Expression of viral envelope glycoprotein and transformation genes in cells transformed by a defective Kirsten murine sarcoma virus.

Concurrent expression of transformation properties and virion envelope glycoproteins has been observed as a property of several clones of normal rat kidney cells transformed by, but not producing, Kirsten murine sarcoma virus. The present studies were carried out to determine whether a genetic linkage exists between the viral sarcoma and envelope genes in these cells. Several alternative models for the possible structure and origin of the sarcoma and envelope genes were considered. One possibility, that the viral envelope gene was derived from an endogenous rat virus, was studied by characterization of the interference properties of the transformed cells. The sarcoma virus genome of envelope-positive clones was efficiently rescued by woolly monkey and murine xenotropic but not by murine ecotropic viruses. Thus, the interference properties of cells producing the envelope glycoprotein are analogous to those of a cell producing murine ecotropic virus, indicating that the envelope was of murine viral origin. In these experiments it was also found that sarcoma viruses rescued from envelope-positive cells upon superinfection with primate and xenotropic murine viruses could transform host cells for both xenotropic and ecotropic viruses, indicating that these superinfecting viruses became phenotypically mixed with the ecotropic envelope expressed in transformed, envelope-positive cells. Possible linkage between the envelope and transformation genes was analyzed by the frequency of concurrent rescue of sarcoma and envelope genes. Transfer of the Kirsten sarcoma viral genome to uninfected cells upon rescue by superinfection with woolly monkey virus showed a high frequency of apparent segregation of the transformation and envelope genes [from 29 to 57% for (KSV env⁺)NRK-6]. The model supported by the present data is that the transformed, envelope-positive cells were infected with a virus which contained both the envelope and the sarcoma genes.     

Origin and biological properties of a new feline sarcoma virus

A new strain of feline sarcoma virus (GR-FeSV) was isolated from a spontaneous sarcoma of an 8-year-old domestic house cat. The virus induced sarcomas at high incidence after a short latent period in fetal and newborn kittens and transformed cat embryo fibroblasts in vitro after 5 days. Compared to Gardner-Arnstein and the Snyder-Theilen strains of FeSV, GR-FeSV induced more pleomorphic sarcomas and larger, more rounded and discrete foci of cell transformation. GR-FeSV was shown to be defective for replication, and nonproducer transformed clones from several species were obtained at limiting GR-FeSV dilution. The defective sarcoma virus could be rescued from such transformants by superinfection with replication competent type-C viruses. The primary translational product of the GR-FeSV genome is a 70,000-dalton polyprotein that contains the amino-terminal domain of the FeLV gag gene precursor protein and a sarcoma virus-specific polypeptide. These results differentiate GR-FeSV from previously isolated FeSV strains, and establish it as an independent spontaneously occurring sarcomavirus isolate of the domestic cat.     

Isolation of a fibroblast nonproducer cell line containing the Friend strain of the spleen focus-forming virus.

A BALB/c spleen homogenate containing a mixture of B-tropic type-C helper virus and the Friend strain of spleen focus-forming virus (SFFV) was found by end point titration to contain equivalent biologic titers of helper virus and SFFV. By infection of BALB/c 3T3 cells with an appropriate dilution of this homogenate, we have isolated two single cell clones which contain SFFV free of replicating type-C helper virus. When these SFFV-containing nonproducer cells were superinfected with a cloned stock of the Friend strain of helper type-C virus,.a virus mixture was obtained which produced abundant splenic foci within 9 days in young adult BALB/c mice. The nonproducer cells containing the SFFV genome are similar in morphology to uninfected BALB/c 3T3 cells, do not release virus particles, and' are virus negative by electron microscopy. Thus, the Friend strain of SFFV has been obtained free of replicating helper virus. The results suggest that SFFV represents a class of replication-defective RNA type-C viruses which are unable to transform mouse fibroblasts but which, like mammalian RNA-containing sarcoma viruses, are associated with a rapid malignant disease in mice.     

Analysis of cells transformed by defective leukemia virus OK10: production of noninfectious particles and synthesis of Pr76gag and an additional 200,000-dalton protein

Nonproducer cells transformed by the defective leukemia virus (DL V), OK10, have been analyzed. Unlike nonproducer cells transformed by the other avian defective leukemia viruses examined so far, the OK10-transformed cells were found to release noninfectious particles. Analysis of these particles indicated that they contained the viral gag gene proteins but lacked env and pol gene products. In agreement with these results analysis of [³⁵S]methio-nine-labeled cell extracts of these nonproducer clones by immune precipitation showed that of the three viral structural protein precursors Pr769^gag, gPr95^env, and Pr180^gag-pol only Pr76^gag could be detected. In addition, a 200,000 molecular weight protein (OK10-200K) was identified in the cell extracts which by using specific antisera, was shown to be related to the gag and pol gene products but not to the product of the env gene. Tryptic peptide analysis of the OK10-200K protein confirmed the immunological data in that the OK10-200K protein was shown to contain all but one of the Pr1809^gag-pol methionine tryptic peptides plus unique peptides which were specific for OK10 and not related to the env gene product. One of these OK10-specific peptides was also shown to comigrate with one of the putative mac gene product tryptic peptides of the MC29-110K protein. These data indicate a novel gene order for a DLV.     

Biological properties and translational products of three independent isolates of feline sarcoma virus

Three transforming retroviruses have been isolated from naturally occurring tumors of outbred cats. In the present report, the biological properties of the SM, GA, and ST strains of feline sarcoma virus (FeSV) have been characterized in tissue culture. Like previous mammalian transforming viruses, each FeSV strain was demonstrated to be replication defective, requiring a type-C RNA virus as a helper. Reproducible differences were demonstrated in the morphology of transformed foci induced by each FeSV strain. These findings were independent of helper virus or assay cell utilized, suggesting that the transforming activities of the three FeSV strains are distinguishable. Nonproducer transformants of SM-FeSV expressed feline leukemia virus (FeLV) gag gene products, p15, p12, and p30. In contrast, the GA-and ST-FeSV genomes coded only for p15 and p12 proteins, further distinguishing SM-FeSV from the other FeSV strains. Each remained stable with respect to viral protein expression upon transmission to new cells, demonstrating the stable association of FeLV gag gene information with each FeSV genome. Analysis of the FeLV structural proteins translated in nonproducer transformants of the three FeSV strains revealed the presence of precursor molecules, whose sizes could not be accounted for solely by the number of FeLV gag proteins present in them.     

Genome structure of the defective avian sarcoma virus PRCIV

The genome of PRCIV, a defective avian sarcoma virus of class II, was studied by nucleic acid hybridization and oligonucleotide mapping. It has a length of about 6.1 kilobases (kb). A stretch of retrovirus replicative sequences encompassing part of the gag, all of the pol, and most of the env region, in all about 5 kb, is deleted. In its place a 2.9 kb substitution that is transformation-specific for PRCIV was found. Flanking the PRCIV-specific substitution are sequences shared with the helper virus, a stretch of 2.0 kb on the 5′-side, including part of the gag region, and a stretch of up to 1 kb on the 3′-side, including the C-region and possibly some env sequences. A PRCIV-specific cDNA probe representing the transformation-specific sequences was prepared. This probe hybridizes only with RNAs of other class II sarcoma viruses but fails to hybridize with either class I or class III sarcoma viruses.     

Murine sarcoma viruses: the helper-independence reported for a Moloney variant is unconfirmed; distinct strains differ in the size of their RNAs.

A variant of Moloney murine sarcoma virus (Mo-MSV) reported to behave like a nondefective sarcoma virus was subjected to biological and biochemical analyses to determine whether its alleged helper-independence could be confirmed. When plated at low multiplicity the virus was shown to readily generate (four out of six) transformed clones which failed to produce virus unless superinfected with helper leukemia virus. The RNA of the parental virus stock was compared electrophoretically to that from clones which produced virus after the initial infection (producer clones) or after superinfection with Mo-murine leukemia virus (MLV) (nonproducer clones). All clones contained a MSV-specific 30 S RNA species. In addition, virus from one producer clone also contained 38 S MLV RNA at a high relative concentration, indicating that the original Mo-MSV stock must have contained such an RNA species. However, the original virus stock as well as virus from another producer clone contained 38 S MLV RNA at a low, uncertain relative concentration. A hypothesis consistent with these and previous data suggests that the Mo-MSV variant investigated here is defective and contains helper leukemia virus at a low concentration. This explains (i) the ready generation of nonproducer clones by infection at low multiplicity, (ii) the difficulty in detecting helper leukemia virus 38 S RNA in the original virus stock, and (iii) the low complexity (approximately 1.9 × 10⁶ daltons) of the MSV-specific 30 S RNA. These results are compatible with the properties reported for a defective MSV genome, but incompatible with those of a nondefective MSV genome. The MSV-specific RNA components of different clonal isolates of Mo-MSV differed from each other in size, ranging between 2.1 and 1.6 × 10⁶. The Harvey sarcoma virus-specific RNA was 1.9 × 10⁶, that of Kirsten sarcoma virus was 2.5 × 10⁶, and the spleen focus forming component of Friend virus was 2.0 × 10⁶. The sarcoma- or transformation-specific RNA components of all transforming viruses tested here were smaller than the 38 S RNA of helper leukemia viruses of 3.1 × 10⁶.     

Identification of a 39,000-dalton protein in cells transformed by the FBJ murine osteosarcoma virus

Two components of the FBJ murine osteosarcoma virus complex have been isolated separately in tissue culture; the FBJ murine leukemia virus (FBJ-MLV) by dilution and the FBJ murine sarcoma virus (FBJ-MSV) by the establishment of nonproducer transformed rat cells. Analysis of these cells using MLV antisera indicated that there were no new proteins related to viral structural proteins specifically associated with the presence of the FBJ-MSV genome. The FBJ-MSV nonproducer cells were used to induce tumors in syngeneic and allogeneic F₁ rats. Sera from tumor-bearing rats were examined for activity against FBJ-MSV-specific antigens. A number of sera were found to precipitate a 39,000-dalton protein, p39, from several producer and nonproducer FBJ-MSV transformed rodent cells, but not from cells transformed by other strains of MSV or cells infected with MLV. Precipitation of p39 was not blocked by the presence of excess viral proteins, indicating that p39 is not related to the viral structural proteins. This conclusion was confirmed by methionine tryptic peptide analysis which showed that the fingerprint of p39 was distinct from those of the viral gag or env gene proteins. The data demonstrate the presence of a unique antigenic protein, unrelated to the MLV proteins, in FBJ-MSV transformed cells.     

Genetic analysis of the defectiveness in strain MC29 avian leukosis virus.

Avian myelocytomatosis virus MC29 is defective in replication. The extent of this defectiveness was analyzed in complementation experiments. Continuous nonproducer quail cell lines transformed by MC29 were superinfected with different helper viruses. Infectious MC29 pseudotypes were formed only with helper viruses which belonged to the avian leukosis-sarcoma virus complex, e.g., Rous-associated virus type 1 (RAV-1) or ring-necked pheasant virus (RPV). Helper viruses of other retroviral species, such as reticuloendotheliosis virus (REV), golden pheasant virus (GPV), or an amphotrophic murine leukemia virus (MuLV-1313), could not rescue MC29 from these nonproducer cells but complemented the envelope-defective Bryan high titer strain of Rous sarcoma virus. Host range and interference patterns of MC29 rescued from nonproducer cells indicated that the envelope specificity of the pseudotypes was determined exclusively by the activating helper virus. Cocultivation or Sendai virus-induced fusion of MC29 nonproducer cells with quail or chicken cells transformed by the defective Bryan high titer strain of Rous sarcoma virus did not result in complementation of the defects in either of the two viruses. Fluorescent antibody staining failed to detect virus-specific antigens at the surface of MC29 nonproducer cells. Temperature-sensitive mutants of Rous sarcoma virus with lesions in the genes coding for the RNA-directed DNA polymerase (pol gene) or the nonglycosylated structural proteins (gag gene) did not acquire wild-type characteristics when grown in MC29 nonproducer cells. It is concluded that MC29 is defective in all three genes essential for the replication of retroviruses, namely, env, pol, and gag.     

Esh avian sarcoma virus codes for a gag-linked transformation-specific protein with an associated protein kinase activity

Esh sarcoma virus, initially isolated from a spontaneous tumor of a chicken, transforms fibroblasts in vitro and induces fibrosarcomas in vivo. It is defective for replication, and infectious viral stocks consist of a mixture of a sarcomagenic virus (ESV) and an a avian leukosis virus of subgroup A (EAV) which serves as helper. Cloned stocks of infectious ESV contain two RNA components of M_r, 3 and 1.5 × 10⁶, respectively, as determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The component of M_r 1.5 × 10⁶ appears to be the genome of defective ESV, since it is not detected in preparations of the helper virus EAV. The size of the ESV genome suggests major deletions of replicative genes, and ESV-transformed nonproducer cells fail to express functional translation products of the gag, pol, and env genes. ESV-transformed producer and nonproducer clones also do not express pp60^src but contain a gag-related protein of M_r 80,000 (p80). Two-dimensional analyses of the [³⁵S]methionine-labeled tryptic peptides of p80 indicate that this protein contains part of the sequences of gag-p19 covalently linked to additional sequences unrelated to gag, pol, and env gene products. These ESV-specific sequences are also unrelated to pp60^src and to gag-linked polyproteins found in cells transformed by defective avian sarcoma viruses PRCII and Fujinami or defective leukemia viruses AEV, MC29, and MH2. P80 is phosphorylated in vivo at two major sites, one involving phosphoserine and the other phosphotyrosine residues. Immunoprecipitates containing ESV-p80 are associated with a protein kinase activity that is specific for tyrosine residues of several acceptor molecules including p80 itself, rabbit immunoglobulin H chain of the immune complex and exogenously added α-casein. p80 is phosphorylated in vitro at the same tyrosine site as in vivo suggesting that the enzyme activity detected in vitro is of physiological significance. The p80-associated protein kinase activity is strictly dependent on the presence of Mg²⁺ or Mn²⁺ but was found independent of known effectors of cellular protein kinases Ca²⁺, cAMP, or cGMP.     

Characterization of a 105,000 molecular weight gag-related phosphoprotein from cells transformed by the defective avian sarcoma virus PRCII

In cells infected with the replication-defective avian sarcoma virus PRCII a single virus-specific product is detectable, a polyprotein of 105,000 molecular weight (p105). P105 can be precipitated with antisera togag proteins of avian leukosis and sarcoma viruses. By two-dimensional tryptic peptide analysis of [³⁵S]methionine-labeled proteins we have shown that p105 contains peptides of helper viriongag proteins p19 and p27, but not of p15. In addition a number of peptides are present in p105 that are not found in any of the helper virus gene products including gPr95^env and Pr180^gag-pol. These p105-specific peptides are not detectable in the p60^src protein of Rous sarcoma virus (RSV) nor in thegag-related polyproteins encoded by avian myelocytoma and carcinoma viruses MC29 and MH2 or avian erythroblastosis virus AEV. P105 is not detectably glycosylated, but is heavily phosphorylated. In this respect it resembles p60^src of RSV rather than the polyproteins of avian leukemia viruses. Since p105 is the only viral gene product detectable in nonproducing cells transformed by PRCII, this protein may be important in the initiation and maintenance of oncogenic transformation. The nonstructural sequences in p105 would then represent a new class of transforming gene in avian oncoviruses.     

Continuous production of Rous sarcoma virus free of transformation defective virus in clones of established RSV-transformed quail cells

Quail embryo fibroblasts were infected with a Schmidt-Ruppin strain RSV × chf recombinant virus. Virus-transformed cells were established as a permanent line and then cloned in methyl cellulose. Out of 140 clones isolated four clones were capable of indefinite growth. These clones were examined for (i) production of sarcoma and td virus particles, (ii) number of integrated virus genome equivalents, and (iii) deletions of the src gene in the provirus. We found that the clones yield about 106 focus-forming units of the sarcoma virus per milliliter of the culture medium. No td virus could be detected by plating of the virus at the endpoint dilution and no 35 S td virus RNA but only 38 S sarcoma virus RNA was found in virions. Hybridization kinetic studies indicated that three different clones contain about 2 virus genome equivalents, and one clone contains about 4 virus genome equivalents per diploid cell. Upon transfection the proviruses of different clones generated sarcoma viruses and no td viruses. Finally digestion with EcoRI restriction endonuclease released in all four clones a 1.9 × 106-dalton fragment characteristic of the complete src gene, while no 0.8 × 10⁶-dalton fragment characteristic of a td provirus could be detected. We concluded that the clones of RSV-transformed quail cells contain only nondefective sarcoma proviruses and produce from these proviruses nondefective focus-forming virions in the absence of any segregant td virions.     

Avian erythroblastosis virus: transformation-specific sequences form a contiguous segment of 3.25 kb located in the middle of the 6-kb genome.

Several foci of chicken embryo fibroblasts transformed by avian erythroblastosis virus (AEV) strain ES-4 were found to produce virus progeny containing the RNA of the replication-defective AEV in excess over the RNA of the helper virus. The size of AEV RNA was determined by methylmercury-agarose gel electrophoresis and electron microscopy to be 28 S or 6 kb. About 40 to 45% of this RNA is homologous by RNA-DNA hybridization to the RNA of other chicken leukosis and sarcoma viruses; the rest of the genome is AEV specific. These AEV specific sequences, which presumably contain the genetic information responsible for transformation form a contiguous stretch of 3.25 kb, located by heteroduplex mapping in the center of the 6 kb genome between two segments of 1.06 kb at the 3′ end and 1.64 kb at the 5′ end which are homologous to the genome of avian sarcoma virus. From the length of the region showing homology between AEV and avian sarcoma virus at the 5′ end of the genome and from the known sequence composition of the AEV-specific 75K protein (Hayman et al., 1979), it can be deduced that the initiation point for the N terminus of the gag protein p19 is located about 1.0 kb from the 5′ end of the genome in avian oncoviruses. Nonproducing AEV-transformed chicken embryo fibroblasts were also isolated. Infectious AEV could be rescued from these cells only with chicken leukosis viruses; unrelated avian retroviruses were ineffective, probably because AEV requires complementation in the gag and pol genes, in addition to env.     

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^?7M 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.     

Immunolatex spheres for cell and virion surface labeling in the electron microscope.

Latex spheres containing aromatic amines have been activated by diazotization and covalently coupled to immunoglobulins to produce immunospecific markers. The uniform size (200 ran), shape, and electron density of these spheres allow for their visualization by means of thin sections in the electron microscope. The utility of these spheres was evaluated in a known system using antisera to the major envelope glycoprotein (gp70) of Rauscher murine leukemia virus. This broadly reactive antiserum bound to the virions and surfaces of cells producing the various envelope types of murine leukemia virus and rat and cat type C viruses, but not to those producing RD114, Mus caroli type C virus, bovine leukemia virus, baboon type C virus, or woolly monkey sarcoma virus. Results obtained with the immunolatex method compared favorably with those obtained using the immunoferritin technique.     

Isolation and characterization of low-molecular-weight DNA-binding proteins from retroviruses

The DNA-binding proteins from Rauscher leukemia virus, baboon leukemia virus, endogenous cat virus, woolly monkey virus, bovine leukemia virus, equine infectious anemia virus, Rous sarcoma virus, Prague strain, mouse mammary tumor virus, and Mason-Pfizer monkey virus were purified to homogeneity by molecular sieving chromatography and DNA affinity chromatography. The amino acid composition shows each to be a basic protein.