Strains Of Murine Sarcoma Virus Research Articles

Mouse cells contain two distinct ras gene mRNA species that can be translated into a p21 onc protein.

The Kirsten (Ki) and Harvey (Ha) strains of murine sarcoma virus encode a 21,000-dalton protein (p21 ras) which is the product of the transforming gene of these viruses. Normal cells express low levels of p21 ras encoded by cellular genes (Ki-ras and Ha-ras) homologous to the Ki and Ha murine sarcoma virus transformation genes. A bone marrow-derived mouse cell line, 416B, has been shown to express unusually high levels of p21 ras. In this manuscript, we investigated the molecular biology of p21 ras gene expression in 416B and other normal mouse cells. We identified four distinct polyadenylated and polysome-associated RNAs, two related to Ki-ras and two to Ha-ras. The levels in 416B cells of the two Ki-ras RNAs, sized 5.2 and 2.0 kilobases, were both elevated approximately 25-fold over levels found in normal mouse cells; there was no corresponding change in 416B cells in the levels of the two Ha-ras RNAs. We partially purified the two Ki-ras mRNAs and separated them by velocity sedimentation in sucrose density gradients. Both the 5.2- and 2.0-kilobase mRNAs could be translated in vitro into p21 ras. These results show that a cellular onc protein can be translated from two distinct cellular mRNA species.

Mouse cells contain two distinct ras gene mRNA species that can be translated into a p21 onc protein.

The Kirsten (Ki) and Harvey (Ha) strains of murine sarcoma virus encode a 21,000-dalton protein (p21 ras) which is the product of the transforming gene of these viruses. Normal cells express low levels of p21 ras encoded by cellular genes (Ki-ras and Ha-ras) homologous to the Ki and Ha murine sarcoma virus transformation genes. A bone marrow-derived mouse cell line, 416B, has been shown to express unusually high levels of p21 ras. In this manuscript, we investigated the molecular biology of p21 ras gene expression in 416B and other normal mouse cells. We identified four distinct polyadenylated and polysome-associated RNAs, two related to Ki-ras and two to Ha-ras. The levels in 416B cells of the two Ki-ras RNAs, sized 5.2 and 2.0 kilobases, were both elevated approximately 25-fold over levels found in normal mouse cells; there was no corresponding change in 416B cells in the levels of the two Ha-ras RNAs. We partially purified the two Ki-ras mRNAs and separated them by velocity sedimentation in sucrose density gradients. Both the 5.2- and 2.0-kilobase mRNAs could be translated in vitro into p21 ras. These results show that a cellular onc protein can be translated from two distinct cellular mRNA species.

Candidate product of the FBJ murine osteosarcoma virus oncogene: characterization of a 55,000-dalton phosphoprotein.

Sera from rat bearing tumors induced by inoculation of FBJ murine osteogenic sarcoma virus (FBJ-MSV) nonproducer rat cells precipitate two proteins with molecular weights of 55,000 (p55) and 39,000 (p39) from FBJ-MSV-transformed cells. These proteins cannot be precipitated from uninfected cells or cells transformed by other strains of murine sarcoma virus, nor can they be precipitated by sera specific for the viral structural proteins. A methionine tryptic peptide mapping analysis showed that p55 and p39 have little or no homology and that they are not related to the helper virus gag and env gene products. p55 could also be detected among the in vitro translation products of 70S RNA from FBJ murine leukemia virus plus FBJ-MSV virions but not among those from FBJ murine leukemia virus alone. This suggests that p55 is encoded by the FBJ-MSV genome, whereas p39, which was not detected among the in vitro translation products, may not be virus encoded. Another difference between p55 and p39 is that p55 is phosphorylated, with most of the phosphate on a serine residue(s), whereas p39 is phosphorylated to a much lesser extent, if at all. No protein kinase activity was associated with p55 and p39 immune complexes under standard conditions. Our data suggest that p55 is a strong candidate for the FBJ-MSV oncogene product.

The p21 src genes of Harvey and Kirsten sarcoma viruses originate from divergent members of a family of normal vertebrate genes.

The Harvey and Kirsten strains of murine sarcoma virus encode enzymatically and serologically related p21 src proteins which are required for virally mediated cellular transformation. The genes in each virus encoding p21 show such extensive divergence from each other that cloned probes from these genes detect distinct sets of cellular genes in the DNA from several vertebrate species. These data suggest that cellular p21 sarc genes constitute a divergent family of vertebrate genes that can regulate the growth of cells.

Murine sarcoma viruses block corticosteroid-induced differentiation of bone marrow preadipocytes associated with long-term in vitro hemopoiesis

A unique population of clonagenic bone marrow cells accumulates neutral fat and differentiates to adipocyte morphology in response to 10 −7 M 17-hydroxy-corticosteroid. Differentiation of these adherent marrow preadipocytes is associated with maintenance of pluripotent hemopoietic stem cell and granulocyte-macrophage progenitor cell proliferation in bone marrow cultures in vitro. The present studies demonstrate that infection of preadipocytes with Harvey, Kirsten, or Moloney strains of murine sarcoma virus (MSV) but not the associated helper leukemia virus blocks corticosteroid-induced lipogenesis. This effect is observed both in whole marrow culture and in a new assay for the individual colony-forming unit adipocyte (CFUa). CFUa are single cells concentrated in light density fractions of an isopycnic density gradient, normally accumulate lipid, and grow to ≥50 cell colonies following suspension in 0.8% methylcellulose-containing medium with horse serum and hydrocortisone. An MSV-specific block of both CFUa formation and insulin-dependent differentiation of the 3T3-L1 embryo preadipocyte cell line is detected by 14 days. In contrast, MSV infection of granulocyte-macrophage progenitor cells in the same marrow does not block differentiation in response to colony-stimulating factor. These data indicate that cells of a specific phenotype can be identified within an organ culture as targets for the early biological effects of a particular class of RNA type-C viruses.

In vitro isolation of stable rat sarcoma viruses

A Sprague-Dawley (SD-1) rat embryo culture, at low passage level, released an endogenous ecotropic type C virus (SD-RaLV) and after about 20 further passages it underwent spontaneous transformation. The SD-RaLV, released from the transformed cells, did not cause rapid transformation of other rat embryo cells. However, when the transformed cells were repeatedly cocultivated with three different chemically transformed and serially transplanted rat tumor cell lines (sarcoma, carcinoma, and hepatoma), rapidly fibroblast-transforming "sarcoma" viruses (RaSV) were recovered after each attempt. RaSV was not recovered from one of these tumor cell lines before transplantation, nor could focus-forming virus be rescued from these same tumor cells by cocultivation with other cells releasing heterologous type C viruses. Foci were induced on normal rat kidney and several other rat embryo cell strains within 7-15 days and both productive and nonproductive NRK clones were derived. The productive clones were positive for rat specific p30 antigen and the RaSVs released were serially transmitted to other rat embryo cells. RaSV genome was rescued from the nonproductive clones by superinfection with SD-RaLV, wild rat type C virus, and several heterologous type C viruses. These observations appear to represent naturally occurring transformation-specific (src) genes being recovered in vitro in the form of stable "sarcoma" viruses. These viruses differ from the Kirsten and Harvey strains of murine sarcoma virus in that they apparently contain no MuLV sequences and are of purely rat origin.

Characterization of dna complementary to nucleotide sequences adjacent to poly(A) at the 3′-terminus of the avian sarcoma virus genome

We have prepared DNA complementary to a sequence of 300–400 nucleotides adjacent to the poly(A) at the 3′-terminus of the genome of avian sarcoma virus (ASV) DNA was transcribed from denatured ASV RNA following initiation on the primer (dT) 12–18. We demonstrated that homopolymer synthesis was not a significant feature of our enzymatic reaction, and we proved that most if not all the initiations on (dT) 12–18 occurred at or near the 3'-terminus of the viral RNA. The presence of oligo(dT) on cDNA 3, accelerates hybridization between cDNA 3, and polyadenylated viral RNA; this artifact can be eliminated by including a competing homopolymer [poly(dT) or poly(A)l in the reaction mixtures. Nucleotide sequences complementary to cDNA 3, occur only once or twice in the genome of avian sarcoma viruses, are present in the genomes of virus strains from subgroups A, B, C, D, E, and F of avian leukosis-sarcoma viruses, and are conserved even when large deletions affect an adjacent viral gene ( src) By contrast, we find little or no complementarity between cDNA 3, and the genomes of golden pheasant virus (subgroup G of avian leukosis-sarcoma viruses), mouse mammary tumor virus, and the Moloney strains of murine sarcoma virus and murine leukemia virus. cDNA 3, can be used to identify the 3′-terminus of the avian sarcoma virus genome by molecular hybridization and will therefore be a useful reagent for the analysis of viral replication.

Tumour-associated transplantation antigens of neoplasms induced by a naturally occurring murine sarcoma virus (FBJ-MSV).

FBJ osteosarcoma virus (FBJ-MSV) isolated originally from a spontaneously arising osteosarcoma in a CF1 mouse is the only known naturally occurring murine sarcoma virus (MSV). It is unique among strains of MSV in producing primarily sarcomata in mice. The capacity of tumour cells transformed in vivo by this agent to elicit specific transplantation immunity in syngeneic hosts was investigated. A low level of resistance (10(4)-10(5) cells) was consistently induced by implantation of x-irradiated (15,000 rad) tumours or surgical excision of developing subcutaneous grafts. By contrast intraperitoneal inoculation of virus containing cellfree extracts of FBJ-MSV sarcomata was a far less effective immunization procedure. Confirmatory evidence for the antigenicity of these neoplasms was obtained in tests in which preincubation of tumour cells with lymphoid cells from specifically immune donors inhibited in vivo outgrowth of the FBJ-MSV cells in untreated syngeneic recipients. The induction of host resistance to FBJ-MSV cells by immunization with identical and independently-induced FBJ-MSV tumours established that FBJ-MSV cells possess common cell surface antigenic specificities in a manner analogous to those of experimental neoplasms induced by other oncogenic DNA and RNA viruses. Since FBJ-MSV cells release infectious virus it was not possible in this system to establish whether the tumour-rejection antigen was cellular or virion in nature. The antigenic weakness of FBJ-MSV cells in syngeneic hosts is comparable with that of virus-induced murine leukaemias of the Gross (G) or "wild" type subgroup to which category FBJ-MSV also belongs. These features suggest that FBJ-MSV exemplifies naturally occurring sarcomagenic viruses more closely than those of the Friend-Moloney-Rauscher-Graffi (FMRGr) subgroup which in general induce highly antigenic neoplasms.

Demonstration of a cell-surface antigen associated with murine sarcoma virus by immunoelectron microscopy.

Cells transformed by murine sarcoma virus have been examined for the presence of a new virus-associated cell-surface antigen by immunoelectron microscopy. A common antigen has been detected on the surface of nonproductively transformed cells that were induced by two different strains of murine sarcoma virus, Kirsten and Moloney. This antigen shows crossreaction with cell lines transformed by murine sarcoma virus that were produced in two different mammalian species, rats and mice. Further, this antigen is distinct from previously described antigens on the surfaces of cells infected by murine leukemia virus, on the viral envelope, and on the surfaces of spontaneously transformed cell lines or cell lines transformed by x-irradiation.

Properties of a murine sarcoma virus isolated from a tumor arising in an NZW-NZB F 1 hybrid mouse. II. Physical and biological characteristics.

AbstractThe properties of a potent murine sarcoma virus (MSV), which was isolated after in vivo passage of a spontaneous tumor arising in an NZW/NZB F1 hybrid mouse, are described. The agent had the morphological and growth characteristics of a type C virus, and contained the group‐specific (gs) antigen of the murine leukemia‐sarcoma viruses. The virus induced discrete, focal lesions of transformed cells in mouse tissue‐culture cells. Focus formation usually followed a “two‐hit” titration pattern, and sarcoma virus preparations contained a large excess of an accompanying non‐transforming type C virus. The MSV was stable when stored at — 70° C; it was ether‐sensitive and had a buoyant density of 1.16g/cm3 when centrifuged in sucrose density gradients. These properties are similar to those of the Moloney (M‐MSV), Harvey (H‐MSV), and Kirsten (Ki‐MSV) isolates of MSV. When inoculated into hamsters, the new strain of MSV rapidly induced sarcomas. While the viruses produced by hamster and mouse tumors both contained the murine gs antigen, the host range of the MSV was altered by hamster passage. It failed to induce focal lesions in mouse and hamster cells, and did not induce sarcomas or leukemias when injected into newborn mice and hamsters. The antigenic properties and infectivity of the virus produced by hamster tumor cells were significantly different from the properties of the hamster leukemia pseudotype viruses obtained from hamster tumors induced by the other strains of MSV.

Cytoplasmic DNA synthesis induced by RNA tumor viruses.

Infection of mouse embryo cells with two strains of murine sarcoma virus and one strain of murine leukemia virus was followed rapidly by synthesis of DNA in the cytoplasm. Persistently infected cells have not shown such synthesis, and ultraviolet-irradiated virus did not induce DNA synthesis. This new DNA presumably represents an intermediate in the virus replication cycle specified by the virion DNA polymerase(s). Failure to observe cytoplasmic DNA synthesis in persistently infected cells suggests, in keeping with the results of inhibitor experiments, that the new "viral" DNA becomes associated with cellular DNA in some form of stable interaction.

The effects of oronasal administration of two strains of murine sarcoma virus in mice.

AbstractOronasal administration of MSV‐H produced pathological changes in the lungs, mediastinum, gastric mucosa, spleen and brain. The early pulmonary changes showed intra‐alveolar haemorrhage, then organization forming a “granuloma” and finally a circumscribed lesion containing predominantly vasoformative tissue. Similar vasoformative lesions were seen in the perinodal areas of the medimtinum. MSV‐M lesions were found only in muscb and were of an inflammatory and reparative type of reaction. The significance of these findings is discussed.

Retention of antigen specificity of murine sarcoma viruses grown in hamster cells in vitro.

TUMOURS can be induced in hamsters by the various strains of murine sarcoma virus (MSV)1–6. Tumours differ, however, in the antigens which are expressed. Whereas the cell line HT-1, derived from early passages of a hamster tumour induced by the Moloney strain of MSV (M-MSV), contains no trace of infectious virus or virion antigen2,7, tumours induced by the Harvey (H), Kirsten (Ki) and later passages of the M-MSV-(GLV) viruses have yielded sarcoma viruses with a hamster-specific host range3–6,8 which do not share envelope4–6,9 or group specific10 antigens with murine viruses. The HT-1 cell does retain the MSV genome which can be rescued by murine leukaemia viruses2. Such rescued viruses are termed pseudo-types and contain the envelope and group-specific antigens of the rescuing virus. The virus preparation from tumours induced by M-MSV(GLV) differed from the other hamster-specific viruses in that a non-sarcomagenic C-type virus could be isolated from cultures infected beyond the cell transformation end point6. This virus was also hamster-specific in host range and antigenic properties and specifically interfered with cell transformation by the various hamster-specific virus strains9. This virus also shared an ether-stable virion-antigen with a C-type virus found in a lymphoma which occurred spontaneously in a hamster10. This shared antigen seems to be the principal structural polypeptide of hamster C-type viruses and is structurally similar but antigenically distinct from its mouse homologue (unpublished work of S. O., C. Foreman, G. K. and R. V. G.). These findings led us to propose that the hamster-specific non-sarcomagenic C-type virus was a hamster leukaemia virus (in the generic but not necessarily the pathological sense) and the virus is therefore designated HaLV9,10. The hamster-specific sarcoma viruses are considered to be pseudotypes of MSV rescued in vivo by HaLV and are abbreviated accordingly; for example, M-MSV(HaLV) represents the hamster-specific sarcoma virus rescued from M-MSV induced tumours. This is plausible because HaLV is able to rescue the MSV genome from HT-1 cells6. (This change in the nomenclature has been made in order to reflect the antigenic composition of the hamster-specific virus more accurately. In addition, to indicate the virus rescued from M-MSV(GLV)-induced hamster tumours, a terminal G is added after the parentheses. This has been done only to distinguish it from the virus obtained from M-MSV induced hamster tumours, for there is no evidence of residual activity from GLV.)

Pathologic and Virologie Studies of Tumors Induced in Mice by Two Strains of Murine Sarcoma Virus<xref ref-type="fn" rid="FN2">2</xref>

Pathologic and Virologie Studies of Tumors Induced in Mice by Two Strains of Murine Sarcoma Virus<xref ref-type="fn" rid="FN2">2</xref>

Studies of hamster-specific oncogenic virus derived from hamster tumors induced by Kirsten murine sarcoma virus.

Hamster tumors induced by the Kirsten strain of murine sarcoma virus (Ki-MSV) were found to contain a virus antigenically distinct from Ki-MSV. The virus had a restricted host-range and was infectious only for the hamster species but not for the mouse species from which the Ki-MSV was originally derived. The hamster-specific virus produced foci of cell transformation in hamster embryo fibroblasts and progressively growing sarcomas in newborn hamsters. One established tissue culture line of in vitro transformed hamster embryo cells (Line SLH) was found to release the hamster-specific focus-forming and sarcomagenic virus. These independent observations confirm and extend the recent observations of other investigators on the presence of such hamster-specific viruses in hamster tumors induced by various strains of murine sarcoma virus.

Studies on Murine Sarcoma Virus; a morphological comparison of tumorigenesis by the Harvey and Moloney strains in mice, and the establishment of tumor cell lines.

AbstractAnatomic lesions produced by the Moloney and Harvey strains of Murine Sarcoma Virus (MSV‐M and MSV‐H) in mice have been compared. Erythroblastic splenomegaly is a distinctive feature of disease produced by MSV‐H. Solid tumors induced by both viruses were quite similar in morphology, although some consistent differences were noted. The tumors appear to arise by massive recruitment of primitive mesenchymal cells rather than by clonal proliferation. The morphological features of these lesions would suggest that, although MSV‐M and MSV‐H are similar entities, they are not quite identical.Four transplant lines have been established from the mouse tumors; three MSV‐H and one MSV‐M. The MSV‐M line (CBA strain) was initiated with difficulty and did not release sarcoma virus. The MSV‐H lines (two CBA and one BALB/c) were easier to initiate and virus release was demonstrated both in vivo and in vitro. In addition to tumor growth at the site of implantation, the BALB/c line also produced splenic erythroblastosis. The MSV‐M and one of the MSV‐H CBA lines underwent a distinctive alteration during the course of in vivo passage in which the original spindle‐cell lesion evolved into an undifferentiated small‐cell tumor.

Studies on Murine Sarcoma Virus: antigenic characterization of Murine Sarcoma Virus induced tumor cells.

AbstractThe antigenicity of Murine Sarcoma Virus (MSV) transformed mouse, rat and hamster cells has been studied by several techniques designed to detect surface antigens. Antigens were readily demonstrated on neoplastic mouse and rat cells. There was complete cross‐reactivity between cells induced by the Harvey (MSV‐H) or Moloney (MSV‐M) strains of MSV and by Moloney Leukemia Virus (MLV). No evidence of a distinct or separate „MSV‐non‐MLV”︁ antigen could be obtained by cross absorptions of MSV/MLV antisera and cells, or by tests designed to break MLV tolerance. Such results favor the view that MSV and MLV are closely related entities.With the 8303 line of MSV‐M transformed hamster cells serological tests revealed little if any reactivity with strongly positive mouse anti‐MSV/MLV antisera. In one test, however, immunization of mice with these cells gave some degree of protection against syngeneic MSV sarcoma.

Different cell culture characteristics of two strains of murine sarcoma virus.

SummaryThe production of different morphological types of foci by two strains of MSV is described. MSV (Harvey) foci contain large clear cells which are absent from the MSV (Moloney) foci. Cells transformed by MSV (Moloney) are apparently restricted in their ability to divide. MSV (Harvey) transformed cells are not subject to the same restriction.