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Late passages of the PFT porcine oviduct cell line spontaneously release a typical type C virus antigenically related to the type C virus released from the PK(15) porcine kidney cell line. The PFT virus was non-infective for a large variety of cell lines. Type C virus can also be induced by BrdU treatment from earlier passages of the PFT cell line.

The morphology and development of four members of the reticuloendotheliosis virus group were studied by transmission electron microscopy. Virions of duck spleen necrosis virus, duck infectious anemia virus, chicken syncytial virus, and reticuloendotheliosis virus strain T are sperical with a diameter of approximately 110 nm. They are covered with surface projections about 6 nm long and 10 nm in diameter. The center-to-center distance of surface projections is about 14 nm. The budding virions contain crescent-shaped electron-dense cores 73 nm in diameter with electron-lucent centers. After release of the virions the cores apparently become condensed to 67 nm in diameter. Virions were found budding at the plasma membrane and into smooth-walled, intracytoplasmic vesicles of productively infected cells. The distribution of budding reticuloendotheliosis viruses on cells appeared random over the cell surface, and occasionally aberrant multiple forms of budding virions were observed. The virions appear to resemble mammalian leukemia and sarcoma viruses more closely than avian leukosis-sarcoma viruses.

Phenotypic expression of the murine intraspecies and interspecies antigenic determinants of the major type C viral structural 30,000-dalton polypeptide, p30, was measured by radioimmunoassay inhibition in cell lines from different species. Uninfected normal rat kidney (NRK) cells did not contain detectable levels of murine intraspecies and interspecies p30 antigen, whereas rat cells transformed by and producing murine sarcoma virus (MSV)-Moloney leukemia virus (M-MSV-MuLV) contained high levels of both murine intraspecies and interspecies p30 antigen. Significant amounts of murine intraspecies and interspecies p30 antigen were detected in wild-type MSV-transformed nonproducer NRK cells. The control of p30 antigen expression was examined in temperature-sensitive MSV-transformed nonproducer cells [NRK(MSV-1b)] which are cold sensitive for maintenance of the transformed phenotype. Both murine intraspecies and interspecies p30 antigens were detected in NRK(MSV-1b) cells when grown at the permissive (39 C) or nonpermissive (33 C) temperature, suggesting that p30 antigen expression is not correlated with maintenance of the transformed phenotype. The results demonstrate that previously undetectable p30 antigens are expressed in MSV-transformed nonproducer NRK cells, and suggest that the expression of p30 antigen may be a useful marker for viral gene expression in mammalian cells.

Quantitation by enumeration of virion particles, measurement of absorbancy at 260 nm, and densitometry on sodium dodecyl sulfate-polyacrylamide gels has shown that mouse L-M cells yielded 7- to 10-fold less endogenous C-type virions than the parental lines, L or L929. The previously noted stimulation of L-M cell virion production by a concomitant 10% increase in fetal calf serum concentration (D. A. Kindig, R. Karp, and W. H. Kirsten, 1968), was not observed.

Most vertebrates contain sets of gene sequences (virogenes) which are an integral part of the chromosomal DNA and which can code, in some instances, for the production of Type C RNA tumor viruses. These genes are transmitted from parent to progeny along with other cellular genes, and their activation from a normally reressed state may be part of the mechanism by which RNA tumor viruses produce cancer. Isolates of endogenous genetically transmitted baboon Type C viruses are morphologically and biochemically related to other mammalian Type C viruses but can clearly be distinguished from the other groups (mouse, rat, cat, etc.) by immunologic and nucleic acid hybridization criteria. Within the primates, Type C viral gene sequences have evolved as the species have evolved, with virogenes from the most closely related genera and families showing the most sequence homology; all higher primate, including man, however, do have detectable virogene sequences in their normal tissues. Type C viruses have also been transferred under natural conditions between species only remotely related phylogenetically. The results show three clear examples where viral genes from one group of animals have become incorporated into the germ line of genetically distant groups of animals (inheritance of acquired genes). Infectious Type C viruses of primates, distinct from the endogenous primate virus group, have also been isolated (woolly monkey and gibbon isolates) and can be shown to produce tumors in other primates. Related viral information (nucleic acid sequences, enzymes, and antigens) have been reported in human tumors. The significance of infectious and/or genetically transmitted viruses in naturally occurring cancer is a major focus of current research. The presence of genetically transmitted viral genes in so many vertebrate species and the evidence that they have been conserved in several distinct vertebrate lineages suggests that they may provide some normal function(s) advantageous to the species carrying them and that their potential to cause cancers is a pathologic manifestation of normal, as yet undefined, physiologic processes.

Mouse cells contain the genetic information for multiple endogenous type-C RNA viruses. The mechanisms by which the cell controls expression of these naturally integrated viruses are not yet known. Recently, chemicals that inhibit protein synthesis have been shown to induce a specific type-C virus at high frequency from BALB/c mouse embryo cells. In the present studies, virus activation in response to a representative translational inhibitor, cycloheximide, is demonstrated to be transient, with virus release primarily occurring within the first 12-24 hr following drug exposure. Analysis of virus-specific RNA in cells by molecular hybridization revealed an absolute increase in viral RNA concentration in cycloheximide-treated cells. This was blocked by simultaneous exposure of the cells to actinomycin D. Further, inhibition of RNA synthesis during but not subsequent to cycloheximide exposure prevented virus activation. These findings show that virus induction by cycloheximide requires de novo RNA synthesis during but not after drug exposure and suggest that the required RNA species may be that of the virus itself. The present results are consistent with the hypothesis that translational inhibitors prevent synthesis of a labile protein whose normal action is to inhibit viral RNA transcription or to cause degradation of viral RNA.

When inhibitors of protein synthesis are added to BALB/c mouse cells in culture, induction of naturally integrated C-type RNA virus occurs in a high percentage of cells. The action of protein synthesis inhibitors differs from that of halogenated pyrimidines, another class of virus inducers, in their effects on biologically distinguishable viruses. The use of such inhibitors to study integrated virus expression provides a means for studying gene regulation in mammalian cells.

Enhancement of mortality rates and symptomatology was observed in isolator-held LSI-SPF chickens concurrently inoculated with MDHV and avian oncornaviruses (RAV-1, RAV-2, RAV-7, RAV-50, or REV). Interference with MD antigen production also was demonstrated in extracts of the feather follicle epithelium from chickens inoculated with both MDHV and RAV-1.

After an initial acute infection with cell killing, chicken or duck embryo fibroblasts infected in culture with reticuloendotheliosis viruses set up a chronic infection with no cell killing or morphological transformation. Essentially all of the chronically infected cells produced virus. The virus production was not sensitive to cytosine arabinoside or mitomycin C as was virus production in an acute infection. The chronically infected cells had a strong group-specific resistancto the c.p.e. of superinfecting reticuloendotheliosis viruses. However, they were sensitive to vesicular stomatitis virus and avian leukosis-sarcoma viruses. After double infection, single cells produced reticuloendotheliosis virus and avian leukosis-sarcoma virus.

Type-C viral polypeptides are expressed at very low levels in C57BL/10 liver and embryo cells as compared to cells of other mouse strains. The 12,000-mol. wt. polypeptide in C57BL/10 cells was purified and shown to possess type-specific antigenic determinants indistinguishable from those of a previously described class of endogenous virus. The very low levels of endogenous virus-specific RNA in cells of C57BL/10 as compared to other strains suggests control of this endogenous virus at the level of transcription. Analysis of viral antigen expression F1 hybrids of C57BL/10 with strains characterized by intermediate and high levels of viral antigen expression indicates that the restriction of this endogenous virus by C57BL/10 cells is not due to simple dominance by a repressor of virus expression.

While C-type RNA viruses are known to induce leukemias and lymphomas, oncogenic transformation of lymphoid cells by them in vitro has not been reported. In this study, splenocytes from female BALB/c mice were infected in vitro with Abelson virus, a C-type RNA virus that induces nonthymic lymphomas and plasmacytomas in mice. The cells were transplanted into recipients of different karyotype, either male BALB/c mice or hybrid BALB/c x AL/N (CALF1) mice, which bear the Rb(5.19)1Wh translocation. Transplants of eight of the resulting tumors (one plasmacytoma and seven lymphomas) contained cells of donor BALB/c karyotype, indicating that transformation of splenocytes occurred in vitro.

T24C, a continuous cell line derived from the pooled thymic tissue of normal inbred OM rats, spontaneously produced type-C virus. The virus genome was expressed cyclically. The amount of RNA-dependent DNA polymerase (RDP) and the number of 1.14 g dense particles/ml fluctuated simultaneously with cultivation. The released virus, RPT24C, did not infect cell lines from the rat, mouse, dog, or human. T31, also a rat thymus line, during its 2.5 years of cultivation did not produce type-C virus. Cocultivation with potentially permissive lines did not rescue any virus. 5-lodo-2'-deoxyuridine treatments at earlier passages yielded negative results. Chemical treatment at passages 111, 116, 123, and 128 yielded varying amounts of 3H-uridine incorporation at a sucrose density of 1.14 g/ml. Enzyme assays on chemically treated T31 cultures tested at passage 111 showed a small but transient burst of RDP activity. T31-B, a subline of T31, which was frozen and thawed once, released rat type-C virus spontaneously at passage 56. Two additional sublines of T31 (NI-T31 and NII-T31) were maintained for 2.5 years in culture without any cell-dispersing treatment. NI-T31, but not NII-T31, spontaneously released type-C virus. Once induced, the type-C viruses from T31-B and NI-T31 were continuously produced.

Nucleic acid base analogues were used to examine a Herpesvirus saimiri (HVS)-infected marmoset lymphoid cell line (MLC-1) for possible association with type C viruses. Synthetic templates poly(rA).d(pT)(10) and poly(dA).d(pT)(10) were used to detect RNA-directed DNA polymerase activity in 100-fold concentrated tissue culture fluids. HVS was monitored by immunofluorescence for early, late, and membrane antigens. MLC-1 cells were exposed to 30 mug of 5-bromo-2'-deoxyuridine (BUdR) per ml for 24 h and examined daily. Similar experiments used 5-iodo-2'-deoxyuridine (IUdR) (20 mug/ml) for 30 h or IUdR (20 mug/ml) for 3 days followed by 2% dimethyl sulfoxide for 4 days. Results of these experiments failed to show any type C virus-like polymerase; however, HVS expression was greatly stimulated. BUdR and IUdR enhanced expression of HVS-associated antigens five- to sevenfold, with maximal stimulation being observed 3 to 4 days after removal of the analogue. IUdR-dimethyl sulfoxide treatment was generally less effective. Although more cells showed HVS antigens, the treatments did not increase cell-free infectious virus. The data suggest that HVS-infected lymphoid cells can be stimulated to express virus in a manner similar to that of the Epstein-Barr virus in Burkitt's lymphoma cells. No evidence of type C virus was found in stimulated cultures.

The existence of the selectively permissive rabbit cell line SIRC allows definition of a new class of endogenous murine type-C virus. Continuous clonal lines of transformed cells derived from the BALB/c mouse-embryo cell line BALB/3T3 contain at least two distinct classes of endogenous type-C viral genomes. Spontaneously released endogenous viruses grow well on the mouse cell line NIH/3T3 (N-tropic viruses) but not on the rabbit cell line SIRC. Type-C viruses induced by treatment with BrdU grow well on SIRC (S-tropic viruses) but not in NIH/3T3 or BALB/3T3. BrdU-treated AKR mouse-embryo cells also release an S-tropic virus. N-tropic and S-tropic viruses both have the mouse intraspecies gs-1 and viral RNA-directed DNA polymerase antigenic determinants. DNA.RNA hybridization techniques reveal that the two host-range classes of endogenous viruses are only partially related to each other. Cell transformation facilitates the spontaneous release of the N-tropic viruses; treatment with thymidine analogues induces the production of the S-tropic viruses. Thus, the two classes of viral genomes appear to be subject to different cellular controls.

Production of particles with the ultrastructural appearance of C-type virions persisted for at least 6 h in actinomycin D-treated cells infected with murine leukemia virus. This phenomenon occurred despite severe inhibition of viral RNA synthesis. Virus particles present in a 6-h harvest sedimented in sucrose gradients with the buoyant density characteristic of RNA tumor viruses (1.16 g/cm(3)) and exhibited high levels of reverse transcriptase activity in response to the exogenous template polyriboadenylic acid.oligo deoxythymidylic acid in the range of untreated controls. However, RNase-sensitive endogenous activity was only (1/5) the level found in controls. This observation correlated with a marked reduction in infectivity. Kinetic studies on the appearance of labeled RNA in banded virions revealed that within the first hour after addition of actinomycin D, particles contained 60 to 70S RNA and two low-molecular-weight RNA species corresponding to 8 and 4S RNA. After approximately 1 h of incubation with actinomycin D, 60 to 70S RNA could not be detected and 4S RNA was the predominant species. These findings suggest that murine leukemia virus particles assembled in the presence of actinomycin D are deficient in 60 to 70S viral RNA.

Cells of mouse and rat origin contain endogenous type C viruses. There are intrinsic regulatory factors which restrict the ability of different classes of these inducible viruses to persist in cells after chemical activation. The cellular block to certain viruses is shown to be overcome under conditions in which the virus-activated cells are allowed continued exponential growth. These findings may help to explain the high frequency at which replicating type C viruses are detectable in naturally occurring tumors of some species.

The capacity of leukovirus RD-114 to replicate in human embryo lung diploid cell cultures and continuous human angiosarcoma cell cultures (AS and 709 lines). Differences in the capacity to support the virus reproduction were observed in the two strains of human embryo lung cells (HEL-1 and HEL-3) and the two continuous angiosarcoma cell lines. No virus reporduction was observed in mouse and rat cell cultures. No cytopathic or transformation changes were caused by the virus in any of the systems examined.

Properties of a chicken lymphoblastoid cell line (MSB-1) from a Marek's disease tumor were studied. The cell line grew well at 41 degrees C in medium RPMI-1640 supplemented with 10% bovine fetal serum and had a doubling time of 8-12 hours. Cells grown in stationary suspension culture did not attach to the vessel and had the morphology of typical lymphoblasts. At 37 degrees C, the cell line grew initially but ceased to divide after several subcultures. In the subcultures maintained for 48-72 hours, 1-2% of the cells produced Marek's disease virus (MDV)-specific intracellular and mambrane antigens and contained herpesvirus particles when examined by the electron microscope. Cocultivation of these cells with duck or chicken embryo fibroblast cultures resulted in transfer of infection and production of microplaques typical of MDV. Peripheral nerve lesions and lymphoid tumors characteristic of Marek's disease were caused by inoculation of susceptible chicks with MSB-1 cells or duck cells infected with strain BC-1 of MDV recovered from the MSB-1 cell line. No specific tumors were produced at the site of inoculation, and infection was readily transmitted to cagemates. Tumors were also produced in the skeletal muscles and seemed to be largely virus induced. MSB-1 cell line was free of C-type virus particles.

We have recently established a cell line, designated FVNC, by infection of non-EBV-productive human lymphoid NC-37 cells with the type-C virus FLV. The FVNC cell line has been maintained free of detectable EBV- and FLV-related immunofluorescent antigens for three years but both viral genomes exist in the cells in a repressed form. The FVNC cells were morphologically similar to the uninfected NC-37 cells but a D-group chromosome change was consistently seen. The colony-forming capacity of FVNC cells in semi-solid agar medium was about 1/10 of that of NC-37, although the former grew rather faster than the latter in fluid culture. FVNC cells were three times more sensitive to EBV superinfection than NC-37, as shown by immunofluorescence. In addition, an obvious induction of FLV antigenic markers occurred in FVNC cells on exposure to EBV. Nucleic acid hybridization experiments showed a striking decrease in the number of EBV genomes in FVNC cells. The number of genomes was too small to be measured, but EBNA was clearly present in all FVNC cells as well as in NC-37 cells. The implications of these findings were discussed from the point of view of a possible co-carcinogenesis in man by EBV and the type-C virus.

Mouse cells transformed by murine sarcoma virus were made resistant to 8-azaguanine. Resistant cells and cell clones isolated from them were deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity. They did not grow in HATG medium, did not incorporate labeled hypoxanthine, and had negligible HGPRT activity. The resistance was genetically stable. The resistant cells were hyperdiploid and contained telocentric chromosomes only. The resistant cells as well as the progenitor cells were slightly tumorigenic in mice, the plating efficiency in soft agar was very low. The parental cells and aza-G resistant cells produced C-type viral particles having RNA-dependent DNA polymerase activity. The resistance to aza-G did not influenced the expression of murine sarcoma virus genome in cells. The resistant cells are suitable for preparation of cell hybrids.

A C-type virus isolated from long term cultures of myeloid cells from a patient with acute myelogenous leukaemia is infectious for a wide variety of cells. The establishment of chronically infected cells enabled us to characterise the virus by biological, immunological, and biochemical tests. The virus is closely related to the simian sarcoma-associated virus isolated from a woolly monkey fibrosarcoma.

The sugar 2-deoxy-D-glucose (2-DG) induced the release of type C virions from an established line of normal rat kidney (NRK) cells. Within 20 h after the addition of 5 mg of 2-DG per ml to exponentially growing NRK clutures, more than 80% of the cells expressed the mammalian type C virus interspecies-specific antigen (p30) as determined by indirect cytoplasmic immunofluorescence. Maximal virion release occurred 1 to 2 days after 2-DG was added for 24 h to the growth medium although a low level of virion production was detected as early as 2.5 h after 2-DG treatment. Studies with inhibitors of RNA synthesis indicated a requirement for de novo RNA synthesis after the addition of 2-DG. Sensitivity of NRK cells to type C virion induction was limited to a relatively short period of in vitro growth and preceded spontaneous virion release by 8 to 10 subculture generations. A model is presented for the sequential derepression of latent type C virus information in serially propagated NRK cells.

The xenotropic mouse type C virus, originally detected in cultured embryo cells from New Zealand Black (NZB) mice, has been recovered from over 50 adult NZB animals and 15 NZB embryos. Its presence is best detected by measuring its ability to rescue a murine sarcoma virus (MSV) genome from a non-virus-producing MSV-transformed rat cell. The virus can serve as a helper for replication of MSV. It has a distinct type-specific coat and is a prototype for a third serotype of mouse type C viruses, NZB. The xenotropic virus may have an evolutionary role since it has a wide host range, including the ability to infect avian cells. It is produced spontaneously by all cells cultivated from NZB tissues and accounts for the high concentration of viral antigens associated with NZB tissues. The extent of virus production is similar in both male and female mice. All cell clones established from embryos also produce the virus. A variability in the intracellular regulation of virus replication is suggested since tissue cells from the same animal differ quantitatively in their ability to produce xenotropic viruses. Since enhanced spontaneous virus production is associated with cells from NZB mice, the virus may play a role in the autoimmune disease of this mouse strain.

Cordycepin (3-deoxyadenosine), an inhibitor of poly (a) synthesis, was found to inhibit the induction of the endogenous type "C" RNA virus by 5-iodo-2'-deoxyuridine in a line of Fisher rat embryo cells (H43) grown in vitro, and when continuously incorporated into the medium at those same concentrations, it was found to protect the cells from transformation by the chemical carcinogen 3-methylcholanthrene.

Leukemia viruses can be activated from within murine hosts by immunological reactions against foreign histocompatibility antigens both during graft versus host reactions (GVHR) and host versus graft reactions (HVGR). Presence of both lymphocyte-mediated graft rejection reactions and immuno suppression appears necessary for maximal virus activation and replication in vitro, although viruses can be activated by mixed lymphocyte reactions (MLR) alone in vitro. During HVGR, viruses are first detectable in lymph nodes draining the graft site, and later reach maximal titers in the spleen. Mice infected with murine leukemia virus (MuLV) as neonates or carrying MuLV secondary to milk transmission (carriers) also develop complex autoaggressive responses. By a sensitive in vitro microcytotoxicity assay, neoplastic or pre-neoplastic thymocytes from carrier mice were found to react vigorously against normal uninfected syngeneic embryonic fibroblasts, whereas they reacted much less vigorously with similarly prepared but MuLV-infected fibroblasts. Thymocytes from uninfected animals did not react with infected or uninfected fibroblasts. Peripheral lymphocytes from carriers reacted against MuLV infected fibroblasts, but not against uninfected fibroblasts, a pattern indistinguishable from deliberately-immunized mice. It is proposed that responses analogous to GVHR are also important in the pathogenesis of leukemia following exogenous leukemia virus infection.

Guinea pig embryo (GEP) cells were transformed in vitro by the Kirsten strain of mouse sarcoma virus (Ki-MSV). The transformed cells were found to release infectious virus continuously and produced high titers of group-specific (gs) complement-fixing (CF) antigen characteristics of the murine sarcoma-leukemia virus complex. Foci of transformed cells were similar in appearance to those obtained with Ki-MSV in mouse and rat cells. The transformed cells produced RNA dependent DNA polymerase and type C virus particles with a density of approximately 1.15 g/ml in sucrose gradients by 3H-uridine labeling. The transformed cells produced tumors when transplanted into newborn guinea pigs. A number of focus-derived clonal lines from Ki-MSV transformed cells were isolated and characterized. All the focus-derived lines were found to be either producers or nonproducers (NP). The NP guinea pig cells produced neither infectious virus nor viral antigens of the murine sarcoma-leukemia virus complex although they were morphologically indistinguishable from virus-releasing MSV transformed GPE lines and produced tumors when transplanted into newborn guinea pigs. However, the sarcoma virus genome could be rescued in these NP cells by cocultivation with "helper" murine leukemia virus (MuLV) releasing GPE cells. Particles resembling guinea pig leukemia virus were activated from guinea pig NP cells or cultured normal guinea pig cells following chemical treatment. These particles were approximately 100 nm in the mature form and had a density of 1.16-1.17 g/ml. They contained RNA dependent DNA polymerase activity.

Two forms of DNA polymerase are present in RD-114-infected human, dog, and mink cells, but are not detectable in uninfected cells. The two enzymes are indistinguishable catalytically and immunologically, but differ with respect to molecular weight and elution position from (dT)12-18-cellulose and phosphocellulose. The large enzyme (equivalent 95,000 daltons) is found in the infected cells, but not the virions produced by these cells. The virions contain only the smaller enzyme (equivalent 70,000 daltons). The larger form may represent a mammalian viral equivalent to the beta subunit of avian RNA tumor virus DNA polymerase.

The information for type C RNA viruses is genetically transmitted within the cellular DNA of the normal mouse cell. These viruses can be induced after exposure of cells to two classes of chemicals, inhibitors of protein synthesis and halogenated pyrimidines. The metabolic requirements for activation of one endogenous virus of BALB/c mouse cells by representatives of each class of drugs were studies. Cycloheximide and iododeoxyuridine each induce virus efficiently from cultures in exponential growth but are inactive on cells in stationary phase. However, cells are maximally sensitive to the actions of each drug at different times within the cell cycle. Further, virus induction in response to each is differentially inhibited under conditions of simultaneous cell exposure to inhibitors of DNA or RNA synthesis. The results provide support for the concept that inhibitors of protein synthesis and halogenated pyrimidines act by different mechanisms to induce type C virus release.