Search

The Friend virus complex contains a helper-independent retrovirus, F- MuLV, and a replication-defective retrovirus, SFFV. Murine erythroleukemia cell lines (MEL) have been isolated previously from the leukemic tissues of mice infected with such stocks of Friend virus complex. Since our laboratory has shown that either F-MuLV or SFFV can induce a lethal erythroproliferative disease, it has been unclear which of the properties of conventional MEL cells were causally related to the infection of these cells by F-MuLV and which properties were related to the infection of these cells by SFFV. In this article, we have focused on the helper-independent component of the Friend virus complex, F-MuLV clone 57, which has recently been purified by molecular cloning. We report a method for the reproducible isolation of a new type of murine erythroleukemia cell that can be isolate from mice infected with F-MuLV clone 57. We have designated these cells as 57 TP- 1, indicating their origin from mice infected with F-MuLV clone 57 and their characteristics as transplantable erythroleukemia cells. Upon subcutaneous, intraperitoneal, or intravenous inoculation of these cells, 57 TP-1 cells grow in the hematopoietic organs of the recipient mice. No subcutaneous or intraperitoneal nodules occur. Both F-MuLV and a Friend MCF virus can be detected in these cells, but no evidence to SFFV can be found.

Immunization of rats with syngeneic cells infected with spleen focus-forming virus (SFFV) but not with its helper, Friend murine leukemia virus (FMuLV), produces antisera which specifically neutralize SFFV, and not FMuLV, in the Friend virus complex. To determine which SFFV-encoded protein molecule bears the antigen recognized by these neutralizing antibodies, we studied different lots of rat anti-SFFV antiserum by immunoprecipitation and virus neutralization assays. The ability of these sera to neutralize SFFV correlated with the titer of antibodies to p45gag and not with the titer of those to gp52, suggesting that the neutralizing antibodies recognize the p45gag molecule. To verify this specificity for p45gag, we tested antisera to various MuLV gag gene-encoded proteins for neutralization of SFFV. Goat anti-Rauscher murine leukemia virus (RMuLV) p30 and goat anti-RMuLV p10 sera neither precipitated p45gag from SFFV-infected nonproducer cells nor neutralized SFFV. In contrast, goat anti-RMuLV Pr65gag and goat anti-RMuLV p12 sera precipitated p45gag from SFFV-infected cells and also specifically neutralized SFFV in the Friend virus complex. These findings suggest that, unlike the gag proteins coded for by FMuLV, the proteins coded for by defective SFFV are incorporated into the envelope of virions carrying the SFFV genome, but not into the envelope of those carrying the helper FMuLV genome.

Friend murine leukemia virus (G-MuLV) is a helper-independent, type C retrovirus isolated from stocks of Friend virus complex (spleen focus-forming virus plus MuLV). In cell culture, F-MuLV has an ecotropic and NB-tropic host range and causes XC cells to fuse. When injected into newborn NIH Swiss mice, F-MuLV produces hepatosplenomegaly, severe anemia, and numerous circulating hematopoietic precursors in the peripheral blood with normal thymus and lymph nodes after 3 to 6 weeks. Recently, we molecularly cloned an 8.5-kilobase pair (kbp) form of F-MuLV DNA from which we could recover the pathogenic F-MuLV virus by DNA transfection of NIH 3T3 cells. From this molecularly cloned F-MuLV DNA, we have now subcloned in pBR322 a 4.1-kbp HindIII fragment which contains in continuity 3.0 kbp from the 3' terminus (env and c region), 0.6 kbp of the terminal repeat sequences, and 0.5 kbp from the 5'terminus of the viral RNA (genome). NIH 3T3 fibroblasts were transfected with this DNA fragment an then infected with the wild mouse amphotropic retrovirus (cl 1504-A). In cell culture, 1504-A is a helper-independent type C virus which has an N-tropic host range and does not cause fusion of XC cells. When injected into newborn NIH Swiss mice, 1504-A does not produce splenomegaly or thymic enlargement in mice held for up to 8 months. The transfection with the F-MuLV fragment and the infection with 1504-A consistently yielded virus preparations that were XC positive. From such virus stocks we were able to isolate both helper-independent and replication-defective XC-positive viruses. The helper-independent virus was shown to be a recombinant virus since it contains a gp70 molecule derived at least in part from F-MuLV and a specific gag precursor derived from 1504-A as determined by radioactive immune precipitation assays. When injected into newborn Swiss mice, the recombinant helper-independent virus caused hepatosplenomegaly in approximately 50% of the mice in 6 to 8 weeks. The histology of the diseased splenic tissue was indistinguishable from that seen in the disease caused by the whole F-MuLV. The replication-defective virus could be pseudotyped with new 1504-A virus, and this viral complex also caused the F-MuLV disease picture when the complex was injected into newborn Swiss mice. We conclude that the genetic information responsible for the pathogenicity of F-MuLV is contained within the 4.1-kbp DNA fragment, which includes env gene sequences, the terminal repeat sequences, and the c region sequences of the F-MuLV genome.

A biologically active subgenomic DNA fragment of a polycythemia-inducing strain of the replication-defective spleen focus-forming virus (SFFV) has been molecularly cloned. The SFFV DNA fragment includes 2.0 kilobase pairs (kbp) from the 3' end of SFFV, the long terminal repeat sequences of SFFV, and 0.4 kbp from the 5' end of SFFV. The fragment contains the previously described env-related gene of SFFV. All the properties associated with SFFV can be assigned to this SFFV DNA fragment by using a two-stage DNA transfection assay with infectious helper virus DNA. The virus recovered from the transfection assays can induce erythroblastosis, splenic foci, and polycythemia in infected mice. Fibroblast cultures transfected with the SFFV DNA fragment synthesize gp52, the known intracellular product of the env-related gene of SFFV. gp52 can also be detected in spleens from diseased mice infected with the virus recovered in the two-stage transfection. The results are consistent with the hypothesis that the env-related gene sequences of SFFV and their product gp52 are required for the initiation of SFFV-induced disease.

Although it has been previously determined that the long terminal repeat (LTR) sequences of several murine retroviruses specify the major tissue tropism of leukemias they induce, data reported here show that the LTR is not responsible for tissue tropism in the case of all leukemogenic viruses. In an effort to determine whether LTR sequences of the acute erythroleukemia-inducing spleen focus-forming virus (SFFV), like those of the other murine leukemia viruses, are uniquely required to confer tissue specificity to the virus, we prepared recombinant SFFVs in which the LTR region containing promoter and enhancer functions was replaced with analogous LTR regions from Friend and Moloney ecotropic and mink cell focus-inducing viruses. It was found that all of the SFFV constructs, even those with a LTR derived from lymphoma-inducing viruses such as Moloney murine leukemia virus, transformed erythroid cells in vitro and induced exclusively an erythroid disease. These results demonstrate that sequences in SFFV that determine the tissue-specific nature of the disease reside outside the LTR.

Under serum-deprived and chemically defined culture conditions, the growth of Rauscher erythroleukemia cells is mediated by an autocrine mechanism. The growth-promoting activity is produced by fresh or irradiated cells and resembles the activity of interleukin-3 (IL-3) in its ability to sustain colony formation from three of four IL-3- dependent cell lines and to induce formation of granulocyte/macrophage (GM) colonies and, in the presence of erythropoietin (Ep), of erythroid bursts and mixed erythroid colonies. IL-3, IL-1, IL-4, IL-6, GM colony- stimulating factor (GM-CSF), G-CSF, M-CSF, Ep, and media conditioned by concanavalin A-stimulated mouse spleen cells or phytohemagglutinin- stimulated LBRM 33 cells were unable to induce proliferation of the Rauscher erythroleukemia cells. Northern analysis of total and polyA- selected RNA extracted from untreated Rauscher cells or from cells 24 hours after irradiation showed the presence of message for M-CSF but not for IL-3, IL-1, GM-CSF, or G-CSF. The production of IL-6 was excluded by a sensitive bioassay. These results indicate that the autocrine growth of the Rauscher cell line is mediated by a growth factor different from IL-3, but with similar biological activity. Activation of the expression of such a growth factor during viral infection may contribute to the generation of leukemic cells that have the property to grow in vitro and generate Rauscher erythroleukemia cell lines.

Yolk-sac-derived hematopoietic cells were infected with a helper-free stock of Abelson virus (A-MuLV). After infection, cells were plated in a clonogenic methylcellulose culture in the absence of exogenous growth factors such as interleukin 3 (IL 3) and erythropoietin (Epo). No colonies were observed in cultures without viral infection, whereas factor-independent colonies were consistently observed with virus- infected cultures. The number of colonies was linearly correlated with the number of cells plated. Erythroid-mix colonies consisting mostly of erythroblasts, macrophages, and mast cells could be observed. Tumorigenic, continuously growing mast cell lines could be generated at high frequency from these erythroid-mix colonies after they were initially passaged in the presence of an irradiated feeder layer for 4 to 8 weeks. Southern blot analysis of the DNA from five of these lines examined were all shown to contain integrated A-MuLV proviral DNA. These data are evidence that A-MuLV can directly infect embryonic multipotent hematopoietic progenitor cells and drive them to differentiate to various progeny cells without exogenous growth factors.

We have recently described a system that supports the development of continuously growing and tumorigenic cell lines after infection of individual multilineage hematopoietic colonies with Abelson murine leukemia virus (A-MuLV). We now provide definitive evidence that these transformed lines express features characteristic of mast cells. Although these lines have been maintained in some cases for more than a year in the absence of exogenous growth factors other than those present in fetal calf serum, colony formation could consistently after 2 months, and variably after 5 months, be shown to be increased several fold when pokeweed mitogen-stimulated spleen cell conditioned medium (CM) was added to the cultures. CM from the A-MuLV-transformed lines was then tested for its ability to stimulate hematopoietic colony formation by cells from both fetal and adult tissues. Four of four randomly selected cell lines produced factors that were active on erythropoietic, granulopoietic, and in some cases pluripotent progenitors. Removal of viral particles from the CM from one of the lines (27d1) by either heat inactivation or high-speed centrifugation did not alter the colony-stimulating activity detected. When CM from 27d1 cells was tested for its ability to stimulate the proliferation of interleukin 3 (IL3) granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent FDC-P1 cells, a positive result was obtained. This stimulatory activity was not reduced in the presence of neutralizing anti-IL 3 immunoglobulin (Ig), suggesting that the activity detected was GM-CSF and not IL 3. This was confirmed by the lack of expression of the IL 3 gene in 27d1 cells as determined by Northern analysis of 27d1 cell RNA. Furthermore, S1 analysis of mRNA from 27d1 cells as well as two other lines indicated that the GM-CSF gene in all three was transcriptionally active. Taken together, these data suggest that A- MuLV transformation of normal mast cells or their precursors under certain conditions commonly activates the production of GM-CSF.

Four phenotypically normal mink cell clones, each containing a transformation-defective provirus of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV), synthesized an 85,000-dalton viral polyprotein (P85) indistinguishable in size and antigenic complexity from that encoded by wild-type transforming ST-FeSV. An additional transformation-defective, ST-FeSV-containing flat cell clone produced a polyprotein of 88,000 daltons (P88). The viral polyproteins immunoprecipitated from cytoplasmic extracts of these cells lacked the tyrosine-specific protein kinase activity associated with the wild-type ST-FeSV gene product. In addition, the products encoded by representative transformation-defective ST-FeSV genomes were poorly phosphorylated in vivo and lacked detectable phosphotyrosine residues. Whereas proteins of ST-FeSV transformants contained elevated levels of phosphotyrosine, those of mink cells containing transformation-defective ST-FeSV exhibited phosphotyrosine levels no higher than those found in uninfected cells. These findings provide genetic evidence that the tyrosine-specific protein kinase activity associated with ST-FeSV P85 is required for virus-induced transformation.

NFS/N mice inoculated at birth with an ecotropic murine leukemia virus (Cas-Br-MuLV) obtained from wild mice developed hind limb paralysis beginning at 7 weeks of age and nonthymic lymphomas beginning at more than 20 weeks of age. Studies of 1- to 7-week-old Cas-Br-M MuLV-infected mice showed the following: (i) a marked increase in nonecotropic MuLV-related antigens on spleen cells but not thymocytes beginning at 2 weeks; (ii) the appearance of dual-tropic mink cell focus-forming (MCF) MuLV-related gp70 in spleen but not thymus or brain cells at 4 weeks; and (iii) the isolation of infectious MCF MuLV from spleen cells of 7-week-old mice. A role for MCF MuLV in Cas-Br-M MuLV-induced nonthymic lymphomas is indicated by these studies, and a role for recombinant MuLV in neurological disease is considered.

Erythroleukemia cell lines HFL/d and HFL/b, derived from tumors induced in vivo in BALB/c (H-2d) and congenic BALB.B (H-2b) mice, respectively, by a polycythemia-inducing strain of Friend virus, produced both spleen focus-forming virus (SFFV) and its native NB-tropic helper virus (Friend murine leukemia virus [FMuLV]) during early-passage generations in culture. Eventually each line ceased production of both infectious viruses but retained its tumorigenic potential in syngeneic hosts. Virus-producer and -nonproducer clones of these cell lines were examined for expression of proteins encoded by the SFFV or FMuLV genomes. Lysates of labeled cells were treated with various antiviral sera, and the precipitates were examined by gel electrophoresis. Expression of the FMuLV env gene-encoded precursor protein, gPr84env, was observed in all producer and most nonproducer clones, but the FMuLV gag and pol gene products, Pr65gag and Pr200gag-pol, were uniformly undetectable in nonproducer clones. All HFL/d and HFL/b clones expressed appreciable amounts of the SFFV-encoded envelope protein, gp52, including one exceptional clone which had ceased to express any FMuLV-encoded proteins. The molecular weight of this SFFV-encoded envelope protein was consistently smaller in all HFL/b clones than in HFL/d clones, regardless of their producer or nonproducer status. The virus-nonproducer phenotype thus appears to be due to shutdown of expression of the 5' portion of the FMuLV genome in two independent cell lines.

In these studies, we have shown data that are consistent with the hypothesis that mink cell focus-inducing viruses (MCF) play an important role in the generation of an erythroproliferative disease developing after injection of certain strains of newborn mice with ecotropic Friend murine leukemia virus (F-MuLV). Resistance to this disease is correlated with the endogenous expression of an MCF/xenotropic virus-gp70-related protein that may interfere with the replication or spread of MCF viruses. These ideas are supported by the following observations: (a) after infection with F-MuLV, only 6/13 strains of mice-developed disease, and studies with crosses between susceptible and resistant strains indicated that resistance was dominant. Although F-MuLV was shown to replicate equally well in all strains tested, viruses coding for MCF-specific viral envelope proteins could be detected only in the spleens of mice from strains that were resistant to F-MuLV-induced disease and not in the spleens of mice from strains that were resistant to F-MuLV-induced disease; (b) a Friend MCF (Fr-MCF) virus isolated from the spleen of an F-MuLV-infected mouse from a susceptible strain induced the same erythroproliferative disease when injected as an appropriate pseudotype into mice from susceptible but not resistant strains of mice; and (c) resistant but not susceptible strains of mice endogenously express MCF/xenotropic virus-related envelope glycoproteins that may be responsible for resistance by blocking receptors for MCF viruses. These results not only indicate that Fr-MCF virus is a crucial intermediate in the induction of disease by F-MuLV, but also suggest that a novel gene, either an MCF/xenotropic virus-related envelope gene or a gene controlling its expression, is responsible for resistance to erythroleukemia induced by F-MuLV.

A nucleotide sequence was determined for the envelope (env) gene of the polycythemia-inducing strain of the acute leukemia-inducing Friend spleen focus-forming virus (SFFV) and from this the amino acid sequence of its gene product, gp52, was deduced. All major elements of the gene were found to be related to genes of other retroviruses that code for functional glycoproteins. Although the carboxyl terminus of gp52 is encoded by sequences highly related to sequences in its putative parent, ecotropic Friend murine leukemia virus, the majority of the protein (69%), including the amino terminus, is encoded by dualtropic virus-like sequences. Nucleotide sequence comparisons suggest that the nonecotropic region may be more closely related to the 5' substitution in dualtropic mink cell focus-inducing viruses that it is to the 5' end of xenotropic virus env genes. A large deletion and two unique insertions have been located in the env gene of polycythemia-inducing SFFV and may account for some of the unusual structural characteristics, aberrant processing, and pathogenic properties of gp52. As a consequence of the deletion, amino-terminal gp70 and carboxyl-terminal p15E-encoding sequences are juxtaposed and it appears that translation from the p15E region, 3' to the deletion, continues in the standard reading frame used by other retroviruses. Insertions of six base pairs and one base pair at the very 3' end of the gp52-encoding region results in a SFFV-unique amino acid sequence and a premature termination codon.

C57BL/6 mice infected with a mixture of murine leukemia viruses (MuLV) develop a syndrome characterized by lymphoproliferation and profound immunodeficiency. Analyses of this viral mixture (LP-BM5 MuLV) showed that it includes replication-competent ecotropic and mink cell focus-inducing MuLV and defective viruses with genome sizes of 3.8-6.5 kilobases. The ecotropic and mink cell focus-inducing MuLV biologically cloned from the mixture did not induce disease, whereas viral preparations containing the ecotropic MuLV and 4.8-kilobase defective virus were active. Cells producing the 4.8-kilobase defective virus expressed an unusual gag-encoded polyprotein of Mr 60,000.

Cas NS-1 is an acutely transforming murine retrovirus that induces pre-B and pro-B cell lymphomas. Molecular cloning showed it was generated from the ecotropic Cas-Br-M virus by sequential recombinations with endogenous retroviral sequences and a cellular oncogene. The oncogene sequence shows no homology with known oncogenes but some similarity to the yeast transcriptional activator GCN4. A 100-kDa gag-cbl fusion protein, with no detectable kinase activity, is responsible for the cellular transformation. The cellular homologue of v-cbl, present in mouse and human DNA, is expressed in a range of hemopoietic lineages.

Monoclonal antibodies which recognize a region common to Friend spleen focus-forming virus encoded gp52 and Friend mink cell focus-inducing viral gp70 were isolated. One such antibody from hybridoma 7C10 was tested extensively in immune precipitation and was found to react with a determinant on envelope gp70s of all mink cell focus-inducing, xenotropic, and amphotropic mouse retroviruses tested, but not with envelope gp70s of ecotropic viruses, including Friend, Moloney, and AKR murine leukemia viruses. Monoclonal antibody from hybridoma 7C10 precipitated a 23,000-molecular-weight fragment, derived by V8 protease digestion of Friend mink cell focus-inducing gp70. This 23,000-molecular-weight peptide was determined to derive from the amino terminus of the molecule. These results correlate well with other genetic data which indicate that endogenously acquired sequences of mink cell focus-inducing viruses are found at the 5' end of the envelope gene.

A series of insertion-deletion mutants was constructed in a molecularly cloned DNA copy of the Friend strain of spleen focus-forming virus (SFFV). The mutants were produced by inserting a synthetic oligonucleotide linker containing the recognition sequence of SalI endonuclease into several different locations of the SFFV DNA. Three classes of mutants were isolated: insertion-deletion mutants in the 5' half of the SFFV genome, in the long terminal repeat of the SFFV genome, and in the env gene of the SFFV genome. The env gene mutant has a deletion of sequences shared in common between the env gene of SFFV and the env genes of mink cell focus-inducing murine leukemia viruses. From analyses of the biological activity of the various mutants and a biologically active subgenomic SFFV DNA fragment described herein, we can deduce that the coding sequence encompassing the env gene of SFFV is required for the biological activity. This region, required for the pathogenic phenotype, cannot be larger than 1.5 kilobase pairs, a size only slightly more than that sufficient to encode the nonglycosylated precursor of the gp52 env gene product.

We have described the isolation of a replication-defective murine leukemia virus from a culture of AKR lymphoma cells [Rein et al., Nature (London) 282:753-754, 1979]. To facilitate the characterization of this murine leukemia virus, we transmitted it to mink cells and analyzed its genome by restriction mapping of the mink cellular DNA. This genome resembled the Akv genome quite closely, but it had an additional KpnI cleavage site at 1.3 kilobase pairs from the 5' end of the provirus and a small (approximately 50-base-pair) deletion between 1.8 and 3.0 kilobase pairs from the 5' end. When we tested these mink cells by immune precipitation or by competition radioimmunoassay, we found that they synthesized gPr82env, but contained no detectable gag or pol proteins. It seems likely that the KpnI cleavage site at 1.3 kilobase pairs reflects an abnormal sequence at or near the beginning of the gag gene, which prevents gag or pol translation by introducing a frameshift or termination codon into this region.

Using a series of BALB/c mice congenic for various DBA/2 genes, we were able to establish that DBA/2 mice carry a gene on chromosome 5, at or near the Rmcfr locus, that plays a major role in resistance to early erythroleukemia induced by injection of Friend murine leukemia virus (F-MuLV) into newborn mice. The fact that this gene controls the replication of mink cell focus-inducing (MCF) viruses strengthens the case for these viruses playing a crucial role in the development of erythroleukemia, since failure to replicate MCF viruses results in resistance to early erythroleukemia. The expression of the Rmcfr gene is correlated with the constitutive expression of an MCF virus-related envelope glycoprotein that apparently blocks the receptor for MCF viruses, preventing their spread. Thus, the Rmcfr gene is either a structural gene for this unique protein, which can block the receptor for MCF viruses, or is a regulatory gene that controls expression of such a structural gene. Although the Rmcfr gene is clearly involved in resistance to the early erythroleukemia induced by F-MuLV, it appears to have no effect on the late myeloid, lymphoid or erythroid diseases that appear in DBA/2 and other strains of mice after injection of F-MuLV, consistent with data indicating that replication of MCF viruses is not required for the development of these late diseases. Our studies with congenic and backcross mice also indicate that, in addition to the Rmcfr gene, other genes of DBA/2 origin may contribute to resistance to F-MuLV-induced early erythroleukemia by mechanisms other than blocking the replication of MCF viruses.

The src gene product of Harvey murine sarcoma virus is a 21,000-dalton guanine nucleotide-binding protein. We have recently shown that a wide variety of vertebrate cell strains and cell lines express much lower levels of an endogenous p21 immunologically related to the Harvey murine sarcoma virus-coded p21. In this report, we have examined the levels of endogenous p21 in a unique hemopoietic precursor cell line, 416B, which was originally described as a continuous cell line of a hemopoietic stem cell, CFU-S. The currently available 416B cells express markedly elevated levels of endogenous p21. The level of endogenous p21 in the 416B cells is 5- to 10-fold higher than the level of p21 in Harvey murine sarcoma virus-infected cells and more than 100 times higher than the level of endogenous p21 that we have observed in a variety of other fresh or cultured cells. The results indicate that marked regulation of the levels of an endogenous sarc gene product can occur, and speculation about a possible role for endogenous p21 in normal hemopoietic stem cells is discussed.

Friend virus complex (FV), which comprises replication-competent Friend murine leukemia virus (FMuLV) plus replication-defective spleen focus-forming virus (SFFV), induces a multistage erythroleukemia. We have examined the role of replication-competent helper virus in the early and late stages of FV disease by replacing FMuLV, the native helper, with Akv, the endogenous ecotropic MuLV of AKR mice. SFFVP/FRE, an established fibroblast line nonproductively infected with the polycythemic strain of SFFV, was superinfected with FMuLV or with Akv. Although supernatants from these cells showed similar titers in the XC plaque assay, supernatants from Akv-infected SFFVP/FRE cells showed 100- to 5,000-fold less activity than did those from FMuLV-infected cells with respect to spleen focus induction in vivo. Since virions isolated from these two supernatants contained similar ratios of SFFV to helper virus genomic RNA, it did not appear that the difference was due to a relative inability of Akv to package SFFV. Although FMuLV- and Akv-rescued SFFV are equally infectious in a mouse fibroblast cell line (NIH 3T3), FMuLV-rescued SFFV was far more efficient in inducing erythroid bursts in cultured primary bone marrow cells. Adding Akv to preparations of FMuLV-rescued SFFV did not significantly interfere with burst induction. Helper-free SFFV induced 50- to 500-fold more spleen foci when coinjected with FMuLV than it did with Akv. Helper virus also affected mortality rates that reflect the late stage of the disease. When FMuLV- or Akv-rescued SFFV was injected into NIH Swiss mice at dosage levels adjusted to give equal numbers of spleen foci, all mice receiving FMuLV-rescued SFFV developed splenomegaly and died, whereas no mice receiving Akv-rescued SFFV died or developed detectable splenomegaly. When FMuLV was coinjected with Akv-rescued SFFV, the mortality rate rose from 0 to 100%. Injection of helper-free SFFV alone did not induce mortality, but coinjection of helper-free SFFV with FMuLV resulted in 100% mortality. Thus, the helper virus used to rescue SFFV plays at least a quantitatively important role in the early stage of FV disease and a crucial role in the late stage of the disease in vivo.

A murine sarcoma virus (MSV) was recovered from an (NFS X NS.C58v-1) F1 mouse which developed splenic sarcoma and erythroleukemia 6 months after inoculation with a mink cell focus-inducing murine leukemia virus (MuLV) isolated from an NFS mouse infected with a wild mouse ecotropic MuLV. The MSV, designated NS.C58 MSV-1, induced foci of transformation in mouse and rat fibroblasts, and inoculation of mice of various strains 2 weeks of age or younger resulted in erythroleukemia and sarcomatous lesions in spleen, lymph node, and brain. The MSV provirus was molecularly cloned from a genomic library prepared from transformed non-producer rat cells. The 8.8-kilobase proviral DNA contained a 1.0-kilobase p21 ras coding segment which replaced most of the gp70-encoding portion of an MuLV, most likely the endogenous C58v-1 ecotropic virus. The ras oncogene is closely related to v-Ha-ras by hybridization, expression of p21 protein, and nucleotide sequence. It is nearly identical in sequence to v-bas, the only previously described transduced, activated mouse c-ras. At position 12 in the p21 coding region, arginine is substituted for the naturally occurring glycine present in c-ras. A second MSV isolate is described which is similar to NS.C58 MSV-1 except for a 100- to 200-base-pair deletion in the noncoding region of the ras-containing insert.

Two variants of the spleen focus-forming virus (SFFV), SFFVP and SFFVA, induce acute erythroleukemia in mice but differ in their effects on erythroid cells as well as in the posttranslational modification of the product of their envelope genes. To localize the region of the SFFV envelope gene responsible for these differences, we utilized a recombinant virus containing the 3' half of the SFFVP env gene, where the vast majority of the differences between SFFVP and SFFVA reside, and the SFFVP long terminal repeat (LTR) on an SFFVA background. Analysis of the recombinant virus indicates that it is capable of inducing all of the biological effects previously associated with SFFVP, including the ability to proliferate and differentiate without the need for erythropoietin. In addition, the env gene product of the recombinant virus can be detected on the cell surface, a property previously associated only with SFFVP. Although the recombinant virus also contains LTR sequences from SFFVP, we do not believe it is likely that the four LTR nucleotides that are unique to SFFVP are responsible for the biological or biochemical differences observed. These results strengthen the argument that the SFFVP env gene product acts at the cell surface to alter the hormonal requirements for erythroid cell growth and differentiation.

Both the polycythemia-inducing and the anemia-inducing strains of Friend spleen focus-forming virus can induce acute erythroleukemia in susceptible adult mice. However, only cells infected with the polycythemia-inducing strain become erythropoietin-independent for proliferation and differentiation. The sequences responsible for the altered erythropoietin responsiveness have previously been localized to a 678-base-pair EcoRI-Cla I fragment at the 3' end of the envelope gene. This region is now further analyzed by dividing it into two fragments by using the Fok I restriction site. Two recombinants were made by replacing either the 558-base-pair EcoRI-Fok I or the 113-base-pair Fok I-Cla I env gene fragments from the anemia-inducing strain of spleen focus-forming virus with sequences derived from the polycythemia-inducing strain. Our results indicate that the 113-base-pair Fok I-Cla I fragment, which encodes primarily the transmembrane domain of the envelope protein, determines erythropoietin-independent growth.

Previous studies in our laboratory and others have been consistent with the hypothesis that the envelope (env) gene of the spleen focus-forming virus (SFFV) is the only gene essential for the induction of acute erythroleukemia. However, no studies have been carried out with the SFFV env gene in the complete absence of other SFFV sequences. To accomplish this goal, we isolated the sequences that encode the envelope glycoprotein, gp52, of SFFVA and expressed them in a Moloney murine leukemia virus-based double-expression vector containing the neomycin resistance gene. The method used to produce retrovirus stocks in tissue culture cells affected the expression of the gp52 gene in the vector and the subsequent pathogenicity of the vector in mice. Highly pathogenic virus stocks were obtained by cotransfection of vector and helper virus DNAs into fibroblasts, followed by virus replication and spread through the cell population. Mice infected with this stock developed a rapid erythroid disease that was indistinguishable from that induced by the entire SFFV genome, and the virus stock transformed erythroid cells in vitro. Spleen cells from the diseased mice expressed the SFFV env gene product but not the SFFV gag gene product. As expected, mice given the virus containing the SFFV env gene in the reverse orientation did not express the SFFV env gene product or develop erythroleukemia. This study, therefore, demonstrated (i) that double-expression retroviral vectors can be used under specific conditions to produce viruses expressing high levels of a particular gene and (ii) that incorporation of the SFFV env gene into such a vector in the absence of other SFFV sequences results in a retrovirus which is as pathogenic as the original SFFV.

Two different strains of Friend spleen focus-forming virus, SFFVP and SFFVA, are known to cause a rapid erythroleukemia. The SFFVP-infected cells can proliferate and differentiate maximally without the addition of the erythroid-specific hormone erythropoietin, whereas the SFFVA-infected cells require erythropoietin for differentiation and for maximum proliferation. We previously reported that a recombinant virus containing sequences from the 3' half of the SFFVP envelope gene and the SFFVP long terminal repeat on an SFFVA background has all of the biological and biochemical characteristics of SFFVP. We are now presenting data on a new recombinant virus to show that only the 3' half of the SFFVP envelope gene is responsible for the differences observed between the two strains.

A nucleotide sequence analysis carried out on the envelope gene of the anemia-inducing strain of the Friend spleen focus-forming virus (F-SFFVA) reveals that its product has some unique features in common with previously described polycythemia-inducing strains of F-SFFV (F-SFFVP). (i) It contains an amino terminus that is highly related to the gp70 of mink cell focus-inducing viruses, (ii) it is a fusion protein containing the amino terminus of gp70 and the carboxy terminus of p15E, and (iii) it lacks the R-peptide normally found at the carboxy end of the p15E region. Although the envelope genes of F-SFFVA and F-SFFVP are quite similar overall, they do show sequence variation, particularly at the 3' end in the p15E-related region. These variations may contribute to previously observed differences in the response of F-SFFVP- and F-SFFVA-infected erythroid cells to regulatory hormone or to differences in the way the envelope glycoproteins are processed. The long terminal repeat regions of F-SFFVA and the Lilly-Steeves strain of F-SFFVP were also sequenced and compared with each other and with a previously published sequence of another F-SFFVP long terminal repeat. The sequences were found to be reasonably similar to each other but different from their ecotropic parent, Friend murine leukemia virus, as a result of a deletion of one copy of the direct tandem repeat in the enhancer regions. The observation that all SFFVS have this common change in the long terminal repeat enhancer region raises the possibility that it is required for pathogenicity.

A new isolate of a murine erythroblastosis-inducing spleen focus-forming virus (Cas SFFV), derived from the wild mouse ecotropic murine leukemia virus Cas-Br-M, was further characterized after the production of a nonproducer cell line. When rescued from the nonproducer cells with a helper murine leukemia virus, the Cas SFFV induced rapid splenic enlargement and focus formation when inoculated into adult NFS/N mice. The Cas SFFV nonproducer cell line was also utilized to compare the envelope-related glycoprotein of Cas SFFV with gp52s from three strains of Friend SFFV. Cas SFFV was found to encode a 50,500-dalton glycoprotein (gp50) distinct in size to the envelope-related glycoproteins of the Friend SFFVs. The Cas SFFV was also compared in RNA blot hybridization studies. The genomic viral RNA of Cas SFFV was found to be slightly larger than two polycythemia-inducing strains of Friend SFFV and markedly larger than the anemia-inducing strain. Further comparisons between the SFFVs were made by examining their transforming capabilities in an in vitro erythroid burst assay. The erythroid bursts induced by Cas SFFV and the anemia-inducing strain of Friend SFFV showed similarities in their erythropoietin requirements. This study supports our recent observations that Cas SFFV is biologically similar to the anemia-inducing strain of Friend SFFV yet biochemically distinct from all Friend SFFVs.

Stage 1 (pre-malignant) and stage 2 (malignant) cells derived from mice infected with Friend murine leukemia virus or polycythemia-inducing Friend virus complex were examined and compared for the expression of a transformation-related cellular protein, p53. Stage 2 cells were found to express high levels of p53, whereas stage 1 cells did not express detectable levels of this protein. These results indicate that p53 may be a marker for transformed cells present in the second stage of diseases induced by Friend murine leukemia virus or polycythemia-inducing Friend virus complex.

Friend murine leukemia virus (F-MuLV) is a replication-competent, ecotropic, NB-tropic retrovirus which produces a rapidly fatal erythroleukemia in susceptible strains of mice. We previously molecularly cloned the entire F-MuLV genome. Transfection of this cloned DNA into NIH 3T3 mouse fibroblasts produces a virus with the same leukemia-inducing characteristics as F-MuLV. To identify which portion of the F-MuLV genome is responsible for causing leukemia, we made recombinant viruses between subgenomic fragments of F-MuLV DNA and another retrovirus--Amphotroph clone 4070. Amphotroph clone 4070 is a replication-competent, amphotrophic, N-tropic virus which does not produce any detectable malignancy in mice. A 2.4-kilobase-pair fragment of F-MuLV DNA was isolated. This DNA fragment encompassed approximately 700 base pairs from the 3' end of the F-MuLV pol gene and 1.7 kilobase pairs of the env gene including all of gp70 and the N-terminal four-fifths of p15E. A molecularly cloned fragment of Amphotroph DNA was ligated to the 2.4-kilobase-pair F-MuLV DNA, and an 8.3-kilobase-pair hybrid F-MuLV-Amphotroph DNA was subcloned into a new plasmid (p5a25-H). Transfection of p5a25-H DNA into fibroblasts resulted in the production of a replication-competent, ecotropic, N-tropic retrovirus--5a25-H virus. Inoculation of this virus into newborn NIH Swiss mice caused leukemia within 4 to 6 months. The disease caused by 5a25-H was pathologically and histologically indistinguishable from the disease caused by F-MuLV. We conclude that the F-MuLV sequences needed to cause disease are contained in these 2.4 kilobase pairs of DNA.

An NFS/N mouse inoculated at birth with an ecotropic murine leukemia virus (MuLV) obtained from wild mice (Cas-Br-M MuLV) developed a lymphoma after 18 weeks. An extract prepared from the lymphomatous spleen was inoculated into newborn NFS/N mice, and these mice developed erythroleukemia within 9 weeks. Spleens from the erythroleukemic mice contained ecotropic and mink cell focus-inducing (MCF) MuLVs; however, when these viruses were biologically cloned and reinoculated into newborn NFS/N mice, no erythroleukemia was induced. In contrast, cell-free extracts prepared from the erythroleukemic spleens induced erythroleukemia within 5 weeks. Analysis of cell-free extracts prepared from the erythroleukemic spleens showed that they contained a viral species that induced splenomegaly and spleen focus formation in adult mice, with susceptibility controlled by alleles at the Fv-2 locus. The spleen focus-forming virus coded for a 50,000-dalton protein precipitated by antibodies specific to MCF virus gp70. RNA blot hybridization studies showed the genomic viral RNA to be 7.5 kilobases and to hybridize strongly to a xenotropic or MCF envelope-specific probe but not to hybridize with an ecotropic virus envelope-specific probe. The virus described here appears to be the fourth independent isolate of a MuLV with spleen focus-forming activity.

Previously, we have molecularly cloned proviral DNA of a polycythemia-inducing strain of the spleen focus-forming virus (SFFVp). In this paper, we report that unintegrated proviral DNA of the anemia-inducing strain of SFFV (SFFVA) has been molecularly cloned into pBR322. This molecularly cloned DNA retains the biological activity of SFFVA, as infectious SFFV can be recovered from the DNA clone by marker rescue using a previously described two-stage cotransfection assay (Linemeyer et al., J. Virol. 35:710-721, 1980). The recovered SFFV retains an important property of the initial SFFVA which distinguishes SFFVA from SFFVP, namely, the ability of SFFVA to cause proliferation of erythroid cells in which hemoglobin synthesis is erythropoietin dependent. By utilizing a marker rescue technique, the splenomegaly and anemia characteristic of SFFVA-induced disease have been traced to a DNA fragment of SFFVA containing sequences coding for the env gene product. gp52. The results suggest that the differences in pathogenicity between SFFVP disease and SFFVA disease are an intrinsic property of the env gene products of these two variants of Friend virus, and future studies with the molecular clones of each strain should allow us to map regions of each env gene responsible for common and distinctive features of the erythroproliferative diseases induced by each virus.

Monoclonal antibodies prepared to epitopes encoded by the transforming gene (v-fms) of the McDonough strain of feline sarcoma virus were used to study v-fms-coded antigens in feline sarcoma virus-transformed rat and mink cells. These antibodies reacted with three different polypeptides (gP180gag-fms, gp140fms, and gp120fms), all of which were shown to be glycosylated. Protein blotting with [125I]-labeled monoclonal immunoglobulin G's was used to determine the relative steady-state levels of these glycoproteins in transformed cells and showed that gp120 and gp140 were the predominant products. Immunofluorescence assays and subcellular fractionation experiments localized these molecules to the cytoplasm of transformed cells in quantitative association with sedimentable organelles. Thus, v-fms-coded glycoproteins differ both chemically and topologically from the partially characterized products of other known oncogenes and presumably transform cells by a different mechanism.