synthesized from 20S AEV RNA. A comparison is drawn between the cell-free products of MC29 and AEV RNAs.     

gag-Related polypeptides encoded by replication-defective avian oncoviruses.

The content of viral structural (gag) protein sequences in polypeptides encoded by replication-defective avian erythroblastosis virus (AEV) and myelocytomatosis virus MC29 was assessed by immunological and peptide analyses. Direct comparison with gag proteins of the associated helper viruses revealed that MC29 110K polypeptide contained p19, p12, and p27, whereas the AEV 75K polypeptide had sequences related only to p19 and p12. Both of these polypeptides contained some information that was unrelated to gag, pol, or env gene products. In addition, no homology was detected between these unique peptides of MC29 110K and AEV 75K. The AEV 75K polypeptide shared strain-specific tryptic peptides with the p19 encoded by its naturally occurring helper virus; this observation suggests that gag-related sequences in 75K were originally derived from the helper viral gag gene. Digestion of oxidized MC29 110K and AEV 75K proteins with the Staphylococcus aureus V8 protease generated a fragment which comigrated with N-acetylmethionylsulfoneglutamic acid, a blocked dipeptide which is the putative amino-terminal sequence of structural protein p19 and gag precursor Pr76gag. This last finding is evidence that the gag sequences are located at the N-terminal end of the MC29 110K and AEV 75K polypeptides.     

Particle and naked RNA mycoviruses in industrially cultivated mushroom Pleurotus ostreatus in China

Many cultivated mushroom strains, such as Pleurotus ostreatus TD300, displayed symptoms of degeneration. A spherical virus POSV and four dsRNA segments were extracted from mycelium of P. ostreatus TD300. POSV had a diameter of 23 nm and encapsidated a 2.5kb dsRNA segment with coat proteins whose molecular weights were 39 kDa and 30 kDa. Four dsRNA segments were 8.2 kb, 2.5 kb, 2.0 kb, and 1.1 kb in size, respectively. The 1.1 kb dsRNA segment often escaped detection. The cDNA and the amino acid sequences of the 8.2 kb dsRNA were homologous to those of RNA-dependent RNA polymerases (RDRP) of ssRNA oyster mushroom spherical virus (OMSV), and contained conserved motifs A to D which were almost identical to those in RDRP of OMSV. The cDNA and amino acid sequences of the 2.5 kb and 2.0 kb dsRNA segments were homologous to that of RDRP and capsid protein of dsRNA virus P. ostreatus virus 1 (PoV1), respectively. In particular, the amino acid sequence of 2.5 kb dsRNA segment had high identity with the conserved motifs A to C in RDRP of PoV1, a Partiviridae virus. After eliminating the viruses in P. ostreatus TD300, the symptoms of degeneration completely disappeared. The results reveal that P. ostreatus TD300 was at least infected by a particle virus POSV, and two naked viruses, one was a dsRNA virus with a 2.0 kb dsRNA segment, the other was an ssRNA virus whose replicating form of genome was an 8.2 kb dsRNA segment. Mycoviruses infection is a causative agent of mushroom strain degeneration.     

Nuclear location of the putative transforming protein of avian myelocytomatosis virus

The putative transforming protein of avian myelocytomatosis virus MC29 is a 110,000 dalton (P110gag-myc) polyprotein comprised of sequences derived from both the gag region and the MC29-specific myc region. Two approaches have been taken to determine the location of the MC29 gag-related proteins in transformed cells: subcellular fractionation and immunofluorescence. Analysis of subcellular fractions of MC29-transformed cells by immunoprecipitation indicates that the majority of the gag-myc polyprotein is found in the nuclear fractions of Q8 cells (a nonproducer line of MC29-transformed quail embryo fibroblasts) and nonproducer cells derived from a liver tumor of MC20-infected quail. This is in contrast to the distribution of gag-related helper virus proteins lacking myc, which are found only in nonnuclear fractions of superinfected Q8 cells. The purity of unlabeled nuclei was assessed by electron microscopy and enzyme assays, revealing little contaminating material from other subcellular fractions. Immunofluorescence experiments using monospecific anti-gag serum showed specific, intense immunofluorescence in the nuclei of fixed Q8 cells. In contrast, the majority of P75gag-erb, a candidate transforming protein produced by avian erythroblastosis virus (AEV), is absent from the nuclei of nonproducer AEV-transformed chick embryo fibroblasts. The nuclear association of the MC29 transforming protein may be related to some of the unique properties of MC29-transformed cells.