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Summary Mouse cells made resistant to 6-azauridine were found to undergo syncytium formation upon infection with the Rauscher pseudotype of Moloney sarcoma virus (M-MSV(RLV)) and the Moloney strain of murine sarcoma virus (M-MSV(MLV)). The formation of syncytia was related to sarcoma virus replication since syncytia were never produced by leukaemia virus alone or by β-propiolactone-inactivated M-MSV(RLV).

BALB/3T3 cells transformed by the Kirsten sarcoma virus (nonvirus producer BALB/3T3 cells) and mutant cell lines derived therefrom by treatment with bromodeoxyuridine (BrdU) were analyzed for expression of virus-specific RNA using single-stranded DNA transcripts of Rauscher leukemia virus (RLV), a virus activated in one of the cell lines (58-2T), and Ki-SV-specific DNA transcript; the latter transcript after removal of all sequences cross-reactive with RLV RNA. The Rauscher virus DNA detected multiple copies of viral RNA in virus-producing cells (∼2.5 x 103/cell) whether infected with RLV or activated to produce virus with BrdU. Nonproducer (NP) cells and normal BALB cells showed small numbers of RNA genomes (70–250/cell) and only partial saturation of the transcript. The intracellular RNA sedimented at 35S (main peak) with a variable minor peak at 20S with the exception of one mutant cell, M-43-2 (main peak at 26–27S). The 58-2T transcript reacted preferentially in NP cells and their derivatives with biphasic kinetics suggesting the possibility of sequences specific for the original transforming virus. The size of Ki-SV specific sequences were 30S in mutant cells whether or not complete virus was being produced and independent of in vivo transplantability.

Human rhabdomyosarcoma cells (RD) productively infected with the transformation-defective feline virus, RD-114, contain viral-specific DNA integrated into the chromosomal DNA which specifies complete information for progency virus formation. This cellular DNA is infectious for permissive cells of human and canine origin, giving rise to progeny virus identical in assessed biochemical and biological properties to the parental virus. The host range for transfection with RD-114 DNA is similar to that of the intact virus. Although progeny virus could first be detected by reverse transcriptase assay at 2 to 4 weeks after transfection, virus is actually released at 3 days post-transfection, as shown by assay of culture media on sensitive indicator cells. The minimum dose of RD-114 DNA required for one infectious event in 2 × 10 6 RD cells is 0.10 to 0.20 μg. This gives a calculated efficiency of infection of 1 infectious unit per 5 × 10 4 to 5 × 10 5 viral gene copies, if there are 2 or 20 copies per infected cell, respectively. RD cells, productively infected with the transformation-defective mammalian viruses FeLV and GaLV, also contain viral-specific DNA, which gives rise to the respective virus of origin upon application to permissive RD cells.

Infection of mouse embryo cells with two strains of murine sarcoma virus and one strain of murine leukemia virus was followed rapidly by synthesis of DNA in the cytoplasm. Persistently infected cells have not shown such synthesis, and ultraviolet-irradiated virus did not induce DNA synthesis. This new DNA presumably represents an intermediate in the virus replication cycle specified by the virion DNA polymerase(s). Failure to observe cytoplasmic DNA synthesis in persistently infected cells suggests, in keeping with the results of inhibitor experiments, that the new “viral” DNA becomes associated with cellular DNA in some form of stable interaction.

Recently we demonstrated the induction of a C-type RNA virus (XCIV) in tissue culture line XC derived from Wistar random-bred rats. In this article two other rat C-type virus isolates (NRKAV and RPLAV) are described. They were associated with, and isolated from, NRK and RPL lines originating in Osborne-Mendel and Lewis rat strains, respectively. The main biological characteristics shared by the three viruses were buoyant densities in sucrose gradients in the range of 1.14 to 1.15 g/ml, endogenous DNA polymerase activity, high molecular weight (60 to 70S) RNA, rat major speciesspecific (gs-1) antigen and gs-3 interspecies antigen. Two of the isolates (NRKAV and RPLAV) were able to rescue mouse sarcoma virus (MSV) genome from a rat MSV-transformed non-productive line. The NRK line was assayed for virus release at various in vitro passage levels. In lower passages (43–73) spontaneous virus release was absent or minimal but the virus could be induced by treatment of cells with 5-bromodeoxyuridine. At higher passage levels the cells produced a large amount of virus spontaneously, but in these treatment with 5-bromodeoxyuridine decreased virus production. The results of karyological analysis suggested that clonal selection was taking place in the NRK line during consecutive in vitro passages and simultaneously with the process of increasing spontaneous virus release.

Summary The group-specific antigen of two murine C-type RNA tumour virus types was purified to homogeneity by isoelectric-focusing of virus disrupted with tween-ether. The antigen has a molecular weight of 25,000 to 26,000 (estimated from sedimentation in sucrose relative to standards) and an isoelectric point of 6.7. The latter value was obtained from preparations of Friend, Moloney, Rauscher and Gross subgroups. The antigen was immunogenic in guinea-pigs inducing the synthesis of monospecific group-reactive antibodies. The antigen was relatively free of nucleic acid and appeared to be the only virus protein, based on labelling studies, with an isoelectric point near neutrality. Envelope antigen was localized at pH 4.5 by the electrofocusing technique.

Gilden , R. V. (Wistar Institute, Philadelphia, Pa.), and R. I. Carp . Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells. J. Bacteriol. 91: 1295–1297. 1966.—Synthesis of the simian virus 40 (SV40) T antigen in primary African green monkey kidney cells was abolished when cycloheximide was added up to 10 hr postinfection. In contrast, puromycin, another inhibitor of protein synthesis, did not suppress antigen production. The basis of this differential effect was the inability of puromycin to inhibit protein synthesis in the cells used. This was shown by the failure of the drug to depress the incorporation of labeled amino acid into protein and also failure to inhibit poliovirus synthesis. The puromycin preparation used was very effective in inhibiting poliovirus synthesis in HeLa cells. Thus, appearance of the SV40 T antigen is dependent on protein synthesis in infected cells.

A cell line (HT-1) derived from a hamster tumour induced by the Moloney sarcoma virus has been extensively used for preparation of murine pseudotype sarcoma viruses (Huebner et al. 1966; Hartley et al. 1969). Infectious virus could not be isolated from these cells nor was the group specific (gs) antigen of the murine C-type RNA viruses detectable in complement-fixation tests (Huebner et al. 1966). In further studies with this cell line, clonal sublines were derived and tested for infectious virus and virus particle production, gs antigen using both complement-fixation and precipitation-ingel assays, and the presence of virus genome by a highly reproducible rescue technique. HT-1 cells in the 80th in vitro passage were seeded into ten 60 × 15 mm. plastic Petri dishes (50 cells/dish). Each culture yielded 5 to 10 individual colonies, of which five were isolated by trypsinization within glass cloning rings. Each of the 50 clones was maintained separately in Eagle's basal medium with 10% foetal bovine serum.

A radioimmunoassay (RIA) for the major internal protein of the bovine leukemia virus (BLV p24) was established using anti-BLV p24 natural antibodies and purified 125I-labelled BLV p24. The final precipitation of the immune complexes was realized by a preparation of inactivated Staphylococcus aureau Cowan I. Sera from 363 cows belonging to (1) leukemic herds, (2) non-leukemic but BLV-exposed herds, and (3) apparently unexposed herds were studied comparatively in BLV p24 RIA, complement fixation and immunodiffusion. The BLV p24 RIA appeared much more senstive than the two other methods in the detection of positive sera. With this method 100% of the leukemic animals, excluding those with juvenile lymphosarcoma, presented very high antibody titers (greater than or equal to 10,000). Practically all cows with persistent lymphocytosis were also positive with slightly lower levels of antibodies, confirming the relationship between BLV infection and the persistent lymphycytosis. Moreover, about two-thirds of the hematologically suspect animals and one-third of the normal animals from BLV-exposed herds were found positive, whereas 100% of the sera from unexposed cows remained negative for anti-BLV p24 antibodies.

A low molecular weight, highly basic DNA-binding protein was purified from several oncornaviruses by the sequential procedures of gel filtration in guanidine-hydrochloride, DEAE-cellulose chromatography and affinity chromatography on single-stranded DNA sepharose. The binding protein from Rauscher and woolly monkey type-C viruses was the fastest migrating of the virion proteins in SDS-polyacrylamide gels and thus is designated p10 according to previous convention although our estimates of molecular weight were 8-9,000 daltons. The binding protein from these two viruses was resolved into two bands by acid-urea electrophoresis although only a single NH2 terminal amino acid was detected (S. Oroszlan, personal communication), thus indicating charge heterogeneity. Antibody to Rauscher virus p10 species-specific in gel diffusion and complement-fixation tests and did not exhibit cross-reactivity with other virion proteins. A DNA-binding protein was also detected in preparations of mouse mammary tumor virus. This purified protein had an apparent molecular weight of 12,500, was the second fastest migrating component in the virus preparations, and was antigenically unrelated to the mouse type-C virus p10.

DNA transcripts were prepared from three related viruses of hamster and analyzed by interviral hybridization and by reaction with cellular DNA. A virus (G-HaLV), isolated from a dimethylbenzanthraceneinduced tumor cell in Graffi hamsters, contained nucleic acid sequences highly specific for hamster cell DNA and did not react with mouse cell DNA nor did its transcript show homology (5%) with mouse or rat viral RNAs. The hamster-specific sarcoma virus, B-34, isolated by Bassin and coworkers from tumors induced by the Harvey strain of murine sarcoma virus, contained mouse-, hamster-, and possibly rat-specific sequences. B-34 transcripts were predominantly mouse-specific. GLOH(-), a lymphomagenic virus derived by dilution beyond the transforming endpoint of a hamster-specific sarcoma virus obtained from tumors induced by the Gross pseudotype of murine sarcoma virus, also contained hamster- and mouse-specific sequences. Only a portion of its hamster sequence (about 50%) was shared with B-34 and G-HaLV viruses. As expected, transcripts of GLOH(-) virus were reactive with mouse and hamster cellular DNA.

Natural and expreimental cat sera were tested in radioimmune precipitation assays vs purified p30s from FeLV, RD114 and MuLV. Antibodies with specificity for FeLV p30 comparable to hyperimmune sera from heterologous species but of low titer were found in a high percentage of normal cats from households with a high incidence of FeLV and neoplasia. Sera from cats with neoplasms were generally negative. Cats immunized with FeLV gave low-level immune response, also of the same general specificity as heterologous hyperimmune sera. Cat sera do not normally show antibody to RD114 p30 although two immunized weanling cats produced low titered but highly specific p30 antibody. Thus, for both classes of feline type-C virus p30s, there is an evident capability of the cat to mount an immune response to naturural or experimental exposure to the respective proteins. The magnitude of the response is between 100 and 1,000 fold below that seen in heterologous species. In contrast, cats immunized with MuLV p30 gave immune responses comparable to those seen in guineapigs, rabbits and goats. Several very old cats with carcinoma had antibody which preferentially precipitated MuLV p30. A competition assay using one such serum and labelled MuLV p30 was inhibited by FeLV, RD114, and MuLV p30s. This indicates that the assay is “interspecies” in nature. Among the possible explanations of this reaction category is that it represents antibody to the p30 of an as yet undefined class of feline type-C virus.

BALB/3T3 cells transformed by the Kirsten sarcoma virus (nonvirus producer BALB/3T3 cells) and mutant cell lines derived therefrom by treatment with bromodeoxyuridine (BrdU) were analyzed for expression of virus-specific RNA using single-stranded DNA transcripts of Rauscher leukemia virus (RLV), a virus activated in one of the cell lines (58-2T), and Ki-SV-specific DNA transcript; the latter transcript after removal of all sequences cross-reactive with RLV RNA. The Rauscher virus DNA detected multiple copies of viral RNA in virus-producing cells ( approximately 2.5 x 10(3)/cell) whether infected with RLV or activated to produce virus with BrdU. Nonproducer (NP) cells and normal BALB cells showed small numbers of RNA genomes (70-250/cell) and only partial saturation of the transcript. The intracellular RNA sedimented at 35S (main peak) with a variable minor peak at 20S with the exception of one mutant cell, M-43-2 (main peak at 26-27S). The 58-2T transcript reacted preferentially in NP cells and their derivatives with biphasic kinetics suggesting the possibility of sequences specific for the original transforming virus. The size of Ki-SV specific sequences were 30S in mutant cells whether or not complete virus was being produced and independent of in vivo transplantability.

DNA was extracted from two human sarcoma cell lines, TE-32 and TE-418, and the leukemic cells from five children with acute myelocytic leukemia, three children with acute lymphocytic leukemia and four adults with acute myelocytic leukemia. The DNAs, assayed for infectivity by transfection techniques, induced no measurable virus by methods which would detect known mammalian C-type antigens or RNA-directed DNA polymerase in TE-32, D-17 dog cells and other indicator cells, nor did they recombine with or rescue endogenous human or exogenous murine or baboon type-C virus. Model systems used as controls were human sarcoma cells, TE-32 and HT-1080, and human lymphoma cells, TE-543, experimentally infected with KiMuLV, GaLV or baboon type-C virus, all of which released infectious virus and whose DNAs were infectious for TE-32 and D-17 dog cells. Other model systems included. two baboon placentas and one embryonic cell strain spontaneously releasing infectious endogenous baboon virus and yielding DNAs infectious for D-17 dog cells but not for TE-32 cells. Four other baboon embryonic tissues and two embryonic cell strains, releasing either low levels of virus or no virus, did not yield infectious DNA.

The defective genome of murine sarcoma virus (MSV) * (moloney isolate) may be rescued from non-producer hamster tumour cells (HT-1) by co-cultivation of the latter and mouse embryo cells with added helper murine leukaemia viruses (MuLV) (Huebner et al. 1966; Huebner, 1967). Rescue of MSV from HT-1 cells by co-cultivation with MuLV-carrier cells has not, however, been described. This report describes the retrieval of MSV from mixed cultures of HT-1 and various MuLV-carrier cells of mouse and hamster origin. Moreover, it was found that the frequency of success and titres of MSV recovered by co-cultivation of HT-1 and F4, one of the MuLV-carrier mouse cell lines, were much higher than those obtained by the conventional method. We have analysed the factors responsible for such high efficiency of genome rescue. Among the MuLV-carrier cell lines used, F4 and F5 were derived from two of nine Rauscher pseudotype MSV (MSV(RLV) transformed foci of NIH Swiss mouse embryo cells isolated by trypsinization within a cloning ring.