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.     

Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA.

A biologically active molecular clone of BALB/Moloney mink cell focus-forming (Mo-MCF) proviral DNA has been reconstructed in vitro. It contains the 5' half of BALB/Moloney murine leukemia virus (Mo-MuLV) DNA and the 3' half of BALB/Mo-MCF DNA. The complete nucleotide sequence of the env gene and the 3' long terminal repeat (LTR) of the cloned Mo-MCF DNA has been determined and compared with the sequence of the corresponding region of parental Mo-MuLV DNA. The substitution in the Mo-MCF DNA encompasses 1,159 base pairs, beginning in the carboxyl terminus of the pol gene and extending to the middle of the env gene. The Mo-MCF env gene product is predicted to be 29 amino acids shorter than the parental Mo-MuLV env gene product. The portion of the env gene encoding the p15E peptide is identical in both viral DNAs. There is an additional A residue in the Mo-MCF viral DNA in a region just preceding the 3' LTR. The nucleotide sequence of the 3' LTR of Mo-MCF DNA is similar to that of the 5' LTR of BALB/Mo-MuLV DNA with the exception of two single base substitutions. We conclude that the sequence substitution in the env gene is responsible for the dual-tropic properties of Mo-MCF viruses.     

Heteroduplex analysis of the sequence relations between the RNAs of mink cell focus-inducing and murine leukemia viruses.

The sequence relationships betwen AKR ecotropic virus and an AKR-derived "mink cell focus-inducing" (MCF) isolate (AKR MCF 247), between Moloney murine leukemia virus (M-MLV) and an M-MLV MCF isolate (M-MLV83), and between AKR and M-MLV were studied by electron microscopic heteroduplex analysis. The MCF-specific sequences were found to map from 1.95 kilobases (kb) to 2.75 kb (+/- 0.15 kb) from the 3' end of the RNAs for both MCF isolates. The major sequence nonhomology regions between AKR and M-MLV lie between 0.9 and 3.5 kb from the 3' end. However, the AKR and M-MLV sequences immediately adjacent to the 1.95- and 2.75-kb junctions with MCF-specific sequences are relatively similar in AKR and M-MLV. Our results suggest that the env gene of MLVs maps from 1 kb to 3 kb from the 3' end of the genomic RNA and that the carboxyl end of the glycoprotein of each MCF strain is similar (or identical) to that of its ecotropic parent.     

Comparison of the restriction endonuclease maps of unintegrated proviral DNAs from four xenotropic murine leukemia viruses.

From purified linear and superhelical DNAs, the restriction endonuclease maps of four xenotropic murine leukemia virus DNAs from NFS, NZB, BALB/c, and AKR mice were determined with ten restriction endonucleases. Each xenotropic proviral DNA was found to be a unique restriction endonuclease map, with differences in the gag, pol, env, and terminal repeated sequence regions. However, type-specific SacI and EcoRI sites in the env region were identical in all four xenotropic murine leukemia virus DNAs and were not found in ecotropic murine leukemia virus DNA. Comparison of the xenotropic murine leukemia virus DNA maps with maps of ecotropic murine leukemia virus DNA showed that the pol and terminal repeated sequence regions were highly conserved. Other similarities in ecotropic and some xenotropic viral DNAs suggest common origins.     

Genetic analysis of myeloproliferative leukemia virus, a novel acute leukemogenic replication-defective retrovirus.

The myeloproliferative leukemia virus (MPLV) is a new acute leukemogenic, nonsarcomatogenic retroviral complex that is generated during the in vivo passage of a molecularly cloned Friend ecotropic helper virus. Examination of viral RNA expression in MPLV-producing cells revealed the presence of two distinct molecular species that hybridized with a long terminal repeat or an ecotropic env-specific probe but not with a xenotropic mink cell focus-forming virus env-specific probe derived from a spleen focus-forming virus: an 8.2-kilobase species corresponding to a full-length Friend murine leukemia virus (F-MuLV) and a deleted species with a genomic size of 7.4 kilobases. This deleted virus was biologically cloned by limiting dilutions and single cell cloning in Mus dunni fibroblasts. Three nonproducer clones with normal morphologies and containing one single integrated copy of the deleted virus were superinfected with F-MuLV, Moloney murine leukemia virus, Gross murine leukemia virus, mink cell focus-forming virus (HIX), or the amphotropic 1504 murine leukemia virus. All pseudotypes caused macroscopic and microscopic abnormalities in mice that were similar to those seen in the parental stock. A comparison of the physical maps of F-MuLV and MPLV, which was deduced from the restriction enzyme digests of unintegrated proviral DNAs, indicated that the MPLV-defective genome (i) is probably derived from F-MuLV, (ii) has conserved the F-MuLV gag and pol regions, and (iii) is deleted and rearranged in the env region in a manner that is clearly distinct from that of Friend or Rauscher spleen focus-forming viruses.     

Nucleotide sequence of the env-specific segment of NFS-Th-1 xenotropic murine leukemia virus.

The sequence of 863 contiguous nucleotides encompassing portions of the pol and env genes of NFS-Th-1 xenotropic proviral DNA was determined. This region of the xenotropic murine leukemia virus genome contains and env-specific segment that hybridizes exclusively to xenotropic and mink cell focus-forming but not to ecotropic proviral DNAs (C. E. Buckler et al., J. Virol. 41:228-236, 1982). The unique xenotropic env segment contained several characteristic deletions and insertions relative to the analogous region in AKR and Moloney ecotropic murine leukemia viruses. Portions of an endogenous env segment cloned from a BALB/c mouse embryo gene library that had a restriction map and hybridization properties typical of xenotropic viruses (A. S. Khan et al., J. Virol. 44:625-636, 1982) were also sequenced. The sequence of the endogenous env gene was very similar to the comparable region of the NFS-Th-1 xenotropic virus containing the characteristic deletions and insertions previously observed and could represent a segment of an endogenous xenotropic provirus.     

Heteroduplex study of the sequence relations between RD-114 and baboon viral RNAs.

The regions of sequence homology and nonhomology between the RNA genomes of RD-114 and baboon endogenous type C viruses have been mapped by an electron microscope heteroduplex study. Short complementary DNA (cDNA) copies (approximately 150 to 200 nucleotides in length) of RD-114 RNA were prepared by an endogenous synthesis; labels of polydeoxythymidylic acid [poly(dT)] were attached to the 3' ends of the cDNA molecules by a reaction catalyzed by deoxynucleotidyl terminal transferase. The cDNA-poly(dT) was hybridized to RD-114 RNA and to baboon viral RNA dimer (50 to 70S) units, and the position- of the poly(dT) labels were observed by electron microscopy. With RD-114, labels were distributed uniformly along the genome. With baboon virus RNA (monomer length, 9.5 kilobases [kb]), the regions of high homology with RD-114 cDNA were observed to lie in the intervals from 1.5 to 2.5 kb and from 3.7 to 5.5 kb from the 5' end. The relations of these heteroduplex maps to the known antigenic similarities and differences among the several viral proteins and to the genetic maps of the viruses are discussed.     

RNA-directed DNA synthesis in Moloney murine leukemia virus: interaction between the primer tRNA and the genome RNA.

Initiation of RNA-directed DNA synthesis in virions of Moloney murine leukemia virus requires a cellular tRNAPro as primer. The site(s) on the Moloney murine leukemia virus genome RNA at which functional primer molecules are bound and at which purified tRNAPro hybridizes has been located near (within 20%) the 5' end of the genome. A relatively stable duplex (temperature at which 50% dissociation has occurred, 76 degrees C) is formed between the amino acid acceptor stem of the tRNAPro and a complementary sequence in the Moloney murine leukemia virus 35S RNA. The interaction involves 19 base pairs, extending from the penultimate nucleotide at the 3' end of the tRNAPro but apparently not including the 3'-terminal adenosine residue. In most respects, the interaction between primer and template in Moloney murine leukemia virus parallels the situation in the avian leukosis-sarcoma viruses.     

Analysis of the myeloproliferative sarcoma virus genome: limited changes in the prototype lead to altered target cell specificity.

The myeloproliferative sarcoma virus (MPSV) derived from Moloney sarcoma virus (MSV-Mol) is a unique sarcoma virus which causes expansion of the hematopoietic stem cell compartment as well as the erythroid and myeloid cell lineages. MPSV also induces spleen focus formation in adult mice as do Friend and Rauscher viruses. Analysis of the MPSV genome on methyl mercury gels showed that the genome size is 7.0 kilobases, which is larger than the defective genome of any known MSV-Mol isolate. Hybridization analysis with specific cDNA probes showed that MPSV is a modified sarcoma virus with no sequences in the unique region of the defective sarcoma genome related to unique Friend virus sequences. The only viral sequences in the defective genome other than helper virus-related sequences are derived from the Moloney sarcoma virus genome with no new cellular sequences added. There was no evidence for induction of xenotropic virus sequences in MPSV-infected spleens of DBA/2J mice, indicating that spleen focus formation can be obtained by different mechanisms.     

Heteroduplex analysis of molecular clones of the pathogenic Friend virus complex: Friend murine leukemia virus, Friend mink cell focus-forming virus, and the polycythemia- and anemia-inducing strains of Friend spleen focus-forming virus.

The pathogenic Friend virus complex is of considerable interest in that, although members of this group are genetically related, they differ markedly in biochemical and biological properties. Heteroduplex mapping of molecular clones of the Friend virus complex, which includes the replication-competent ecotropic Friend murine leukemia virus (F-MuLV) and mink cell focus-forming virus (F-MCF) and replication-defective polycythemia- and anemia-inducing strains of spleen focus-forming virus (SFFVp and SFFVa, respectively), was employed to provide insight into the molecular basis of their relationships. In heteroduplexes of F-MuLV X F-MCF, a major substitution of 0.89 kilobases in the env gene of F-MCF was discerned. Heteroduplexes of SFFVp X F-MuLV or F-MCF and SFFVa X F-MuLV or F-MCF showed several major deletions in the pol gene region and a single major deletion in the 3' half of the env gene region of SFFVp and SFFVa. A major substitution of 0.89 kilobases was mapped to the 5' end of the env deletion of SFFVp and SFFVa in heteroduplexes with F-MuLV, similar to that seen in F-MuLV X F-MCF heteroduplexes. In contrast, this env gene region was totally homologous in F-MCF X SFFVp or SFFVa and SFFVp X SFFVa heteroduplexes. Our results suggest that (i) both SFFVp and SFFVa lack part of the env gene at its 3' end, corresponding to the p15(E) coding region, (ii) major deletions occur in the pol and env genes which account for the replication defectiveness of SFFVp and SFFVa, (iii) minor substitutions occur in the gag gene region of SFFVa that are not present in SFFVp, F-MuLV, or F-MCF, (iv) a major substitution exists in the gp70 region of the env gene between F-MuLV and F-MCF that probably accounts for the differences in their host range specificities, (v) this substitution in F-MCF is identical to the gp70 part of the gp52 coding region of SFFVp and SFFVa, and (vi) heteroduplexes to F-MCF show unambiguously that no additional large substitutions are present in SFFVp or SFFVa that could account for differences in their leukemogenicity.     

Avian reticuloendotheliosis virus: characterization of genome structure by heteroduplex mapping

The genome structure of defective, oncogenic avian reticuloendotheliosis virus (REV) was studied by heteroduplex mapping between the full-length complementary DNA of the helper virus REV-T1 and the 30S REV RNA. The REV genome (5.5 kilobases) had a deletion of 3.69 kilobases in the gag-pol region, confirming the genetic defectiveness of REV. In addition, REV lacked the sequences corresponding to the env gene but contained, instead, a contiguous stretch (1.6 to 1.9 kilobases) of the specific sequences presumably related to viral oncogenicity. Unlike those of other avian acute leukemia viruses, the transformation-specific sequences of REV were not contiguous with the gag-pol deletion. Thus, REV has a genome structure similar to that of a defective mink cell focus-inducing virus or a defective murine sarcoma virus. An additional class of heteroduplex molecules containing the gag-pol deletion and two other smaller deletion loops was observed. These molecules probably represented recombinants between the oncogenic REV and its helper virus.     

Biochemical characterization of the amphotropic group of murine leukemia viruses.

The recently described amphotropic group of murine leukemia viruses constitutes a distinct biological group, differing from the ecotropic and xenotropic groups in host range, cross interference, and serological reactivity. Viruses of this group have been detected only in wild mice from certain areas in California. By using a [3H]DNA probe synthesized in an endogenous reaction from detergent-lysed amphotropic virus (strain 1504-A), it was demonstrated that the amphotropic murine leukemia viruses are distinct biochemically, in that 20% of the viral genome sequences are not shared by AKR-type ecotropic or nay of three types of xenotropic murine leukemia virus tested. A subset of these amphotropic unique sequences, comprising one half of them, is present in the genome of wild mouse ecotropic viruses and in Moloney and Rauscher viruses as well. Sequences homologous to the entire genome of 1504-A amphotropic virus are present in the cellular DNA of all eight inbred mouse strains tested, as well as in wild Mus in Asia, in amounts varying from three to six complete viral genomes per haploid cell genome. Evidence is presented that at least 20% of the DNA sequences in both mouse- and mink-grown murine leukemia virus probes are of host-cell origin.     

Characterization of the env gene in avian oncoviruses by heteroduplex mapping.

The genome of ring-necked pheasant virus, an avian oncovirus, is largely homologous to the genomes of chicken oncoviruses except for a specific nonhomology in env, the gene coding for the surface glycoprotein of the virion (J. Tal, D. J. Fujita, S. Kawai, H. E. Varmus, and J. M. Bishop, J. Virol. 21:497--505, 1977). We have used this nonhomology between ring-necked pheasant virus and chicken oncoviruses in electron microscopic studies of heteroduplex molecules. The env-specific region of nonhomology is 1.5 to 1.7 kilobases in length. Its 3' boundary is located 0.6 to 0.7 kilobases from the 3' end of the genome in transformation-defective viruses and 2.5 kilobases from the 3' end in nondefective avian sarcoma viruses. Comparison of several strains of avian oncoviruses shows that the 3' half of this env region is conserved, while the 5' half is more diverged. A small area at the very 3' end of env also shows divergence between different avian oncoviruses. We found no evidence for the presence of a previously unrecognized gene between env and src. An electrophoretic comparison of the glycoproteins from various avian oncoviruses shows that those of ring-necked pheasant virus and Chinese quail virus differ in molecular weight from the glycoproteins of the chicken oncoviruses.     

Relationship Between Moloney Murine Leukemia and Sarcoma Virus RNAs: Purification and Hybridization Map of Complementary DNAs from Defined Regions of Moloney Murine Sarcoma Virus 124

Complementary DNAs (cDNA's) specific for various regions of the Moloney murine sarcoma virus (MSV) 124 RNA genome were prepared by cross-hybridization techniques. A cDNA specific for the first 1,000 nucleotides adjacent to the RNA 3' end (cDNA 3') was prepared and shown to also be complementary to the 3'-terminal 1,000 nucleotides of a related Moloney murine leukemia virus (MLV) genome. A cDNA complementary to the "MSV-specific" portion of the MSV 124 genome was prepared. This cDNA was shown not to anneal to Moloney MLV RNA and to anneal to a portion of the viral RNA of about 1,500 to 1,800 nucleotides in length, located 1,000 nucleotides from the 3' end of MSV RNA. A cDNA common to the genome of MSV and MLV was also obtained and shown to anneal to the 5'-terminal two-thirds, as well as to the 3'-terminal 1,000 nucleotides, of the MSV RNA genome. This cDNA also annealed to the RNA from MLV and mainly to the 5'-terminal half of the MLV genome. It is concluded that the 6-kilobase Moloney MSV 124 RNA genome has a sequence arrangement that includes (i) a 3' portion of about 1,000 nucleotides, which is also present at the 3' terminus of MLV; (ii) an MSV-specific region, not shared with MLV, which extends between 1,000 and 2,500 nucleotides from the 3' terminus; and (iii) a second "common" region, again shared with MLV, which extends from 2,500 nucleotides to the 5' terminus. This second common region appears to be located in the 5' half of the 10-kilobase MLV genome as well. Experiments in which a large excess of cold MLV cDNA was annealed to (3)H-labeled polyadenylic acid-containing fragments of MSV RNA gave results consistent with this arrangement of the MSV genome.     

Avian acute leukemia virus OK 10: analysis of its myc oncogene by molecular cloning.

Several DNAs representing the genome of the avian acute leukemia virus OK 10 were isolated by molecular cloning from a transformed quail cell line, 9C, which contained at least six OK 10 proviruses. Recombinant lambda phages harboring the OK 10 genome and additional flanking cellular DNA sequences were studied by restriction endonuclease mapping and hybridization to viral cDNA probes. Six of the clones represented complete proviruses with similar, if not identical, viral sequences integrated at different positions in the host DNA. The organization of the OK 10 genome was determined by electron-microscopic analysis of heteroduplexes formed between the cloned OK 10 DNA and DNAs representing the c-myc gene and the genomes of two other avian retroviruses, Rous-associated virus-1 and MC29. The results indicated that the OK 10 proviral DNA is about 7.5 kilobases in size with the following structure: 5'-LTR-gag-delta polmyc-delta env-LTR-3', where LTR indicates a long terminal repeat. The oncogene of OK 10, v-mycOK 10, forms a continuous DNA segment of around 1.7 kilobases between pol and env. It is similar in structure and length to the v-myc gene of MC29, as demonstrated by restriction endonuclease and heteroduplex analyses. Two of the OK 10 proviruses were tested in transfection experiments: both DNAs gave rise to virus with the transforming capacities of OK 10 when Rous-associated virus-1 was used to provide helper virus functions.     

Fv-1 determinants in xenotropic murine leukemia viruses studied with biological assay systems: Isolation of xenotropic virus with N-tropic Fv-1 activity in the cryptic form.

By a biological assay system using phenotypically mixed ecotropic and xenotropic murine leukemia viruses, we investigated whether in the virions of a xenotropic virus there is N- or B-tropic Fv-1 determinant in active form. The existence of N-tropic Fv-1 determinant was demonstrated in SL-XT-1 xenotropic virus isolated from the spleen of a 3-month-old SL mouse, and the N-tropic Fv-1 tropism was confirmed by analysis of the phenotypically mixed viruses harvested from clonal SC-1 cells doubly infected with the SL-XT-1 and B-tropic ecotropic viruses. However, neither N- nor B-tropic Fv-1 determinant was demonstrated in any xenotropic viruses isolated from embryo cells of BALB/c, NZB, or DBA/2 mice, or Cas E #1-IU, and xenotropic-like virus isolated from a wild mouse.     

Characterization of mouse cellular deoxyribonucleic acid homologous to Abelson murine leukemia virus-specific sequences.

The genome of Abelson murine leukemia virus (A-MuLV) consists of sequences derived from both BALB/c mouse deoxyribonucleic acid and the genome of Moloney murine leukemia virus. Using deoxyribonucleic acid linear intermediates as a source of retroviral deoxyribonucleic acid, we isolated a recombinant plasmid which contained 1.9 kilobases of the 3.5-kilobase mouse-derived sequences found in A-MuLV (A-MuLV-specific sequences). We used this clone, designated pSA-17, as a probe restriction enzyme and Southern blot analyses to examine the arrangement of homologous sequences in BALB/c deoxyribonucleic acid (endogenous Abelson sequences). The endogenous Abelson sequences within the mouse genome were interrupted by noncoding regions, suggesting that a rearrangement of the cell sequences was required to produce the sequence found in the virus. Endogenous Abelson sequences were arranged similarly in mice that were susceptible to A-MuLV tumors and in mice that were resistant to A-MuLV tumors. An examination of three BALB/c plasmacytomas and a BALB/c early B-cell tumor likewise revealed no alteration in the arrangement of the endogenous Abelson sequences. Homology to pSA-17 was also observed in deoxyribonucleic acids prepared from rat, hamster, chicken, and human cells. An isolate of A-MuLV which encoded a 160,000-dalton transforming protein (P160) contained 700 more base pairs of mouse sequences than the standard A-MuLV isolate, which encoded a 120,000-dalton transforming protein (P120).     

Murine Mammary Tumor Virus Expression During Mammary Tumorigenesis in BALB/c Mice

Steady-state levels of murine mammary tumor virus (MuMTV) RNA were quantitated during mammary tumorigenesis in BALB/c mice by molecular hybridization with a representative MuMTV complementary DNA (cDNA) probe. Hyperplastic alveolar nodule (HAN) lines are preneoplastic mammary lesions that were induced in BALB/c mice by hormones alone or in combination with 7,12-dimethylbenz(a)anthracene and give rise to mammary tumors. The hormone-induced HAN lines D1 and D2 contained detectable amounts of hybridizable MuMTV sequences. MuMTV RNA sequences were also observed in five of the six transplanted BALB/c mammary tumors that were examined. Similar levels of hybridizable MuMTV RNA were observed between the D1 or D2 HAN line and mammary tumors derived from each HAN line. The D2 HAN line as well as D2, C4, and CD8 mammary tumors accumulated RNA that was apparently homologous to most of the MuMTV genome. Thermal denaturation of hybrids indicated extensive sequence homology between the MuMTV cDNA and hybridizable RNA in the BALB/c HAN lines and mammary tumors. A low level of type C viral RNA was observed in the BALB/c HAN lines and most mammary tumors by molecular hybridization with a cDNA to Moloney murine leukemia virus. These data demonstrate that MuMTV sequences are frequently expressed in hormone-induced BALB/c HAN lines and mammary tumors derived from HAN lines or ductal hyperplasias induced in BALB/c mice by hormones and/or a chemical carcinogen. The transition from the preneoplastic to the neoplastic state in BALB/c mice does not appear to be due to a change in the steady-state levels of MuMTV RNA since the hormone-induced HAN lines and mammary tumors had similar levels of hybridizable MuMTV RNA.