Infectivity of Proviral DNA from Avian Sarcoma Virus-Transformed Mammalian Cells

The number of Rous viral genomes in the cellular DNA from two subclones (RS2/3, RS2/6) derived from the same clone of hamster BHK-21 cells transformed by Rous sarcoma virus was determined by hybridization with viral complementary DNA made in vitro, and the capacity of the cellular DNA to infect (transfect) chicken embryo fibroblasts was compared before and after shearing this DNA to about the size of the provirus (6 x 10(6) to 7 x 10(6) daltons). The two subclones differed widely both in their capacity to give rise to virus (inducibility) after fusion with chicken embryo fibroblasts and in level of expression of viral proteins. It was shown that cells of both subclones contain a single copy of Rous DNA and yield infectious DNA. However, whereas transfection of chicken embryo fibroblasts was successful with both unsheared (>/=18 x 10(6) daltons) and sheared DNA from the most inducible subclone (RS2/3 subclone), which also expresses viral proteins to an appreciable amount, transfection with DNA from the least inducible subclone (RS2/6 subclone), in which viral proteins are not expressed, succeeded only with sheared DNA. It was then about as successful as with sheared or unsheared RS2/3 DNA. The lack of infectivity of unsheared RS2/6 DNA may be explained by the hypothesis proposed by Cooper and Temin (G. M. Cooper and H. T. Temin, J. Virol. 17:422-430, 1976) to explain the lack of infectivity of DNA from certain chicken cells producing spontaneously low amounts of RAV-0 and resistant to exogenous RAV-0 infection, that is, that the viral genome (proviral DNA) is linked to a cis-acting control element which blocks its expression. This linkage might originate, in RS2/6 cells, from translocation of cellular DNA containing the single proviral copy.     

Membrane Proteins of Uninfected and Rous Sarcoma Virus-Transformed Avian Cells

A method for preparing large membrane fragments and cell ghosts was developed for uninfected and Rous sarcoma virus-transformed chicken embryo fibroblasts in culture. Membrane proteins were analyzed by electrophoresis in acrylamide gels containing sodium dodecyl sulfate. A major amino-acid-containing component of uninfected cell membranes was greatly diminished in amount or absent in membranes of virus-transformed cells. This component, called MP-1, had an electrophoretic mobility in sodium dodecyl sulfate-containing gels similar to that of a protein of a mol wt of 1.42 x 10(5). MP-1 was not altered by changes in cell growth rate or in cells infected with the nontransforming virus RAV-1.     

Production of Virus by Mammalian Cells Transformed by Rous Sarcoma and Murine Sarcoma Viruses

Cultured cells of mammalian tumors induced by ribonucleic acid (RNA)-containing oncogenic viruses were examined for production of virus. The cell lines were established from tumors induced in rats and hamsters with either Rous sarcoma virus (Schmidt-Ruppin or Bryan strains) or murine sarcoma virus (Moloney strain). When culture fluids from each of the cell lines were examined for transforming activity or production of progeny virus, none of the cell lines was found to be infectious. However, electron microscopic examination of the various cell lines revealed the presence of particles in the rat cells transformed by either Rous sarcoma virus or murine sarcoma virus. These particles, morphologically similar to those associated with murine leukemias, were found both in the extracellular fluid concentrates and in whole-cell preparations. In the latter, they were seen budding from the cell membranes or lying in the intercellular spaces. No viruslike particles were seen in preparations from hamster tumors. Exposure of the rat cells to (3)H-uridine resulted in the appearance of labeled particles with densities in sucrose gradients typical of virus (1.16 g/ml.). RNA of high molecular weight was extracted from these particles, and double-labeling experiments showed that this RNA sedimented at the same rate as RNA extracted from Rous sarcoma virus. None of the hamster cell lines gave radioactive peaks in the virus density range, and no extractable high molecular weight RNA was found. These studies suggest that the murine sarcoma virus produces an infection analogous to certain "defective" strains of Rous sarcoma virus, in that particles produced by infected cells have a low efficiency of infection. The control of the host cell over the production and properties of the RNA-containing tumorigenic viruses is discussed.     

Purification By Oligo(dT)-Cellulose of Viral-Specific RNA from Sarcoma Virus-Transformed Mammalian Nonproducer Cells

Viral-specific RNA has been purified by oligo(dT)-cellulose chromatography from sarcoma virus-transformed nonproducer cells. This RNA comprises approximately 3% of the purified RNA, as judged by RNA-DNA hybridization.     

Comparison of Rous Sarcoma Virus-Specific Deoxyribonucleic Acid Polymerases in Virions of Rous Sarcoma Virus and in Rous Sarcoma Virus-Infected Chicken Cells

Labeled virions of Rous sarcoma virus (RSV) were disrupted with detergent and analyzed on equilibrium sucrose density gradients. A core fraction at a density of approximately 1.24 g/cc contained all of the (3)H-uridine label and about 30% of the (3)H-leucine label from the virions. Endogenous viral deoxyribonucleic acid (DNA) polymerase activity was only found in the same location. Additional ribonucleic acid (RNA)- and DNA-dependent DNA polymerase activities were found at the top of the gradients. RNA-dependent and DNA-dependent DNA polymerase activities were also found in RSV-converted chicken cells. Particles containing these activities were released from cells by detergent and were shown to contain viral RNA. These particles were analyzed on equilibrium sucrose density gradients and were found to have densities different from virion cores.     

Hybridization of Rous Sarcoma Virus Deoxyribonucleic Acid Polymerase Product and Ribonucleic Acids from Chicken and Rat Cells Infected with Rous Sarcoma Virus

Rous sarcoma virus (RSV)-specific ribonucleic acid (RNA) in virus-producing chicken cells and non-virus-producing rat cells infected with RSV was studied by hybridization with the endogenous deoxyribonucleic acid (DNA) product of the RSV virion DNA polymerase system. By hybridizing the total DNA product with excess virion RNA, the product DNA was separated into hybridized (“minus”) and nonhybridized (“plus”) DNA. The “minus” DNA was complementary to at least 20% of the RNA from RSV which remained of high molecular weight after denaturation. A maximum of approximately 65% hybridization was observed between “minus” DNA and RSV RNA or RSV-infected chicken cell RNA. A maximum of about 60% hybridization was observed between “minus” DNA and RSV-infected rat cell RNA. RSV-infected chicken cells contained RSV-specific RNA equivalent to about 6,000 virions per cell. RSV-infected rat cells contained RSV-specific RNA equivalent to approximately 400 virions per cell. Neither cell type contained detectable RNA complementary to virion RNA. The RSV-specific RNA in RSV-infected rat cells did not appear to be qualitatively different from that in RSV-infected chicken cells.     

Virus-Specific Antigens in Hamster Cells Transformed by Rous Sarcoma Virus

Virus-specific antigens were studied in hamster cells transformed by Rous sarcoma virus (RSV). Antigens were localized in the cytoplasm, as demonstrated by fluorescent antibody staining of fixed cells as well as by complement fixation (CF) following subcellular fractionation. Cytoplasmic extracts were analyzed by velocity and isopycnic centrifugation. CF antigens were found in a soluble form and in association with membranes and polyribosomes. Isolated plasma membranes had no CF antigen. Both soluble and particulate fractions with CF activity contained the same antigenic determinants by Ouchterlony analysis. These antigenic determinants were identical to those released by ether treatment of RSV.     

Virus Recovery in Chicken Cells Tested with Rous Sarcoma Cell DNA

DNA from non-virus-producing RSV transformed mammalian cells converts chicken fibroblasts into Rous sarcoma cells producing infectious RSV particles. The recovered virus is the same biologically and antigenically as the virus which originally transformed the mammalian cells.     

Virion-Associated RNA Primer for Rous Sarcoma Virus DNA Synthesis: Isolation from Uninfected Cells

Uninfected chicken, duck, rat, and human fibroblast cells in culture contained a tRNA-like RNA molecule which was structurally identical to a virion-associated RNA primer for in vitro Rous sarcoma virus DNA synthesis. This primer RNA appeared to be a normal tRNA of these cells. It was not found in a number of lower eukaryotic cells or in Escherichia coli.     

Infectious Rous Sarcoma Virus and Reticuloendotheliosis Virus DNAs

An efficient and quantitative assay for infectious Rous sarcoma virus and reticuloendotheliosis virus DNAs is described. The specific infectivities of viral DNA corresponded to one infectious unit per 10(5) to 10(6) viral DNA molecules. Infection with viral DNA followed one-hit kinetics. The minimal size of infectious Rous sarcoma virus DNA was approximately 6 million daltons, whereas the minimal size of infectious reticuloendotheliosis virus DNA was larger, 10 to 20 million daltons.     

Temperature-Dependent Transformation of Cells Infected with a Mutant of Bryan Rous Sarcoma Virus

Chick embryo cells infected with a mutant (Ta) of the Bryan high-titer strain of Rous sarcoma virus (RSV-BH) are morphologically transformed at 36 C but appear similar to uninfected cells at 41 C. When cells infected with RSV-BH-Ta are switched from 41 to 36 C, morphological changes characteristic of transformation are observable within 10 min. The transformation is reversible; cells shifted from 36 to 41 C have been observed to lose their transformed morphology within 1 hr. The transformation after a shift in temperature is unaffected by inhibition of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein synthesis, demonstrating that the proteins involved in the morphological change are already present. Transformed cells infected with RSV-BH or RSV-BH-Ta take up hexose and synthesize hyaluronic acid at higher rates than uninfected cells or RSV-BH-Ta-infected cells grown at 41 C. However, inhibition of either protein or RNA synthesis, but not DNA synthesis, prevented the induction of increased hexose uptake and hyaluronic acid synthesis after a shift of RSV-BH-Ta-infected cells from 41 to 36 C. Therefore, these biochemical changes are secondary to a more basic change responsible for morphological transformation.     

Characterization of Morphologic Revertants of Murine and Avian Sarcoma Virus-Transformed Cells

Morphologic revertants which contain avian or murine sarcoma viruses have previously been isolated at low frequency from clonal lines of transformed mammalian cells. In the present study, these lines have been further characterized. They are indistinguishable from nontransformed parent cell lines with respect to parameters such as saturation density and colony formation in depleted medium or on monolayers of contact-inhibited cells. The rate of glucose uptake had also reverted to normal. The malignant potential of one of the revertant lines was examined and found to be markedly reduced compared to that of the corresponding transformed cells. The differences in the susceptibilities of revertant cells to retransformation by the same or other oncogenic viruses suggest that different cellular genes may be involved in expression of transformation by various tumor viruses.     

Virus-Specific Ribonucleic Acid in Cells Producing Rous Sarcoma Virus: Detection and Characterization

Cells producing Rous sarcoma virus contain virus-specific ribonucleic acid (RNA) which can be identified by hybridization to single-stranded deoxyribonucleic acid (DNA) synthesized with RNA-directed DNA polymerase. Hybridization was detected by either fractionation on hydroxyapatite or hydrolysis with single strand-specific nucleases. Similar results were obtained with both procedures. The hybrids formed between enzymatically synthesized DNA and viral RNA have a high order of thermal stability, with only minor evidence of mismatched nucleotide sequences. Virus-specific RNA is present in both nuclei and cytoplasm of infected cells. This RNA is remarkably heterogeneous in size, including molecules which are probably restricted to the nucleus and which sediment in their native state more rapidly than the viral genome. The nature of the RNA found in cytoplasmic fractions varies from preparation to preparation, but heterogeneous RNA (ca. 4-50S), smaller than the viral genome, is always present in substantial amounts.     

Genetic Determinants of Rous Sarcoma Virus Particle Size

The Gag proteins of retroviruses are the only viral products required for the release of membrane-enclosed particles by budding from the host cell. Particles released when these proteins are expressed alone are identical to authentic virions in their rates of budding, proteolytic processing, and core morphology, as well as density and size. We have previously mapped three very small, modular regions of the Rous sarcoma virus (RSV) Gag protein that are necessary for budding. These assembly domains constitute only 20% of RSV Gag, and alterations within them block or severely impair particle formation. Regions outside of these domains can be deleted without any effect on the density of the particles that are released. However, since density and size are independent parameters for retroviral particles, we employed rate-zonal gradients and electron microscopy in an exhaustive study of mutants lacking the various dispensable segments of Gag to determine which regions would be required to constrain or define the particle dimensions. The only sequence found to be absolutely critical for determining particle size was that of the initial capsid cleavage product, CA-SP, which contains all of the CA sequence plus the spacer peptides located between CA and NC. Some regions of CA-SP appear to be more important than others. In particular, the major homology region does not contribute to defining particle size. Further evidence for interactions among CA-SP domains was obtained from genetic complementation experiments using mutant ΔNC, which lacks the RNA interaction domains in the NC sequence but retains a complete CA-SP sequence. This mutant produces low-density particles heterogeneous in size. It was rescued into particles of normal size and density, but only when the complementing Gag molecules contained the complete CA-SP sequence. We conclude that CA-SP functions during budding in a manner that is independent of the other assembly domains.     

Chromatographic and Electrophoretic Analysis of Viral Proteins from Hamster and Chicken Cells Transformed by Rous Sarcoma Virus

Several methods have been explored for the detection and characterization of viral proteins from soluble extracts of cells transformed by Rous sarcoma virus (RSV). Viral antigens have been analyzed after gel filtration in several solvents. In addition, immune complexes formed with virus-specific sera have been isolated by agarose gel filtration and by high- or low-speed centrifugation through sucrose solutions. Radioactive proteins from these immune complexes have been analyzed by gel filtration in 6 m guanidine hydrochloride or by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Comparison with proteins from purified virus indicates the presence of two viral core proteins (gs1 and gs2) in the soluble fraction from virus-producing chicken cells. In the same fraction from RSV-transformed hamster cells (which do not produce virus), three gs proteins (gs1, gs2, and gs3) could be identified. The soluble viral gs proteins are strongly bound to at least two larger polypeptides in cell extracts. These polypeptides do not appear to be viral in origin and have the property of undergoing a time-dependent aggregation in the extracts. One of these cell-derived proteins, which is present in a variety of uninfected cell types, closely resembles actin.     

Concentration of Rous Sarcoma Virus from Tissue Culture Fluids with Polyethylene Glycol

Concentration of Rous sarcoma virus from tissue culture fluids with polyethylene glycol, with and without NaCl or dextran sulfate, resulted in significant and highly variable losses caused by entrapment of virus particles in proteinaceous debris. Treatment of concentrated preparations with Pronase greatly enhanced the recovery of virions. Maximum recovery of virus particles was obtained by the addition of 8% polyethylene glycol and 0.4 M NaCl to tissue culture fluids, followed by Pronase treatment of the concentrated virus preparations.