Nature of Ribonuclease-resistant Nucleic Acid of Chick Embryo Cells transformed by Schmidt–Ruppin Strain of Rous Sarcoma Virus

THE mode of replication of RNA or RNA-containing tumour viruses is not understood. The recent studies on Rous sarcoma and other RNA-containing oncogenic viruses suggest that the replicative cycle of the RNA of these viruses might not be associated with ribonuclease-resistant structures (double stranded RNAs), but might involve the synthesis of a DNA intermediate specific to viral RNA^1–3. Two groups of workers, however, presented evidence for the presence of a double stranded RNA in 78 Al cell line of rat embryo fibroblasts which had been transformed and chronically infected with the murine sarcoma-leukaemia virus complex (MSV-MLV)^4,5 and it was suggested that the mode of replication of oncogenic viral RNAs was the same as that of non-oncogenic viral RNAs⁴. This apparent discrepancy prompted me to look for ribonuclease-resistant RNA structures in the chick embryo cells transformed by Schmidt-Ruppin Rous sarcoma virus (SR-RSV).     

Cell Cycle-Dependent Activation of Rous Sarcoma Virus-Infected Stationary Chicken Cells: Avian Leukosis Virus Group-Specific Antigens and Ribonucleic Acid

Stationary chicken embryo fibroblasts exposed to Rous sarcoma virus (RSV) remained stably infected for at least 5 days, but they did not release infectious virus or become transformed until after cell division. These infected stationary cells did not contain avian leukosis virus group-specific antigens or ribonucleic acid (RNA) hybridizable to deoxyribonucleic acid (DNA) made by the RSV endogenous RNA-directed DNA polymerase activity.     

Replication of Reticuloendotheliosis Viruses in Cell Culture: Acute Infection

Replication of reticuloendotheliosis viruses (REV) in cultures of chicken and duck fibroblasts leads to some cell death soon after infection. This cell killing was used to develop a plaque assay for Trager duck spleen necrosis virus (TDSNV) on duck embryo fibroblasts. A normal replicative cell cycle was required for normal virus production and the development of cytopathic effects in chicken cells exposed to TDSNV. The latent period was about two days. Stationary chicken embryo fibroblasts could be infected by REV; DNA synthesis was required, but protein synthesis was not.     

Adsorption of Rous sarcoma virus to genetically susceptible and resistant chicken cells studied by laser flow cytometry.

Quantitative binding of Rous sarcoma virus (RSV) of different antigenic subgroups to chicken cells was examined by using a laser flow cytometer/cell sorter. RSV of subgroups A, C, and E, labeled with the fluorescent membrane probe rhodamine-18, bound 2 to 10 times more to genetically susceptible chicken embryo fibroblasts than to resistant cells, as measured by flow cytometry on a single-cell basis. This suggested that susceptible cells possess both specific and nonspecific receptors for virus adsorption, whereas resistant cells bind virus only by means of nonspecific sites. Polybrene at low concentration increased eightfold the binding of virus. Higher levels of Polybrene inhibited adsorption. Cell binding sites were saturable, and attachment of labeled virus could be partially blocked by preexposure of cells to unlabeled RSV. Virus surface glycoproteins played an important role in adsorption, since their removal with bromelain decreased binding of virus to susceptible cells. Maximal binding of RSV to both susceptible and resistant cells occurred within 10 min, although the level of binding was up to 10-fold higher for susceptible cells. Binding to all cell types showed a broad distribution. This implies that there are considerable differences in the number of virions bound per cell.     

The family of env genes of avian retroviruses: molecular analysis of Rous sarcoma virus adapted to duck cells

For the elucidation of the molecular basis of RSV adaptation to conditionally permissive host from the genome library of duck embryo fibroblasts, transformed by Rous sarcoma virus in 30 passages on these cells, recombinant bacteriophages that include provirus sequences, were obtained. Complete and transformation-defective proviruses were characterized, nucleotide sequences of their env-genes were compared with their counterparts the original RSV (Pr-RSV-C) and with viruses of other subgroups (A, B, D and E). The possible relation of the revealed changes in domains coding gp85 and gp37, with the changes of chicken RSV characteristics during adaptation to duck cells is discussed.     

Role of human beta-defensin-2 during tumor necrosis factor-alpha/NF-kappaB-mediated innate antiviral response against human respiratory syncytial virus

Human respiratory syncytial virus (RSV) constitutes a highly pathogenic virus that infects lung epithelial cells to cause a wide spectrum of respiratory diseases. Our recent studies have revealed the existence of an interferon-alpha/beta-independent, innate antiviral response against RSV that was dependent on activation of NF-kappaB. We demonstrated that NF-kappaB inducing pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF) confers potent antiviral function against RSV in an NF-kappaB-dependent fashion, independent of interferon-alpha/beta. During our efforts to study this pathway, we identified HBD2 (human beta-defensin-2), a soluble secreted cationic protein as an antiviral factor induced during NF-kappaB-dependent innate antiviral activity in human lung epithelial cells. Our results demonstrated that HBD2 is induced by TNF and RSV in an NF-kappaB-dependent manner. Induction of HBD2 in infected cells was mediated by the paracrine/autocrine action of TNF produced upon RSV infection. HBD2 plays a critical role during host defense, because purified HBD2 drastically inhibited RSV infection. We also show that the antiviral mechanism of HBD2 involves blocking of viral cellular entry possibly because of destabilization/disintegration of the viral envelope. The important role of HBD2 in the innate response was also evident from loss of antiviral activity of TNF upon HBD2 silencing by short interfering RNA. The in vivo physiological relevance of HBD2 in host defense was apparent from induction of murine beta-defensin-4 (murine counterpart of HBD2) in lung tissues of RSV-infected mice. Thus, HBD2 functions as an antiviral molecule during NF-kappaB-dependent innate antiviral immunity mediated by the autocrine/paracrine action of TNF.     

Ultrastructure of the Surfaces of Cells Infected with Avian Leukosis-Sarcoma Viruses

When stained with ruthenium red (RR), chick embryo cells infected with various strains of Rous sarcoma virus (RSV) and with avian leukosis viruses RAV-1 and RAV-3 showed an increase in the layer of acid mucopolysaccharides (AMPS) at their surfaces as compared with uninfected cells. This increase was most prominent in cells infected with the Fujinami strain of RSV. The layer was resistant to digestion with neuraminidase or trypsin but was readily removed by exposure to hyaluronidase. The thickness of this AMPS layer was not correlated with the varying degree of loss of contact inhibition exhibited by cells infected with the different strains of virus. The staining of the cell envelope with a solution of phosphotungstic and chromic acids (PTA-CR) suggested the presence of glycoproteins. The outer surface of the virions showed the same staining as the cell surface with RR and PTA-CR, and the budding virus particle was seen to incorporate the RR layer of the cell into its structure. The RR layers of cells and virions appeared to fuse, as did those between virus particles, suggesting that these layers play a role in the aggregation of virus particles and in their adherence to the surface of the cell.     

Transformation of Rat Liver Cells with Chicken Sarcoma Virus B77 and Murine Sarcoma Virus

Rat liver cells in vitro were transformed with chicken sarcoma virus B77, giving RL(B77) cells, and with murine sarcoma virus (Harvey), giving RL(MSV) cells. Rat liver cells transformed spontaneously in vitro were designated RL cells. In addition, the RL(MSV) cell line was adapted for growth in culture fluid containing 25 mug of 5-bromodeoxyuridine per ml. All cell lines were tumorigenic in 1-wk-old rats. The number of cells needed for induction of tumor growth was 1,000-fold higher in the case of RL(B77) cells in comparison with RL(MSV) cells and RL cells. No production of viral particles from any of the cell lines investigated was detected by plating concentrated supernatant fluid of the cultures on different secondary embryo cells with and without fusion by Sendai virus, by labeling with uridine-5-(3)H, or by assay for deoxyribonucleic acid polymerase activity. The viral genome was rescued by fusion of RL(B77) cells with chicken cells. Chicken sarcoma virus rescued from (RL(B77) cells differed in plating efficiency on duck cells from B77 virus rescued from transformed rat embryo cells. No virus was rescued after fusion of RL(MSV) and RL cells with mouse, rat, or chicken embryo cells. Infectious murine sarcoma virus can be induced by 5-bromodeoxyuridine from RL(MSV) cells.     

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.     

Transformation of chicken myelomonocytic cells by a retrovirus expressing the v-myb oncogene from the long terminal repeats of avian myeloblastosis virus but not Rous sarcoma virus.

To test the effect of long terminal repeat (LTR) regulatory sequences on the transforming capability of the v-myb oncogene from avian myeloblastosis virus (AMV), we have constructed replication-competent avian retroviral vectors with nearly identical structural genes that express v-myb from either AMV or Rous sarcoma virus (RSV) LTRs. After transfection into chicken embryo fibroblasts, virus-containing cell supernatants were used to infect chicken myelomonocytic target cells from preparations of 16-day-old embryonic spleen cells. Both wild-type AMV and the virus expressing v-myb from AMV LTRs (RCAMV-v-myb) were able to transform the splenocyte cultures into a population of immature myelomonocytic cells. The transformed cells expressed the p48v-Myb oncoprotein and formed compact foci when grown in soft agar. In contrast, the virus expressing v-myb from RSV LTRs (RCAS-v-myb) was repeatedly unable to transform the same splenocyte cells, despite being able to infect fibroblasts with high efficiency. This difference in the transforming activities of v-myb-expressing viruses with different LTRs most likely results from the presence of a factor (or factors) within the appropriate myelomonocytic target cell that promotes specific expression from the AMV but not from the RSV LTR.     

Fusion of Rous sarcoma virus with host cells does not require exposure to low pH.

We investigated whether Rous sarcoma virus (RSV) infects cells through a pH-independent or a low-pH-dependent pathway. To do this, the effects of lysosomotropic agents and acid pretreatment on RSV infectivity of, and fusion with, chicken embryo fibroblasts (CEFs) were studied. High concentrations of lysosomotropic agents (ammonium chloride and monensin) did not inhibit virus infectivity: equal titers of RSV were produced in the presence and absence of these agents. Similarly, low-pH pretreatment did not inhibit RSV infectivity. In parallel experiments, lysosomotropic agents and acid pretreatment completely abolished the ability of influenza virus to infect CEFs. To monitor the fusion activity of RSV directly, the viral membrane was labeled with the fluorescent lipid probe octadecyl rhodamine at a self-quenching concentration. Upon fusion with a host cell, the probe is diluted in the cell membrane, resulting in fluorescence dequenching (D. Hoekstra, T. de Boer, K. Klappe, and J. Wilschut, Biochemistry 23:5675-5681, 1984). In this assay, fusion of RSV with CEFs was found to occur in both a time-dependent and a strictly temperature-dependent fashion. No fusion occurred unless cells with prebound virus were warmed to temperatures greater than 20 degrees C. Fusion, but not binding, was abolished if virus was pretreated with low concentrations of glutaraldehyde. High concentrations of ammonium chloride had no effect on fusion of RSV with CEFs but greatly diminished the ability of influenza virus and Semliki Forest virus to fuse with CEFs. Similarly, acid pretreatment of RSV had no effect on fusion with CEFs while markedly inhibiting fusion of both influenza and Semliki Forest viruses. Collectively, our results show that RSV fusion with and hence infection of CEFs does not require exposure of the virus to low pH. In this respect, RSV resembles another retrovirus, human immunodeficiency virus.     

Cells transformed by Rous sarcoma virus release transforming growth factors

Chicken embryo fibroblasts and hamster BHK cells transformed by Rous sarcoma virus (RSV) release in their culture media growth factors which enhance markedly anchorage-independent colony formation in gelified medium, at the restrictive temperature (41 degrees 5 C), of chicken embryo fibroblasts (CEF) infected by RSV mutants with a ts mutation of the src gene. This action is not observed with uninfected CEF, and, therefore, appears to require some expression of the viral src gene in the target cells. The enhancing factors are proteins related to the family of the transforming growth factors (TGFs) by their molecular weight (about 20 kd), their heat and acid resistance, and their sensitivity to dithiothreitol. They do not compete with 125I EGF for binding on the EGF receptors of the membrane of A431 cells. As chicken embryo fibroblasts are devoid of EGF receptors, their activity is not potentiated by EGF.     

Use of chicken cell line LSCC-H32 for titration of animal viruses and exogenous chicken interferon

The chicken embryo cell line LSCC-H32 was tested for the propagation and titration of several animal viruses of the families Toga-, Reo-, Rhabdo-, Herpeto-, Orthomyxo-, Paramyxo-, and Poxviridae and compared with secondary chicken embryo cells. The LSCC-H32 cells were demonstrated to be as susceptible for most of the tested viruses as were secondary chicken embryo cells. Both produced comparably sized virus plaques. The titers of Sindbis and Semliki Forest viruses in LSCC-H32 cells were 5- to 40-fold higher than in secondary chicken embryo cells or BHK-21 cells, respectively. Furthermore, exogenous chicken standard interferon was titrated in the LSCC-H32 cells, and a 50% plaque titer reduction of the challenging vesicular stomatitis virus was achieved by 0.12 IU of a standard chicken interferon preparation. Endogenous chicken interferon could not be induced by treatment of the cells with polyinosinic acid-polycytidylic acid. Due to its high plating efficiency and metabolic activities, the LSCC-H32 cell line provides a useful cell system for the titration and large-scale production of the tested animal viruses and for the titration of exogenous chicken interferon.     

Use of chicken cell line LSCC-H32 for titration of animal viruses and exogenous chicken interferon.

The chicken embryo cell line LSCC-H32 was tested for the propagation and titration of several animal viruses of the families Toga-, Reo-, Rhabdo-, Herpeto-, Orthomyxo-, Paramyxo-, and Poxviridae and compared with secondary chicken embryo cells. The LSCC-H32 cells were demonstrated to be as susceptible for most of the tested viruses as were secondary chicken embryo cells. Both produced comparably sized virus plaques. The titers of Sindbis and Semliki Forest viruses in LSCC-H32 cells were 5- to 40-fold higher than in secondary chicken embryo cells or BHK-21 cells, respectively. Furthermore, exogenous chicken standard interferon was titrated in the LSCC-H32 cells, and a 50% plaque titer reduction of the challenging vesicular stomatitis virus was achieved by 0.12 IU of a standard chicken interferon preparation. Endogenous chicken interferon could not be induced by treatment of the cells with polyinosinic acid-polycytidylic acid. Due to its high plating efficiency and metabolic activities, the LSCC-H32 cell line provides a useful cell system for the titration and large-scale production of the tested animal viruses and for the titration of exogenous chicken interferon.     

Cell fusion for genetic analysis of two nonconditional Rous sarcoma virus replication mutants.

Procedures for characterizing replication-defective viruses in nonpermissive mammalian cells were developed and applied to three nonvirogenic Rous sarcoma virus (RSV)-transformed mammalian cell lines--B4, a line of Bryan virus-transformed hamster cells, and two SRD-RSV transformed rat cell lines, LR3/1 and LR3/2. Cell fusion was used to study virus complementation. The three cell lines (i) fused with helper virus-infected chicken cells and the host range of the rescued virus examined, (ii) tested for complementation by fusion with chicken cells exhibiting various patterns of endogenous virus expression, (iii) fused with chicken cells infected with the temperature-sensitive replication mutant LA334 and assayed for complementation at permissive and nonpermissive temperatures, and (iv) tested for complementation of defective viruses in other RSV-transformed mammalian cell lines by fusing pairs of nonvirogenic cell lines and permissive chicken cells. Based upon these complementation studies, we concluded that B4 virus is defective only in the env gene, LR3/) virus is an absolute mutant in the gag and/or pol genes, and LR3/2 virus is a leaky env mutant. Clones of LR3/1 and LR3/2 virus-infected chicken cells were established, and the results obtained from the characterization of these viruses in permissive avian cells substantiates the conclusions reached in the fusion-rescue studies.     

Biosynthesis of subviral oncogenic particles (virosomes) in mitochondria of rous sarcoma and Rauscher murine leukemia cells

Summary Experimental evidence for the presence and biosynthesis of subviral, leukemogenic particles in the isolated mitochondria of spleen cells of mice infected with Rauscher murine leukemia (RML) virus is presented. These subviral particles sediment at a density of 1.27–1.29 g/ml and induce splenomegaly and RML three weeks after i.v. or i.p. administration to white mice. Virosomes have been labelled with [³²P]phosphate in the isolated mitochondria from RML spleen cells and high molecular weight (70S) [³²P]RNA has been isolated from these subviral, leukemogenic particles. Rauscher virus group specific antigens were detected by immunodiffusion in the inner membrane and matrix fraction of the mitochondria of RML spleen cells. These results together with our earlier findings strongly suggest that mitochondria of the transformed cells participate in the biosynthesis of RNA tumor viruses. Possible mechanism of the penetration of viral genetic information of RNA tumor viruses into mitochondria of tumor cellsin vivo is discussed.     

Production of Sindbis,influenza, and vesicular stomatitis viruses in chicken embryo and rat embryo cell suspensions

Studies were carried out on the production of Sindbis, influenza and vesicular stomatitis viruses in suspensions of chicken embryo and rat embryo cells. The yield of Sindbis virus in chicken embryo cell suspensions was independent of the multiplicity of infection over the range 0.0001 to 0.01 although reduction in multiplicity caused a delay in virus production. With influenza virus the use of higher multiplicities gave increased virus yields possibly due to the very slow production at low multiplicities. In both monolayer and suspension cultures of chicken embryo cells addition of serum or use of media richer than minimum essential medium (Eagle) had little effect on Sindbis virus production, but if the glucose were omitted the virus yield was markedly reduced. In cell suspensions, a marked reduction in virus yield occurred if infection were delayed more than 24 hr after cell preparation whereas in monolayers the delay of infection allowed cell propagation and hence a higher yield of virus. It was also shown that vesicular stomatitis virus can be produced in chicken embryo cell suspensions, and that rat embryo primary cell suspensions can be used to prepare both Sindbis and vesicular stomatitis viruses. Sindbis virus obtained from chicken embryo cell suspensions was purified by polyethylene glycol precipitation and sucrose density gradient centrifugation and shown to contain only those proteins previously identified as viral, without any contamination from chicken cell proteins. The relative ease and economics of virus production by cell suspension and monolayer methods is compared.     

Generation of transforming viruses in cultures of chicken fibroblasts infected with an avian leukosis virus.

During serial passages of an avian leukosis virus (the transformation-defective, src deletion mutant of Bratislava 77 avian sarcoma virus, designated tdB77) in chicken embryo fibroblasts, viruses which transformed chicken embryo fibroblasts in vitro emerged. Chicken embryo fibroblasts infected with these viruses (SK770 and Sk780) had a distinctive morphology, formed foci in monolayer cultures, and grew independent of anchorage in semisolid agar. Bone marrow cells were not transformed by these viruses. Another virus (SK790) with similar properties emerged during serial subcultures of chicken embryo fibroblasts after a single infection with tdB77. The 50S to RNAs isolated from these viruses contained a tdB77-sized genome (7.6 kilobases), 8.7- and 5.7-kilobase RNAs, and either a 4.1-kilobase RNA or a 4.6-kilobase RNA. These RNAs did not hybridize with cDNA's representing the src, erb, mac, and myb genes of avian acute transforming viruses. Cells transformed by any one of the Sk viruses (SK770, SK780, or SK790) synthesized two novel gag-related polyproteins having molecular weights of 110,000 (p110) and 125,000 (p125). We investigated the compositions of these proteins with monospecific antiviral protein sera. We found that p110 was a gag-pol fusion protein which contained antigenic determinants, leaving 49,000 daltons which was antigenically unrelated to the structural and replicative proteins of avian leukosis viruses. An analysis of the SK viral RNAs with specific DNA probes indicated that the 5.7-kilobase RNA contained gag sequences but lacked pol sequences and, therefore, probably encoded p125. The transforming ability, the deleted genome, and the induced polyproteins of the SK viruses were reminiscent of the properties of several replication-defective acute transforming viruses.