The "sarcoma-specific" region of Moloney murine sarcoma virus 124.

Labeled, purified 30S RNA from Moloney murine sarcoma virus was annealed to an excess of Moloney murine leukemia virus complementary DNA. Upon treatment of the resulting DNA.RNA hybrids with RNase H followed by sucrose gradient sedimentation, and undigested 18S RNA molecule was recovered. This RNA molecule was shown to represent the "sarcoma-specific" region of the virus. The unintegrated linear DNA provirus of murine sarcoma virus 124 was isolated from newly infected cells and a physical map of the sarcoma-specific region was obtained. First, unintegrated full-length linear proviral DNA molecules were cleaved by several restriction endonucleases. The reciprocal position and orientation with respect to the viral RNA of the resulting fragments were established. The location of the sarcoma-specific region was determined by competition-hybridization with 125I-labeled viral genomic RNAs and proviral DNA fragments. A 1500-base-pair fragment was obtained by cleavage with HindIII + Bgl II. This fragment mapped between 750 and 2250 base pairs from the right end of the proviral DNA (corresponding th the 3' terminus of the viral RNA) and contained the whole set of the sarcoma-specific information. This murine sarcoma virus proviral restriction fragment is approximately of the same size and map position as the isolated 18S sarcoma-specific RNA.     

Abelson murine leukemia virus: molecular cloning of infectious integrated proviral DNA.

The integrated proviral genome of Abelson murine leukemia virus (A-MuLV) was cloned in lambda gtWES . lambda B bacteriophage after EcoRI endonuclease digestion and enrichment of proviral sequences by sequential RPC-5 column chromatography and agarose gel electrophoresis. Recombinant DNA clones containing a 7.8-kilobase-pair EcoRI insert were shown to have the entire integrated A-MuLV genome with both 5' and 3' ends flanked by mink cellular DNA sequences. This DNA fragment was shown to induce focus transformation upon transfection of NIH/3T3 mouse cells. Moreover, focus-forming virus could be rescued from transformed nonproducer cells upon superinfection with a type C helper virus. A polyprotein of molecular weight 120,000 (p120) containing murine leukemia virus gag gene determinants was invariably deteced by immunoprecipitation analysis of individual transformants induced by the 7.8-kilobase-pair DNA. Molecularly cloned integrated A-MuLV in its infectious form should be of use in elucidating the mechanisms involved in transformation by this virus.     

Friend and Moloney murine leukemia viruses specifically recombine with different endogenous retroviral sequences to generate mink cell focus-forming viruses.

A group of mink cell focus-forming (MCF) viruses was derived by inoculation of NFS/N mice with Moloney murine leukemia virus (Mo-MuLV 1387) and was compared to a similarly derived group of MCF viruses from mice inoculated with Friend MuLV (Fr-MuLV 57). Antigenic analyses using monoclonal antibodies specific for MCF virus and xenotropic MuLV envelope proteins and genomic structural analyses by RNase T1-resistant oligonucleotide finger-printing indicated that the Moloney and Friend MCF viruses arose by recombination of the respective ecotropic MuLVs with different endogenous retrovirus sequences of NFS mice.     

Presence and expression of Friend erythroleukemia virus-related sequences in normal and leukemic mouse tissues

The nature and distribution of sequences related to the murine erythroleukemia virus, Friend spleen focus-forming virus (SFFV), have been analyzed by using a radioactive cDNA probe specific for the SFFV genome (cDNA(sff)). From the proportion of high molecular weight viral [(32)P]RNA which hybridized to cDNA(sff), it was estimated that these sequences represent about 50% of the SFFV genome, indicating a genetic complexity of about 3300 nucleotides. cDNA(sff) hybridized extensively (80-95%) to SFFV virion RNA and to cellular RNA from murine and rat cells productively or nonproductively infected with SFFV. Only background homology was detected between cDNA(sff) and viral RNA from a number of murine [Friend murine leukemia virus (MuLV), Moloney-MuLV, and Kirsten sarcoma virus] and nonmurine (Rous sarcoma virus, feline leukemia virus, baboon endogenous virus, and Mason-Pfizer mammary tumor virus) retroviruses. Limited homology was also detected to a number of murine xenotropic and mink cell focus-inducing viruses (20-35%) as well as Rauscher leukemia virus (50%). Nucleotide sequences homologous to cDNA(sff) were also detected in the DNA of normal cells of several mouse strains as single or a few copies per cell. Thermal denaturation analysis indicated that duplexes formed between cDNA(sff) and normal DBA/2J cellular DNA have a reduction in melting temperature of 2 degrees C when compared with the dissociation of hybrids between cDNA(sff) and homologous sequences in SFFV-infected mouse spleen cell DNA. Examination of cellular RNA from uninfected mouse cells indicated that SFFV-related RNA sequences were also expressed in varying degrees in different tissues of adult DBA/2J mice. The highest amounts were observed in cells from bone marrow and spleen, whereas considerably lower amounts were found in cells from the thymus and kidney. No SFFV-related sequences could be detected in RNA extracted from liver, muscle, or fibroblasts. The presence of these SFFV-related sequences in normal, uninfected mouse cell DNA and their differential expression in hematopoietic tissues suggest that these sequences may be an integral part of the program of both normal and leukemic hematopoietic cell differentiation.     

Rat cells infected with anemia-inducing Friend leukemia virus contain integrated replication-competent but not defective proviral genomes.

The integrated proviral DNA in five murine cell lines transformed by the anemic strain of Friend leukemia virus (FLV-A) was examined by Southern hybridization to a cloned Friend virus (F-MuLV) probe. Kpn I fragments 9 kilobases (kb) and 5.7 kb long were observed for each cell line. However, the number of copies of each fragment in the cell genome varied according to the cell type. As compared to the adherent epithelioid cell lines, the anchorage-independent erythroleukemic cell lines contained more copies of the 5.7-kb fragment than of the 9-kb fragment, suggesting that the former may be biologically significant and perhaps related to the growth of erythroid cells. The presence of Kpn I fragments of the same sizes, albeit in fewer copies, in normal mouse spleen DNA made it difficult to distinguish exogenous virus from endogenous viral sequences. Therefore, rat 3Y1 cells, which contained no murine endogenous viruses, were infected with FLV-A stock virus prepared directly from the spleens of leukemic mice. Only the 9-kb Kpn I fragment, representing replication-competent Friend virus component, was detected in the infected rat cell DNA. No hybridization was observed to a 0.6-kb fragment of the spleen focus-forming virus env gene that is specific for xenotropic and dual-tropic mink cell focus-forming viruses. Since the virus synthesized by the infected rat cells was leukemogenic in adult mice, these data suggest that the wild-type FLV-A is replicative and fully pathogenic in the absence of other competent virus components.     

Replication-competent Moloney murine leukemia virus carrying a bacterial suppressor tRNA gene: selective cloning of proviral and flanking host sequences.

A bacterial suppressor tRNA gene was introduced into the long terminal repeat of the Moloney murine leukemia virus (Mo-MuLV) proviral genome to construct a retrovirus that allows easy cloning of the provirus with flanking host sequences. A replication competent virus, Mo-MuLV sup containing a tRNA amber suppressor gene, was derived that replicates to high titers in tissue culture cells and stably transduces the bacterial gene. The recombinant virus can efficiently replicate in vivo when microinjected into midgestation embryos or when injected into newborn mice and displays the same tissue tropism as wild-type Mo-MuLV. The suppressor gene in Mo-MuLV sup is functional in bacteria and allows efficient recovery of proviral genomes. This was shown by ligation of DNA from infected cells to phage lambda Charon 4A arms and selective growth of recombinant phages on su- host cells. All recovered phages contained Mo-MuLV proviral sequences and, because of the high cloning capacity of phage lambda, 1-11 kilobases of flanking host DNA. This virus should facilitate studying virus-host interactions in tissue culture cells and in animals.     

A novel murine oncornavirus with dual eco- and xenotropic properties.

Infection of Swiss mouse 3T3FL cells with a clonal isolate of Moloney leukemia virus (MLV-IC) resulted in virus progeny composed of at least three different murine helper oncornaviruses. Each entity was purified in appropriate cells by several sequential terminal dilution isolations and was grouwn to high titers. Besides ecotropic MLV-IC there was a pure xenotropic virus and a third novel virus with properties of both eco- and xenotropic viruses. The purified xenotropic virus had a wide host range, was restricted in mouse cells, and was inactivated by normal mouse sera like other xenotropic isolates. The purified virus with hybrid properties (HIX) could infect a wide range of mammalian cells, which included both N and B mouse cells. HIX gave single-hit titrations with equal titers on both mouse and cat indicator cells. Envelope properties of HIX were examined by virus preinfection interference, by interference involving viral glycoprotein, and by neutralization with specific antisera. Both xenotropic and MLV-IC type ecotropic determinants were found on the virus coat. The origins of HIX and the xenotropic virus were investigated in detail. The original MLV-IC stock had HIX type virus in low titer but no detectable pure xenotropic virus. Infection of mouse cells with a single infectious unit of the ecotropic virus from the MLV-IC virus stocks could at times give rise to HIX type virus. HIX type virus, passed once through heterologous rat cells, was subjected to long-term passage either in infected mouse or cat cells. After several months HIX type virus disappeared from some mouse and cat cell systems. The possible hybrid nature of HIX and the origins of newly appearing xenotropic viruses are discussed.     

De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells.

We have investigated the block to expression of Moloney murine leukemia virus in murine embryonal carcinoma (EC) cells. Infected EC cells were found to contain up to 100 integrated proviral genomes. However, expression of virus as measured by XC plaque and virus-specific RNA synthesis did not occur at significant levels, in contrast to productively infected differentiated cells. Analysis of the DNA in the infected EC cells revealed that the proviral genomes were highly methylated, as shown by their resistance to cleavage by Sma I. Integrated proviral genomes in infected differentiated cells were readily cut by Sma I and thus were not methylated at these sites. Transfection of DNA from infected EC cells to cells permissive for virus expression failed to induce virus expression. The proviral genomes, however, were potentially infectious because they induced XC plaques when the recipient cells for transfection were treated with 5-azacytidine. This drug is believed to interfere with DNA methylation. We conclude that expression of proviral genomes introduced into EC cells is suppressed and that this inactivation can be correlated with the de novo methylation of the viral DNA. De novo methylation activity thus may be a characteristic of early embryonic cells.     

Structural markers on core protein p30 of murine leukemia virus: functional correlation with Fv-1 tropism.

Tryptic peptide maps from more than 50 isolates of murine leukemia virus (MuLV) have shown that, in general, the structure of core protein p30 is highly conserved. However, a structurally variable region of p30 has been identified that is functionally associated with Fv-1 tropism. On the basis of this structural variability, MuLV strains can be classified as B-tropic, N-tropic, xenotropic, and/or as being derived from wild mice. Certain xenotropic viruses have a p30 like that of B-tropic MuLV and presumably would be subject to restriction in cells containing an Fv-In allele. Other p30 structural markers serve to distinguish the exogenous Friend, Moloney, and Rauscher viruses from endogenous MuLV. Furthermore, some MuLV strains have structural differences in their p30s that are useful as strain-specific markers. Finally, a possible sarcoma-associated alteration in the structure of p30 has been noted in the ml clone of Moloney murine sarcoma virus.     

Identification of Rauscher murine leukemia virus-specific mRNAs for the synthesis of gag- and env-gene products.

Polyadenylylated mRNA isolated from cells infected with Rauscher murine leukemia virus was fractionated by centrifugation in in a denaturing sucrose gradient into different sizes. Each RNA fraction was injected into oocytes of Xenopus laevis and the virus-specific products were analyzed by immunoprecipitation with polyvalent and monospecific antisera against polypeptides of Rauscher murine leukemia virus, and then by gel electrophoresis and scintillation autoradiography. It was shown that a 35S mRNA species directs the synthesis of a precursor of the internal or group-specific antigens of the virion (the gag-gene products). A 22S mRNA species directs the synthesis of two viral envelope polypeptides and their precursor polypeptide (env-gene products). The results indicate that the gag- and env-related polypeptides of Rauscher murine leukemia virus are synthesized uncoordinately and provide evidence for open and closed cistrons on the virus-specific mRNAs.     

Molecular cloning of infectious integrated murine leukemia virus DNA from infected mouse cells.

The lack of an endonuclease EcoRI site in the AKR murine leukemia virus (MuLV) DNA genome was utilized to molecularly clone, in Charon 4A lambda DNA, integrated infectious AKR MuLV DNA isolated from productively infected mouse cells. Three lambda-mouse recombinants (clones 614, 621, and 623) were selected by virtue of their reactivity with AKR MuLV [32P]cDNA. Clones 614 and 623 contained the complete AKR MuLV DNA flanked by nonviral cell sequences of which no more than 100 base pairs beyond the viral DNA appear to be shared. DNAs from both clones 614 and 623 were highly infectious for mouse cells and yielded N-tropic ecotropic MuLV; the specific infectivity of the DNA and the titer of the derived virus was more than 10-fold higher with 623. Clone 621 contained only some viral DNA and was not infectious under similar conditions.     

In vitro construction of a B-tropic virus by recombination: B-tropism is a cryptic phenotype of xenotropic murine retroviruses.

A B-tropic virus was isolated in vitro from the progeny of mouse cells doubly infected with N-tropic and xenotropic murine leukemia viruses. Biological and structural evidence is presented suggesting that the phenotypically silent structural marker for B-tropism, expressed by the xenotropic virus p30, was transferred to an N-ecotropic virus via recombination, thus resulting in the expression of a B-ecotropic murine leukemia virus.     

Infectious murine leukemia virus from DNA of virus-negative AKR mouse embryo cells.

DNA from virus-negative AKR mouse embryo cells is infectious for NIH 3T3 cells if the transfected cells are treated with 5-iododeoxyuridine (IdUrd) either before or after addition of the DNA. The virus isolated after transfection of the AKR DNA is an ecotropic murine leukemia virus (MuLV) indistinguishable from endogenous AKR MuLV. The AKR DNA is not infectious in the absence of IdUrd treatment of the recipient cells. DNA from AKR cells treated with IdUrd before DNA isolation is not infectious unless the recipient cells have been treated with IdUrd. Transfected DNA from NIH mouse cells is not infectious even when the recipient cells have been treated with IdUrd. DNA from cells chronically infected with AKR MuLV or Rauscher MuLV is infectious with or without IdUrd treatment of the recipient cells, but the infectivity is not enhanced by IdUrd. The results indicate that the endogenous AKR MuLV DNA genome is potentially infectious. The data are consistent with an IdUrd-sensitive restriction in the recipient NIH 3T3 cells that prevents viral replication from endogenous AKR MuLV DNA but does not prevent viral replication from MuLV DNA of productively infected cells.     

Structure of endogenous murine leukemia virus DNA in mouse genomes.

By using molecularly cloned ecotropic AKR murine leukemia virus (MuLV) DNA, a 400-base-pair ecotropic type-specific segment in the env region has been identified. This DNA segment and other defined viral subgenomic fragments have been used as 32P-labeled probes to identify and analyze the structure of integrated ecotropic viral DNA sequences in uninfected mouse genomes. Those mice from which endogenous ecotropic MuLV of the AKR type have been isolated contained at least one virtually complete linear copy of the viral genome. Strains from which ecotropic MuLV has not been isolated lacked ecotropic-specific sequences. All inbred mouse strains tested also contained MuLV DNAs of genomic length whose restriction endonuclease digestion pattern was characteristic of xenotropic viruses.     

Tumours induced by Moloney murine sarcoma virus are clonal in rats,not clonal in mice

Summary Moloney murine sarcoma virus (M-MSV) induces rapidly growing tumours in adult mice of most conventional strains. Rats are less susceptible to M-MSV oncogenesis, but the few rhabdomyosarcomas that do develop after viral inoculation of newborn animals closely resemble conventional malignancies: they develop after a long latency, grow progressively, and metastasize to regional lymph nodes and lungs. Southern blot analysis with a v-mos-specific probe of M-MSV-induced tumours in both species demonstrated an oligo-, monoclonal pattern of exogenous v-mos integration only in the rat system, while mouse tumours were not clonal in origin. Furthermore, the same type of analysis of lymph node and lung metastases showed that cell clones already present in the primary rat lesion colonized secondary sites during tumour progression. Apparently, Moloney murine leukemia virus (M-MuLV) was not involved in rhabdomyosarcoma pathogenesis since M-MuLV-specific DNA sequences could not be demonstrated in three of the six rat tumours. Finally, in all mouse tumours, unintegrated linear M-MSV proviruses could be readily detected.Abbreviations M-MSV Moloney murine sarcoma virus - M-MuLV Moloney murine leukemia virus     

Friend strain of spleen focus-forming virus is a recombinant between ecotropic murine type C virus and the env gene region of xenotropic type C virus.

The spleen focus-forming virus (SFFV), a replication-defective murine leukemia virus that causes the rapid transformation of certain hematopoietic target cells, has acquired specific xenotropic viral genetic information not contained in Friend helper virus. In the current studies, it is shown that a cDNA that represents a xenotropic virus portion of SFFV detects genetic sequences derived from the env gene region of murine xenotropic virus. The significance of the acquisition of these xenotropic viral sequences by SFFV is discussed with regard to their possible role in the rapid leukemogenicity of SFFV, and an analogy is drawn between the formation of SFFV and the formation of the Kirsten and Harvey sarcoma viruses.