Comparison of myeloproliferative sarcoma virus with Moloney murine sarcoma virus variants by nucleotide sequencing and heteroduplex analysis.

The myeloproliferative sarcoma virus (MPSV) was derived by passage of Moloney sarcoma virus (Mo-MuSV) in adult mice. Mo-MuSV variants transform fibroblasts. However, MPSV also affects erythroid, myeloid, and hematopoietic stem cells. The MPSV proviral genome, two temperature-sensitive mutants derived from it, Mo-MuSV variant M1, and Moloney murine leukemia virus (Mo-MuLV) were compared by heteroduplex mapping. MPSV wild type was found to have 1 kilobase pair deleted from the pol gene and to contain v-mos-related sequences. The 3' end of MPSV, including the oncogene-helper junctions, the v-mos gene, and the 3' long terminal repeat, was sequenced and compared with sequences of Mo-MuLV, MSV-124, and the mouse oncogene c-mos. From these data, MPSV appears to be either closely related to the original Mo-MuSV or an independent recombinant of Mo-MuLV and c-mos. Five possible explanations of the altered specificity of MPSV are considered. (i) The MPSV mos protein has properties inherent in c-mos but lost by other Mo-MuSV mos proteins. (ii) The MPSV mos protein has altered characteristics due to amino acid changes. (iii) Due to a frameshift, MPSV codes for a mos protein truncated at the amino terminal and also a novel peptide. (iv) A second novel peptide may be encoded from the 3' env region. (v) MPSV has long terminal repeats and an enhancer sequence more like Mo-MuLV than Mo-MuSV, with a consequently altered target cell specificity.     

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.     

Molecularly cloned c-mos(rat) is biologically active.

A unique rat cellular gene, c-mos(rat), homologous to the transforming sequences, v-mos, of Moloney murine sarcoma virus (M-MSV) was detected by hybridization to a v-mos specific probe. The c-mos(rat) gene was cloned together with its flanking sequences in an 11-kbp EcoRI DNA fragment inserted in vector Charon 4A. Two probes were used to investigate the position and orientation of c-mos(rat) in the clone examined ( D3e ), namely pMSV -31 which contains the sequences specific for the transforming sequences of M-MSV and pCS-1 which harbors 0.5 kbp of 5'-terminal sequences of c-mos(mouse) as well as 0.7 kbp of its flanking sequences. After ligation of a restriction fragment of clone D3e containing c-mos(rat) to a fragment containing the long terminal repeat of M-MSV and transfection of the DNA onto rat cells, we detected foci of transformed cells, thus showing that c-mos(rat) is biologically active. Using DNA framents derived from clone D3e , we studied the conservation of c-mos and of its flanking sequences in several species. c-mos(rat) as well as some of its flanking sequences appeared to be highly conserved in the species studied.     

Harvey sarcoma virus genome contains no extensive sequences unrelated to those of other retroviruses except ras.

The Harvey murine sarcoma virus genome contains two rat-derived sets of genetic information recombined with the Moloney mouse leukemia virus. The rat sequences represent a ras oncogene and a rat VL30 element. The VL30 sequences have several discrete regions of similarity with retroviral sequences which were detected by searching a protein database for similarities with predicted polypeptide sequences from the VL30 regions. On the 5' side, the most similar sequences were those of feline sarcoma viruses; on the 3' side, murine leukemia viruses were the most similar. Some of the regions of similarity could also be detected directly by searching a nucleic acid sequence database with the viral DNA sequences. The most extensive region of similarity was that which corresponded to the endonuclease in the pol gene of a murine leukemia virus. The majority of the rat-derived sequences present in the Harvey sarcoma virus genome can now be attributed exclusively to ras or retrovirus- or retrotransposon-related sequences.     

A generalized method of subcloning DNA fragments by restriction site reconstruction: application to sequencing the amino-terminal coding region of the transforming gene of Gazdar murine sarcoma virus

The technique of restriction site reconstruction was generalized so as to allow the subcloning of any DNA fragment and its subsequent reexcision with EcoRI, XbaI, XhoI or HindIII. After excision, the 3' terminus of each strand will be derived from the starting nucleic acid, permitting the use of such fragments as primers for nucleotide sequencing by primer extension methods. The technique was used to subclone a 56 base pair BstNI-DdeI fragment of Moloney murine sarcoma virus (Mo-MSV) as a unique HindIII-HindIII fragment. This fragment then served as a primer to sequence a portion of the RNA genome of Gazdar murine sarcoma virus (Gz-MSV). The nucleotide sequence which was obtained indicated that the transforming gene of Gz-MSV arose by at least two recombination events involving murine leukemia virus (MLV) and the cellular homologue c-mos. This analysis suggests that a virus indistinguishable from Mo-MSV was an intermediate in the formation of Gz-MSV.     

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.     

Action of temperature-sensitive mutants of myeloproliferative sarcoma virus suggests that fibroblast-transforming and hematopoietic transforming viral properties are related

The myeloproliferative sarcoma virus is molecularly related to the Moloney sarcoma virus (Pragnell et al., J. Virol. 38:952-957, 1981), but causes both fibroblast transformation in vitro and leukemic changes--including spleen focus formation--in adult mice. The fibroblast transforming properties of myeloproliferative sarcoma virus were used to select viral temperature-sensitive mutants at 39.5 degrees C, the nonpermissive temperature. These mutants are temperature sensitive in the maintenance of the transformed state. This was also shown by cytoskeletal changes of the infected cells at permissive and nonpermissive temperatures. Viruses released from cells maintained at both the permissive and nonpermissive temperature are temperature sensitive in fibroblast transformation functions. All temperature-sensitive mutants show only a low reversion rate to wild-type transforming function. The myeloproliferative sarcoma virus temperature-sensitive mutants are inefficient in causing leukemic transformation (spleen enlargement, focus formation) in mice at the normal temperature. A method to maintain a low body temperature (33 to 34 degrees C) in mice is described. One temperature-sensitive mutant was checked at low body temperature and did not induce leukemia. These data thus indicate that the same or related viral functions are responsible for hematopoietic and fibroblast transformation.     

Myeloproliferative virus, a cloned murine sarcoma virus with spleen focus-forming properties in adult mice.

Myeloproliferative virus, derived from Moloney sarcoma virus, causes erythroleukemia and myeloid leukemia in adult mice. This virus is also capable of fibroblast transformation in vitro. The virus consists of two separable biological entities which have been cloned. The helper virus component caused no visible changes in adult mice, whereas the defective virus induced both spleen focus formation and a large increase in erythroid precursor cells but retained the sarcoma virus property of transforming fibroblasts in vitro. Thus, myeloproliferative virus is the first murine sarcoma virus which induces erythroleukemia in adult animals.     

Transfection of Fibroblasts by Cloned Abelson Murine Leukemia Virus DNA and Recovery of Transmissible Virus by Recombination with Helper Virus

A cloned, permuted DNA copy of the Abelson murine leukemia virus (A-MuLV) genome was capable of eliciting the morphological transformation of NIH/3T3 fibroblasts when applied to cells in a calcium phosphate precipitate. The efficiency of the process was extremely low, yielding approximately one transformant per microgram of DNA under conditions which give 10(4) transfectants per microgram of other DNAs (e.g., Moloney sarcoma virus proviral DNA). The DNA was able to induce foci, even though the 3' end of the genome was not present. The transforming gene was thus localized to the 5' portion of the genome. The transformed cells all produced viral RNA and the virus-specific P90 protein. Transmissible virus could be rescued from these cells at very low frequencies by superinfection with helper virus; the rescued A-MuLV virus had variable 3' ends apparently derived by recombination with the helper. Dimerization of the permuted A-MuLV cloned genome to reconstruct a complete provirus did not improve transformation efficiency. Virus could be rescued from these transformants, however, at a high efficiency. Cotransfection of the permuted A-MuLV DNA with proviral M-MuLV DNA yielded a significant increase in the efficiency of transformation and cotransfection of dimeric A-MuLV and proviral M-MuLV resulted in a high-efficiency transformation yielding several thousand more transformants per microgram than A-MuLV DNA alone. We propose that helper virus efficiently rescues A-MuLV from transiently transfected cells which would not otherwise have grown into foci. We hypothesize that multiple copies of A-MuLV DNA introduced into cells by transfection are toxic to cells. In support of this hypothesis, we have shown that A-MuLV DNA sequences can inhibit the stable transformation of cells by other selectable DNAs.     

Construction and isolation of a transforming murine retrovirus containing the src gene of Rous sarcoma virus.

Recombinant murine retroviruses containing the src gene of the avian retrovirus Rous sarcoma virus were isolated. Such viruses were isolated from cells after transfection with DNAs in which the src gene was inserted into the genome of the amphotropic murine retrovirus 4070A. The isolated viruses had functional gag and pol genes, but they were all env defective since the src gene was inserted in the middle of the env gene coding region. Infectious transforming virus could be isolated only from cells transfected with DNA constructions in which the src gene was in the same polarity as that of a long terminal repeat of the amphotropic viral genome. These recombinant viruses encoded a pp60src protein with a molecular weight similar to that of the Schmidt-Ruppin strain of Rous sarcoma virus. In addition, the src protein(s) of these recombinant viruses was as active as protein kinases in the immune complex protein kinase assay. Intravenous injection of helper-independent Moloney and Friend murine leukemia virus pseudotypes of the src recombinant viruses into 6-week-old NIH Swiss mice resulted in the appearance of splenic foci within 2 weeks, splenomegaly and, later after infection (8 to 10 weeks), anemia. Infectious transforming virus could be recovered from the spleens of diseased animals. Such viruses encoded pp60src but not p21ras or mink cell focus-forming virus-related glycoproteins.     

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.     

Molecular cloning of Snyder-Theilen feline leukemia and sarcoma viruses: comparative studies of feline sarcoma virus with its natural helper virus and with Moloney murine sarcoma virus

Extrachromosomal DNA obtained from mink cells acutely infected with the Snyder-Theilen (ST) strain of feline sarcoma virus (feline leukemia virus) [FeSV(FeLV)] was fractionated electrophoretically, and samples enriched for FeLV and FeSV linear intermediates were digested with EcoRI and cloned in lambda phage. Hybrid phages were isolated containing either FeSV or FeLV DNA "inserts" and were characterized by restriction enzyme analysis, R-looping with purified 26 to 32S viral RNA, and heteroduplex formation. The recombinant phages (designated lambda FeSV and lambda FeLV) contain all of the genetic information represented in FeSV and FeLV RNA genomes but lack one extended terminally redundant sequence of 750 bases which appears once at each end of parental linear DNA intermediates. Restriction enzyme and heteroduplex analyses confirmed that sequences unique to FeSV (src sequences) are located at the center of the FeSV genome and are approximately 1.5 kilobase pairs in length. With respect to the 5'-3' orientation of genes in viral RNA, the order of genes in the FeSV genome is 5'-gag-src-env-c region-3'; only 0.9 kilobase pairs of gag and 0.6 kilobase pairs of env-derived FeLV sequences are represented in ST FeSV. Heteroduplex analyses between lambda FeSV or lambda FeLV DNA and Moloney murine sarcoma virus DNA (strain m1) were performed under conditions of reduced stringency to demonstrate limited regions of base pair homology. Two such regions were identified: the first occurs at the extreme 5' end of the leukemia and both sarcoma viral genomes, whereas the second corresponds to a 5' segment of leukemia virus "env" sequences conserved in both sarcoma viruses. The latter sequences are localized at the 3' end of FeSV src and at the 5' end of murine sarcoma virus src and could possibly correspond to regions of helper virus genomes that are required for retroviral transforming functions.     

Structure of the Abelson murine leukemia virus genome and the homologous cellular gene: studies with cloned viral DNA

Circular double-stranded DNA produced after infection of mouse cells with Abelson murine leukemia virus (A-MuLV) was isolated and cloned in the phage vector Charon 21A. The resulting clones of the A-MuLV genome show homology to the ends of Moloney MuLV and to a 3.5 kb central region containing sequences unique to Abelson virus. A 2.3 kb restriction fragment containing only A-MuLV-specific sequences was subcloned in the plasmid vector pBR322 and used as a probe for the cellular gene that had been acquired by the virus. DNA from all inbred mouse lines examined contains an identical region of homology spread out over 11 to 20 kb. The cellular gene contains intervening sequences which are lacking in the viral genome. Rat, Chinese hamster, rabbit, chicken and human DNA also show homology to the viral probe.     

Analysis of mutant Moloney murine leukemia viruses containing linker insertion mutations in the 3'' region of pol.

Twelve linker insertion mutations have been constructed in the 3' part of the pol gene of Moloney murine leukemia virus. This region of the Moloney murine leukemia virus genome encodes IN or p46pol, which is required for integration of the retroviral DNA into the host cell chromosome. Viral proteins synthesized by these mutants were used to pseudotype a neo-containing retroviral vector. Ten of twelve linker insertion mutant pseudotypes were unable to generate stable proviruses in infected mouse cells, as measured by the formation of G418-resistant colonies. Two mutants mapping at the 3' terminus of the IN-encoding region were competent for the formation of stable vector proviruses (hundreds of G418-resistant colonies per mutant pseudotype-infected plate). Representative linker insertion mutants were also tested for the ability to synthesize viral unintegrated DNA in newly infected cells. All assayed mutants were capable of synthesizing all normal forms of viral unintegrated DNA. The structure of integrated vector proviruses generated by defective and nondefective linker insertion mutants was also analyzed. All replication-competent mutants generated normal proviruses, while the few obtainable proviruses generated by replication-defective mutants were sometimes aberrant in structure. These results argue strongly (and confirm previous data) that the IN-encoding region of pol does not play a significant role in DNA synthesis, but is absolutely required for the formation of normal proviral DNA.