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.     

Mutants of Rous sarcoma virus with extensive deletions of the viral genome.

Deletion mutants of Rous sarcoma virus (RSV) have been isolated from a stock of Prague RSV which had been irradiated with ultraviolet light. Quail fibroblasts were infected with irradiated virus and transformed clones isolated by agar suspension culture. Three clones were obtained which did not release any virus particles. Analysis of DNA from these non-producer clones with restriction endonucleases and the Southern DNA transfer technique indicated that the clones carry defective proviruses with deletions of approximately 4 × 10⁶ daltons of proviral DNA. The defective proviruses, which retain the viral transformation (src) gene, contain only 1.7–2.0 × 10⁶ daltons of DNA. Multiple species of viral RNA containing the sequences of the src gene were detected in these clones; some of these RNAs may contain both viral and cellular sequences. The protein product of the src gene, p60^src (Brugge and Erikson, 1977), was also synthesized in the nonproducer clones. However these clones did not contain the products of the group-specific antigen (gag), DNA polymerase (pol), or envelope glycoprotein (env) genes, nor did they contain the 35 and 28 S RNA species which are believed to represent the messengers for these viral gene-products. The properties of these mutants indicate that expression of the src gene is sufficient to induce transformation. These clones may represent useful tools for the study of the expression of this region of the genome.     

Size and genetic content of virus-specific RNA in myeloblasts transformed by Avian Myeloblastosis Virus (AMV)

Radiolabeled complementary DNA probes representing sequences specific for avian myeloblastosis virus (AMV) and sequences of different regions of avian leukosis virus (ALV) genome were used to analyze the gene content and size distribution of viral-specific RNA in AMV-transformed myeloblasts. The producer myeloblasts contained 8–10 times more copies per cell of virus-specific RNA than those of nonproducer (NP) myeloblasts. In the producer myeloblast, 35 S, 34 S, and 21 S virus-specific RNAs were detected. The 35 S RNA contained gag, pol, env, and c sequences; representing both the genomic and the messenger RNA of the helper virus. The 34 S RNA contained gag, pol, AMV, and c sequences, representing the genomic and the messenger RNA of AMV. The 21 S RNA of producer cells contained two different species: one contained env and c sequences and represented the glycoprotein messenger RNA of the helper virus; another contained AMV and c sequences and represented the subgenomic messenger RNA of AMV. In NP cells, two virus-specific RNAs were found; a 34 S RNA containing gag, pol, AMV, and c sequences and a 21 S RNA containing AMV and c sequences. Both RNAs hybridized to a probe specific for 5′ sequences of ALV. About 15% of pol sequences of virus-specific RNA in NP cells were found to be deleted.     

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.     

A comparison of the high molecular weight RNAs of visna virus and Rous sarcoma virus

Visna virus RNA consists of a high molecular weight and low molecular weight species. The size, subunit composition, complexity, and secondary structure of high molecular weight RNA have been studied and compared with Rous sarcoma virus RNA. Visna high molecular weight RNA cosediments in 0.1 M sodium chloride wiih the 70S UNA of Rous sarcoma virus consistent with a molecular weight of 10–12 × 10 daltons. On dissociation with heat, subunit structures of 2.8 × 10⁶ daltons are released. Both the size and heterogeneity of the subunit RNA are identical with the subunit RNA of transforming Schmidt-Ruppin Rous sarcoma virus. Comparison of the complexity of visna RNA with poliovirus RNA indicate unique nucleotide sequences of 7–10 × 10⁶ daltons, in accord with the physical data. The evidence also indicates that there is lack of significant reiteration of nucleotide sequences in the visna RNA. Major differences in the secondary structure of visna and Rous sarcoma virus RNA were observed. Both RNAs possess an exceptional degree of secondary structure when assayed by chromatography on cellulose at various temperatures. Reduction of ionic srength markedly decreases the secondary structure of visna RNA relative to Rous sarcoma virus RNA and leads to anomalous migration in polyacrylamide gels. The differences in secondary structure and in the low molecular weight RNA species associated with 70S RNAs of these viruses indirectly implicate the 4 and 5S RNA in the maintenance of secondary structure of 70S RNA of Rous sarcoma virus.     

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.     

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.     

FBR murine osteosarcoma virus. I. Molecular analysis and characterization of a 75,000-Da gag-fos fusion product

The FBR murine osteosarcoma virus complex induces bone tumors with a similar latency and pathology to those induced by the FBJ virus complex. FBR murine sarcoma virus ( FBR -MSV) has been isolated from its helper virus(es) by the establishment of transformed nonproducer cells. These cells were found to express a 75,000-Da protein (P75) which was antigenically related to the p55 oncogene product of the FBJ murine osteosarcoma virus ( FBJ -MSV). P75 also contained antigenic determinants of murine leukemia virus (MLV) gag gene p15, p12, and p30 proteins, and is therefore a gag- fos fusion protein ( P75gag - fos ). P75gag - fos is a phosphoprotein and is found primarily in the nucleus. Only a single species of RNA, of 3.3 kb, was identified in FBR -MSV-transformed nonproducer cells using both fos and MLV probes, which suggested that P75gag - fos was expressed from genome-sized RNA. Chromosomal DNA from one nonproducer cell line was found to contain a single EcoRI restriction fragment of 12 kb pairs (kbp) which encompassed the FBR -MSV provirus. This DNA fragment was molecularly cloned into bacteriophage Charon 30 (lambda FBR -1), and a 7.5-kbp HindIII restriction fragment containing the entire provirus was subsequently subcloned into pBR322 ( pFBR -1). DNA from pFBR -1 was capable of inducing morphological transformation of mouse and rat fibroblasts in tissue culture. In addition, transfected cells expressed the FBR -MSV P75gag - fos protein.     

RNA in mammalian sarcoma virus transformed nonproducer cells homologous to murine leukemia virus RNA

Mammalian sarcoma virus transformed nonproducer clones of mouse and rat cells contain RNA which hybridizes to the DNA product made from virus preparations containing leukemia virus, or sarcoma and leukemia virus. The hybridization of this virus-specific [³H]thymidine-labeled DNA to either total cellular or polysomal RNA extracted from these cells was detected with an enzymatic assay using a nuclease preparation either from mung beans or from Aspergillus oryzae, or by centrifugation in Cs₂SO₄ density gradients. The enzymatic assays were found to be more sensitive than Cs₂SO₄ for detection of such hybrids.     

Isolation of complementary DNA unique to the genome of avian myeloblastosis virus (AMV)

Avian myeloblastosis virus (AMV) can transform avian cells of hemopoietic origin, such as bone marrow, embryonic yolk sac, and circulating macrophages. The AMV is defective in its replication and can only replicate in the presence of helper viruses. This defectiveness in replication is probably due to a deletion or substitution of nucleotide sequences in its genome. The AMV genome contains no sequences homologous to the src gene, which is responsible for the transforming function of the sarcoma viruses. We attempted to identify the AMV sequences that may contain sequences which are responsible for its transforming function. We isolated a complementary DNA (cDNA_AMV) that hybridized preferentially to the RNA of the transforming AMV but not to the RNA of the helper virus. Using this cDNA_AMV as a probe, we determined the size of the AMV genome to be 33–34 S with a molecular weight of 2.6 × 10⁶. A similar molecular weight estimation of the AMV genome size was obtained by methylmercury-agarose gel electrophoresis of AMV RNA. In AMV-producer myeloblasts we can detect about 6000–7000 copies per cell of AMV-specific RNA, whereas fewer than 2 copies per cell of AMV RNA are found in helper virus-infected cells. In AMV nonproducer myeloblasts, about 2000 copies of AMV-specific RNA are detected. Furthermore, we find that RNA of AMV NP myeloblasts can only hybridize to 55% of cDNA complementary to helper virus genome. In uninfected hemopoietic cells, e.g., bone marrow cells, about 20 copies per cell of AMV-specific RNA are present, whereas in uninfected chick embryo fibroblasts less than 1 copy per cell is found.     

Analysis of the expression of spleen focus-forming virus (SFFV)-related RNA and gp55, a friend and rauscher virus-specific protein

A 55,000-dalton glycoprotein, gp55, is the major intracellular species precipitable with anti-envelope glycoprotein (gp70) sera in murine erythroleukemia cells transformed by either the anemia- or the polycythemia-inducing strains of Friend virus. Similarly, all Friend erythroleukemia cell lines studied contained similar levels of spleen focus-forming virus (SFFV)-specific RNA in the cytoplasm as assessed by hybridization to a Friend SFFV-specific eDNA probe. Neither SFFV-specific RNA nor gp55 is detectable in helper virus-infected cells or in chemically induced rat erythroleukemia cell lines. SFFV non-producer cell lines were isolated and analyzed for viral gene expression by immunoprecipitation and molecular hybridization. SFFV nonproducer cells could be grouped into two classes: those which constitutively synthesize gp55 and those which synthesize gp55 after superinfection with type-C helper virus. No gag gene-related proteins were detected in any of these nonproducer cell lines. Characterization of Friend virus-specific RNAs in SFFV nonproducer cells indicates that they can express both a 32 S and a 21 S RNA species related to Friend virus. SFFV nonproducer cells expressing 21 S SFFV-specific RNA constitutively synthesize gp55.     

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.     

A transfection assay for transformation by feline sarcoma virus proviral DNA

We report that fibroblast lines derived from mouse (NIH 3T3) and mink (CCL-64) can be transformed via transfection with the total cellular DNA extracted from cells transformed with feline sarcoma virus. Replication of helper virus is not required in the recipient cell. Transformation of recipient cells is observed when the donor DNA is extracted from either virus producer or nonproducer transformed cell lines. A transforming virus can be rescued from nonproducer transformed recipients upon superinfection with a replication competent helper virus.     

Avian oncovirus MH2: preferential growth in macrophages and exact size of the genome.

Pseudotype preparations of the defective avian oncovirus MH2 contain a large excess of helper virus when grown in fibroblasts but relatively more MH2 when replicated in macrophages. Such macrophage grown virus was used for an accurate size determination of the genomic RNA of MH2. This 28 S RNA species contains between 5700 and 5900 nucleotides and has a molecular weight of 1.9 × 10⁶.     

Electron microscopy of viral RNA: avian tumor virus RNA.

We have investigated by electron microscopy the structure of native, partially denatured, and heated and quick-cooled RNA from avian myeloblastosis virus (AMV) and Schmidt-Ruppin Rous sarcoma virus (SR-RSV). Native 60–70 S RNA has a highly folded structure, while partially denatured 60–70 S RNA is more extended but retains much secondary and tertiary structure and has multiple free ends. 60–70 S RNA that has been heated and quick-cooled appears smaller than unheated RNA and retains some secondary structure when prepared for electron microscopy under nondenaturing conditions. The appearance of 60–70 S RNA before and after heating indicates a dissociation of the 60–70 S RNA on heating rather than a change in conformation of the RNA. Molecular weight of large subunit RNA has been determined by electron microscopy of formaldehyde-formamide treated RNA. The largest RNA subunit of AMV has a molecular weight of 2.9 × 10⁶. The largest subunit of the transforming virus SR-RSV is 20% larger with a molecular weight of 3.5 × 10⁶.     

Evidence of precursors of defective measles virus

Cytoplasmic extracts of Vero cells infected with wild strain Edmonston measles virus were found to contain at least two distinct nucleocapsid species. The two most prominent species of nucleocapsids sedimented at 200S and 110S and contained RNA of molecular weight 6.0×10⁶ and 0.6×10⁶ daltons respectively. Both species of nucleocapsids had a density of 1.31 g/cm³ in CsCl. A third species sedimenting at 170S was not present in all experiments and was not characterized in detail. Infection of cells with undiluted-passage virus usually resulted in production of mostly 110S nucleocapsids while both 110S and 200S species were found when diluted-passage virus was used. These results suggest that measles virus may produce distinct classes of defective virus which contain segments of RNA representing as little as 10% of the complete viral genome.