Point mutations in the U3 region of the long terminal repeat of Moloney murine leukemia virus determine disease specificity of the myeloproliferative sarcoma virus.

The myeloproliferative sarcoma virus (MPSV) is made up entirely of sequences derived from the Moloney murine leukemia virus (Mo-MuLV) and the cellular mos oncogene. As other members of the Moloney murine sarcoma virus (Mo-MuSV) family, MPSV transforms fibroblasts in vitro and causes sarcomas in vivo. In addition, however, MPSV also causes an acute myeloproliferative disease in adult mice. The mos oncogene is essential for its transforming capacity, but sequences specific to the long terminal repeat (LTR) U3 region of MPSV account for its expanded target specificity as compared to Mo-MuSV (C. Stocking, R. Kollek, U. Bergholz, and W. Ostertag, Proc. Natl. Acad. Sci. USA 82, 5746-5750 (1985)). The U3 region of the LTR of MPSV is, however, closely related to that of the Mo-MuLV, and it appeared likely that the difference between MPSV and Mo-MuSV was caused by a divergent evolution of Mo-MuSV LTRs. In this paper, we show that this is not the case. The few nucleotide differences in the LTR between Mo-MuLV and MPSV are crucial for the expanded host range of MPSV. Moreover, Mo-MuLV-related gag sequences retained in MPSV are not essential for the distinctive biological properties of MPSV.

The myeloproliferative sarcoma virus retains transforming functions after introduction of a dominant selectable marker gene.

The dominant neomycin resistance gene (neoR) was introduced into the genome of the myeloproliferative sarcoma virus (MPSV), a replication-defective retrovirus carrying the mos oncogene. The resulting selectable neoR-MPSV virus did not lose its acute transforming property, unlike the results of attempts by other groups to insert marker genes into oncogenic viruses. NeoR-MPSV DNA was used to generate infectious virus by transfection followed by rescue with Friend or Moloney murine leukaemia virus. Infection of fibroblasts with this virus resulted in morphologically transformed cells which were resistant to the neomycin analogue G418. Segregation of the two functions (transformation and G418 resistance) was not observed in more than 500 independent viral transfers to fibroblasts. Furthermore, neoR-MPSV retained the leukaemogenesis-inducing properties of the wild-type virus. Myeloproliferation and G418-resistance transfer did not segregate after passage in mice.

Viral transfer, transcription, and rescue of a selectable myeloproliferative sarcoma virus in embryonal cell lines: expression of the mos oncogene.

A derivative of the myeloproliferative sarcoma virus (Neor-MPSV) carrying the mos oncogene and dominant selection marker for neomycin resistance (Neor) was introduced into embryonal carcinoma and embryo-derived cell lines by transfection and infection using pseudotypes with Friend helper virus (Friend murine leukemia virus [F-MuLV]). Cells resistant to G418 (a neomycin analog) were cloned and expanded. Transductants retained an undifferentiated phenotype as judged by morphology, tumorigenicity, and cell-surface antigen analyses. Nucleic acid analysis of infectants revealed both Neor-MPSV and F-MuLV proviruses, although no virus was released. G418-resistant transductants remained nonpermissive for the expression of other proviruses and for subsequent superinfection. Northern analysis showed expression of full-length Neor-MPSV, as well as mos-specific subgenomic RNA. mos sequences were deleted from Neor-MPSV (Neor mos-1), and pseudotypes were used to infect embryonal carcinoma cells. No morphological differences were observed in either mos+ or mos- transductants as compared with parental cell lines. However, mos+ transductants showed an enhanced anchorage-independent growth compared with that of mos- transductants in agar cloning. PCC4 transductants were induced to differentiate with retinoic acid and superinfected with F-MuLV. Infection with viral supernatant in fibroblasts and in mice confirmed the rescue of biologically active Neor-MPSV.

Long terminal repeat sequences impart hematopoietic transformation properties to the myeloproliferative sarcoma virus.

The myeloproliferative sarcoma virus not only transforms fibroblasts but also causes extensive expansion of the hematopoietic stem cell compartment on infection of adult mice. Similar to the Moloney sarcoma virus, it carries the mos oncogene. Moloney sarcoma virus, however, does not induce myeloproliferation and leukemia in adult mice. The difference between the two viruses was explored by using their molecularly cloned genomes and the cellular mos oncogene to construct recombinant genomes. It was shown that the U3 region of the viral long terminal repeat (LTR) has a decisive function in determining the target cell specificity of the myeloproliferative sarcoma virus. Any mos gene, whether of cellular or viral origin, is sufficient in conjunction with the proper LTR to induce myeloproliferation. Our results indicate that the pathogenicity of acutely transforming viruses is determined not only by the oncogene but also by sequences in the viral LTR.

Molecular cloning of Rauscher spleen focus-forming virus and biological properties of the cloned virus.

Rauscher virus (RV) induces acute erythroleukaemia and a myeloproliferative disease in adult mice. It consists of a replication-competent murine leukaemia virus (R-MuLV) which acts as a helper virus and a defective transforming component which causes spleen focus formation, Rauscher spleen focus-forming virus (R-SFFV). The integrated proviral DNA of R-SFFV was cloned molecularly. The cloned R-SFFV was compared to that of other viral components which are associated with RV-induced disease and also the cloned Friend SFFV (F-SFFV) and the myeloproliferative sarcoma virus (MPSV), both of which expand the erythroid (F-SFFV, MPSV) and myeloid (MPSV) compartment on infection of adult mice. The genome of R-SFFV differs, if analysed by restriction enzymes, from R-MuLV in the 3' end of the genome between the env gene and the long terminal repeat. The difference is most likely an alteration in the 3' part of the gp70-coding region of the env gene. Comparison with Rauscher mink cell focus-inducing virus (R-MCF) suggests that R-SFFV is derived from R-MCF by substitution of the 3' half of the env gene with a sequence of unknown origin. The molecularly cloned R-SFFV pseudotyped with Friend MuLV induces an increase in late erythroid precursor cells which still require erythropoietin for maturation. Host range studies of the molecularly cloned R-SFFV prove that the Fv-2r locus is required but not sufficient to restrict RV-induced haemopoiesis in adult mice, thus suggesting that R-SFFV has a different target cell range than F-SFFV and is similar to MPSV.

Molecular cloning and characterization of a leukemia-inducing myeloproliferative sarcoma virus and two of its temperature-sensitive mutants.

The myeloproliferative sarcoma virus (MPSV) induces extensive hematopoietic changes, including spleen foci in adult mice, and transforms fibroblasts in vitro. NRK nonproducer cell lines of MPSV and ts temperature-sensitive mutants were analyzed by restriction enzyme digestion and Southern blotting. EcoRI fragments containing the proviral DNAs of MPSV and two temperature-sensitive mutants and rat cellular sequences homologous to c-mos were molecularly cloned. By comparing restriction enzyme cleavage sites, it was shown that the MPSV genome consists only of sequences related either to Moloney murine leukemia virus or to the c-mos mouse oncogenic sequences. Two regions of fragment heterogeneity were observed: (i) in the defective pol gene, where MPSV and the two cloned temperature-sensitive mutants were different from Moloney murine sarcoma virus and from each other, although MPSV wild-type retained more of the pol gene than any of the Moloney murine sarcoma virus isolates; (ii) in the area 3' to the mos gene, which was identical in MPSV and its temperature-sensitive mutants but different from other Moloney murine sarcoma virus variants. Transfection of cloned MPSV DNA in RAT4 cells and virus rescue on infection with Friend murine leukemia virus yielded MPSV which transformed fibroblasts in vitro and also induced spleen foci in adult mice, thus proving that both properties are coded by the same viral genome.

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.

DNA polymerase requirements for parvovirus H-1 DNA replication in vitro.

An in vitro system using nuclei from parvovirus H-1-infected cells was used to characterize the influence of inhibitors of mammalian DNA polymerases on viral DNA synthesis. The experiments tested the effects of aphidicolin, which is highly specific for DNA polymerase alpha, and 2',3'-dideoxythymidine-5'-triphosphate (ddTTP), which inhibits cellular DNA polymerases in the order gamma greater than beta greater than alpha. Both aphidicolin and ddTTP were inhibitory, indicating that both polymerase alpha and a ddttp-sensitive enzyme are required for viral DNA synthesis. This was seen more clearly in kinetic measurements, which indicated an initial period of rapid DNA synthesis with the participation of polymerase alpha, followed by a period of less rapid, but more sustained, rate of DNA synthesis carried out by a ddTTP-sensitive enzyme, probably polymerase gamma. One interpretation of the results is that polymerase alpha functions in a strand displacement stage of the viral DNA replication mechanism, whereas polymerase gamma serves to convert the displaced single strands back to double-strand replicative form.

Synthesis of parvovirus H-1 replicative form from viral DNA by DNA polymerase gamma.

The initial event in the replication cycle of parvovirus H-1 is conversion of the single-stranded linear viral DNA to the double-stranded linear replicative form. We describe here detection of an activity in uninfected cell extracts that carries out this reaction. The activity was purified and identified as DNA polymerase gamma.

Copy number control and incompatibility of plasmid R1: identification of a protein that seems to be involved in both processes.

Investigations into the genetic determinants for incompatibility of miniplasmids and hybrid replicons constructed from wide type and mutant R1 revealed the presence of an incompatibility function at the junction f two small PstI fragments. These two fragments were not distinguished in earlier experiments since they have the same mobility on agarose gels. This incompatibility function is distinct from other inc-determinants of R1 (Kollek and Goebel 1979; Molin and Nordström, 1980) and independent of R1-type replication. By means of specific deletions and subcloning of DNA fragments, the location of this new inc-determinant could be determined further. After deletion of this inc-determinant from inc-determinant from miniplasmids, a 5-fold increase in copy number was observed which could then be reduced to a copy number of about 1 plasmid per cell by complementation with hybrid plasmids having this function. Incompatibility of miniplasmids deleted in this determinant is not reduced, whereas analogous deletions introduced into recombinant plasmids nearly abolished their incompatibility. This determinant seems to exert strong incompatibility only when cloned on pBR322. Therefore, its main function is plasmid R1 is probably restricted to copy control. The appearance of low copy numbers of of miniplasmids carrying this determinant and of trans-acting copy control and strong incompatibility exerted by hybrid plasmids is consistently correlated with the presence of a protein of 11,000 molecular weight, synthesized in relatively large amounts in Escherichia coli minicells.

Site-specific deletion at the replication origin of the antibiotic resistance factor R1.

The recombinant plasmid pRK101 carrying the complete replication origin of the antibiotic resistance factor R1 suffers frequently a deletion of 218 base pairs, removing parts or all of the origin sequence. This deletion seems to occur always when the Pst-E fragment carrying the replication origin is inserted into the cloning vector pBR322 in an orientation where the direction of R1 replication is the same as that of the vector plasmid and frequently when it is inserted in the opposite direction. DNA sequence analysis around the junction site generated by the deletion in three independently isolated deletion mutants reveals that the deletion occurs at a specific site, namely the end of a 22 bp sequence which is repeated almost identically at the other end of a segment of 197 bp. During the deletion one repeat unit is removed whereas the other is retained. The DNA sequence included by the two repeats contains high symmetric structures, i.e. inverted repeats, direct repeats and palindromes which may represent regulatory sites of the origin.

The nucleotide sequence of a DNA fragment from the replication origin of the antibiotic resistance factor R1drd19.

The recombinant plasmid pRK101 contains a DNA fragment which carries the complete replication origin of the antibiotic resistance factor R1drd-19 inserted into the vector plasmid pBR322. In a spontaneously arising mutant of this plasmid (pRK103) a deletion of about 215 base pairs (bp) has been detected by heteroduplex analysis and mapping with restriction endonucleases. Essential parts of the replication origin must be located in the deleted sequence. The deletion mutant pRK103, in contrast to its parent plasmid pRK101 is not replicated under the control of the R1 replicon, even when the R1 factor or copy mutants of it are present within the same cell. These latter plasmids can complement a plasmid-specific protein not coded by pRK101 but essential for R1-directed replication. The nucleotide sequence of a 252 bp HpaII fragment covering about 170--200 bp of the deletion was determined. This piece of DNA is rich in G and C and contains a series of small palindromes, symmetrically arranged repeated sequences and short selfcomplementary structures which may be of significance for the initiation of the DNA replication. The possiblity that the sequenced DNA fragment comprises a major part of the replication origin of R1drd-19 is discussed.

Isolation and characterization of the minimal fragment required for autonomous replication ("basic replicon") of a copy mutant (pKN102) of the antibiotic resistance factor R1.

The mini plasmids deriving from pKN102, a copy mutant of the antibiotic resistance factor R1drd-19 of E. coli, share a common DNA sequence of 2.6 kb, which carries the minimal functions for autonomous replication. By cloning of two PstI fragments of this region it could be demonstrated that the "basic replicon" is a DNA segment not larger than 1.8 kb, which carries the orgin of replication and the genetic information for at least two proteins. Protein F (NW=11.000 dalton) seems to be synthesed in larger amounts in minicells of E. coli than protein C (20.000). Plasmids containing this isolated replicon of R1 are completely compatible with the parental plasmid R1drd-19.

Escherichia coli membrane proteins with an affinity for deoxyribonucleic acid.

From the membrane fraction of Escherichia coli K-12 strain, four protein fractions (peaks I, IIa, IIb, and III) which have affinity for deoxyribonucleic acid (DNA) have been isolated. The molecular weights of these proteins are between 12,000 and 8,000. Only the peak III fraction contains a protein that binds preferentially to single-stranded DNA, whereas the others contain proteins that bind also to double-stranded DNA. The binding activity of the peak IIb protein is inhibited in the presence of polyuridylic acid. Peak I and peak IIa protein fractions behave like hydrophobic proteins.