Search for antigens and antibodies crossreactive with type C viruses of the woolly monkeys and gibbon ape in animal models and in humans.

Several reports have indicated the presence of type-C viral antigens in human tumors and of viruses closely related to those of the woolly monkey and gibbon ape in cultured human cells. In the present studies, attempts to detect woolly monkeys viral antigens in human tissues, or antibodies directed against structural polypeptides of woolly monkey viruses in human sera, were unsuccessful, In contrast, it was possible to demonstrate viral antigens in tissues and antibodies reactive to viral components in several animal and even primate model systems. Further evidence against the presence of woolly monkey viruses in humans is our failure to identify spontaneous or chemically induced viruses of this group in more than 200 individual cultures of human origin examined. These findings argue against the likelihood that viruses closely related to the woolly monkeys virus are associated with human tumors or are common infectious agents of man.     

Identification of virion polypeptides in hamster cells transformed by herpes simplex virus type 1.

Ten polypeptides were detected on the surface of the virion of herpes simplex virus type 1. Of these ten polypeptides, three were detected in hamster cells transformed by herpes simplex type 1.     

Hexose transport in sarcoma virus transformed cells.

Avian and mammalian fibroblast cultures transformed by type C sarcoma viruses show a dramatic enhancement of the rate of hexose transport at the beginning of transformation which is quantitatively and qualitatively different from that seen by variation in culture conditions of nontransformed control cells. The identification of this change as being a transport alteration independent of total glucose metabolism has been shown by use of nonmetabolizable analogues, 2-deoxyglucose, 3-O-methylglucose, and L-glucose. Increased transport rates were not dependent on levels of hexokinase activity. Transport studies of 3-O-methylglucose confirmed these conclusions and further revealed an additional altered nature of hexose transport after transformation by sarcoma virus. 3-O-methylglucose was not only transported more rapidly in the transformed cells than in the parental nontransformed cells, but the sugar "infiltrated" into the transformed cells despite the inhibitory effect of cytochalasin B. This was not seen with control cells. The sarcoma cells were also able to transport L-glucose in contrast to lack of uptake by nontransformed cells. Under conditions in which cell toxicity was not a factor, 2-deoxyglucose and several other sugars present in culture media inhibited transformation by sarcoma viruses. These same sugars reduced the incidence of sarcomas produced by virus in vivo when administered daily to test animals. The transport changes also correlate well with the transformed state as found by other laboratories using temperature-sensitive mutants and revertant cell lines. Collectively these data suggest that manipulation of transport systems may prove useful for control of certain malignancies.     

Two tumor antigens and their polypeptides in adenovirus type 12-infected and transformed cells

A tumor (T) antigen, designated T antigen g, was visualized as fine fluorescent granules in nuclei of adenovirus type 12 (Ad12)-infected cells by immunofluorescence with sera from rats bearing HY cell tumors (H sera). HY cells are rat cells incompletely transformed by the Acc I-H endonuclease fragment (0-4.7 map units) of Ad12 DNA. The antigen is different from the usually described T antigen, designated T antigen f, which is visualized as fluorescent flecks or filaments in both nucleus and cytoplasm of Ad12-infected cells when tested with narrowly reacting T sera. Extracts of [³⁵S]methioninelabeled infected cells were immunoprecipitated with H sera, and the resultant precipitate was analyzed by the two-dimensional gel electrophoresis technique of O'Farrell. The autoradiogram showed the presence of a cluster of several polypeptides (M_r 35,000-40,000, pI 5.0-5.5) that was absent in extracts of mock-infected cells. A similar autoradiogram of infected cells analyzed with narrowly reacting T sera showed the presence of a small polypeptide (M_r 10,000, pI 6.4), that was absent in extracts of mock-infected cells. The results show that M_r 35,000-40,000 polypeptides are components of T antigen g and a M_r 10,000 polypeptide is a component of T antigen f. Ad12-transformed cells showed a similar result. T antigen g was present and T antigen f was absent in HY cells. Both T antigen g and T antigen f were present in CY cells, which are rat cells completely transformed by the EcoRI-C endonuclease fragment (0-16 map units) of Ad12 DNA. The possible functions of these proteins are discussed.     

Analysis of viral DNA sequences in hamster cells transformed by herpes simplex virus type 2.

Herpes simplex virus type 2 (HSV-2) DNA was treated with four restriction endonucleases (EcoRI, HincIII, Bgl II, and Xba I) and eight fragments were purified and labeled with 32P in vitro. The kinetics of renaturation of each of the fragments was measured in the presence of DNA extracted from 333-8-9, a hamster cell line transformed by UV light-inactivated HSV-2 strain 333, and from a series of cloned derivatives and their tumor lines. All of the lines examined contained a partial set of viral sequences present at only a few copies per cell. Passage of the cell lines in tissue culture or in animals resulted in partial loss of viral DNA. Two blocks of sequences were present in most of the lines examined; those mapping at positions 21--33 of the HSV-2 genome were detected in seven of seven cell lines tested and those at positions 60--65 were detected in six of eight. Other sequences from the L component can also be present in the DNA of HSV-2-transformed hamster cells.     

Control of reversion of Moloney virus sarcoma virus transformed cells.

Mouse cells transformed by the Moloney murine sarcoma virus (MSV) native to the species can give rise to revertants which are supersusceptible to a second cycle of transformation. The MSV retransformed cells can give rise to several complex functional states even after several cycles of cloning: a) Formation of sarcoma positive, leukemia negative (S+Lminus) type cells of which some sublines may be inducible for MSV by halogenated pyrimidines; b) detection of initially SminusLminus cells which spontaneously become transformed and S+Lminus; c) the derivation of flat murine leukemia virus positive (MuLV+) cells which have an atypical MuLV and may become MSV+ as well as MuLV+; d) the release of free MSV from some cell clones with an apparent absence of MuLV. A general feature of all the above variants is a failure of detection of spontaneous reversion occurring after the second cycle of transformation. The nature of MuLV spontaneously released is that of a poorly replicating MuLV which exhibits cross-interference with other MuLV pseudotypes of MSV and the envelope which is most similar to that of Moloney leukemia virus (MLV). The examination for spontaneous reversion in human S+Lminus cells transformed by the same type of genome capable of good frequency of reversion in mouse cells, did not yield human revertant cells.     

Temperature-sensitive tumorigenicity of cells transformed by a mutant of Moloney sarcoma virus.

Normal rat kidney cells were nonproductively infected either with CP27, a mutant of Moloney sarcoma virus that is temperature-sensitive for maintenance of transformation, or with the parental wild-type virus. The nonproducer cells were inoculated into the tails of athymic nude mice that were subsequently incubated at 28 or 36 degrees C. CP27-infected cells induced tumors only at 28 degrees C, whereas cells infected with wild-type Moloney sarcoma virus were tumorigenic at both temperatures. Tumors induced at 28 degrees C by wild-type virus-infected cells grew faster after shift of the mice to 36 degrees C. In contrast, tumors induced by CP27-infected cells regressed upon shift to 36 degrees C, indicating that continuous expression of viral functions is required for persistence and growth of the tumors. After regression, secondary tumor growth was observed late after upshift of temperature-sensitive tumors. Cells recovered from these late-appearing tumors were tumorigenic at the nonpermissive temperature, and tumors induced by these cells did not regress after upshift. Virus rescued from these recovered cells retained the temperature-sensitivity for focus formation, indicating that the occurrence of the phenotypically wild-type cells was due to host cell modifications rather than to reversion of the CP27 genome.     

Integration of avian sarcoma virus DNA sequences in transformed mammalian cells.

DNA from six avian sarcoma virus (ASV)-transformed mammalian cell lines was digested with the restriction endonucleases EcoRI, Xho I, or Sal I, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filter strips, and hybridized with specific ASV [32P]cDNA probes. DNA from all of the ASV-transformed cell lines yielded three common virus-specific DNA fragments (2.4, 1.8, and 1.3 X 10(6) daltons) upon cleavage with EcoRI. Xho I appeared to cleave at least once within the integrated provirus and yielded a common fragment of 3.3 X 10(6) daltons as well as a second virus-specific DNA fragment whose size varied from 4.0 to 5.0 X 10(6) daltons in the different transformed cell lines. Sal I did not cleave within the provirus and yielded a single major virus-specific fragment of about 11 X 10(6) daltons in all transformed lines examined. Using specific cDNA probes, we show that the 1.8 X 10(6)-dalton EcoRI fragment contains sequences homologous to the 3' end of the viral RNA as well as to the src region of the viral genome. These studies clearly demonstrate that the same region on the ASV genome is utilized for provirus integration in different ASV-transformed cell lines.     

Alterations in surface proteins of rat cells transformed by avian sarcoma virus B77.

Cell membrane proteins of avian sarcoma virus B77V--transformed cells LWF B55 and LWF B77 and uninfected rat embryo fibroblasts were analyzed by SDS acrylamide gel electrophoresis. Following alterations in cell surface proteins of LWF B55 and LWF B77 cells were found: one, a slight decrease of two high molecular weight proteins the larger of which corresponds presumably to "LETS" protein and a decrease of a protein with approximative molecular weight 50 000; two, increase in content of proteins with molecular weight of about 90--95 000 and 70--75 000 as well as a marked increase of a protein with molecular weight of 30--35 000.     

Study of the avian sarcoma virus infection of chemically transformed cells.

BHK-21/13 cells transformed with various chemical carcinogens and mutagens were tested for susceptibility to avian sarcoma virus infection. The chemically transformed BHK cells were highly tumorigenic. The treatment of these cells in vitro with avian sarcoma virus strain Schmidt-Ruppin D resulted in chemically transformed and viral infected cells with different rescuability of the integrated virus genome. The different rescuability is not due to the difference in virus penetration as was shown by vesicular stomatitis virus Schmidt-Ruppin virus pseudotype VSV(SR-D) technique. The sarcoma virus genome in the doubly transformed cells was not inducible by various virus inducers.     

Neoplastic transformation of mouse embryo cells by virus released from tumorigenic mouse cells transformed by avian sarcoma virus B77.

Mouse C3H embryo cells were transformed in vitro by avian sarcoma virus Bratislava 77 (B77) released scantily from a mouse cell line transformed earlier by the same virus. B77 virus transformed C3H embryo cells contained B77 viral genome and were transplantable into syngeneic as well as allogeneic DBA/2J young mice in which autochthonous sarcomas were induced. Tumors in both strains of mice were virogenic. The probable reasons for an increased transformation capacity of B77 virus in mammals are discussed.     

Infection with paramyxoviruses stimulates synthesis of cellular polypeptides that are also stimulated in cells transformed by Rous sarcoma virus or deprived of glucose

Infection of several types of cultured cells with the paramyxoviruses simian virus 5 and Sendai virus stimulates synthesis of four polypeptides (I-IV) with molecular weights of approximately 99,000, 97,000, 86,000, and 78,000, respectively. That these are host polypeptides encoded in cellular mRNAs has been shown by the inhibition of their synthesis by actinomycin D and by the similarity of the peptide maps of them and of polypeptides with the same electrophoretic mobility from uninfected cells. Peptide mapping as well as identical migration in polyacrylamide gels has also indicated that polypeptides I, II, and IV are the same as plasma membrane polypeptides whose synthesis is enhanced in cells transformed by Rous sarcoma virus and in normal cells by glucose deprivation or treatment with 2-deoxyglucose. Polypeptides I and II appear to be the same polypeptides, with the observed differences in migration reflecting the glycosylation of polypeptide I, a relationship previously shown to exist between polypeptides in glucose-deprived and glucose-fed cells. Infection with paramyxoviruses does not significantly increase the transport of glucose by cells, and the maintenance of a high concentration of glucose in the medium does not prevent the enhanced synthesis of these polypeptides. This is in contrast to the situation in transformed cells in which stimulation of synthesis of these polypeptides is secondary to depletion of glucose in the medium due to increased glucose uptake by the cells. Thus, although paramyxovirus infection and transformation by Rous sarcoma virus result in stimulation of the synthesis of the same membrane polypeptides, the mechanism of stimulation differs.     

Instability of integrated viral DNA in mouse cells transformed by simian virus 40

The state and organization of simian virus 40 (SV40) DNA in tsA mutant-transformed mouse clones were examined early after agar selection in an attempt to elucidate the mechanisms that actively generate the diverse integration patterns found in transformed cells. Although recently selected as a cloned population from agar, A21 cells displayed extremely heterogeneous SV40 DNA patterns when analyzed by agarose gel electrophoresis and Southern blot hybridization. Reselection of clones in agar from A21 at 33 degrees C or 39.5 degrees C and DNA analysis by hybridization demonstrated (i) simplification of the number of integration sites in the new clones; (ii) new sites of integrated SV40 DNA in high molecular weight cell DNA fragments generated by digestion with restriction endonuclease Bgl II; (iii) relatedness between clones with respect to integrated viral sequence arrangement; and (iv) persistence of free viral DNA forms. The majority of free viral DNA appeared to be full length, nondefective SV40 DNA, although a subpopulation of defective viral molecules was also detected. No detectable free SV40 DNA could be observed in A21 clonal derivatives isolated by growth in agar at 39.5 degrees C, indicating that the persistence of free viral forms was regulated by the A gene. These results suggest that the heterogeneity in viral sequences in the A21 cells was generated within a cloned population from which new clones can be derived with different transformed phenotypes and integration patterns.     

The mechanism of viral carcinogenesis by DNA mammalian viruses. VI. A new class of virus-specific RNA molecules in cells transformed by group C human adenoviruses

A new class of virus-specific RNA molecules was found in cells transformed by group C human adenovirus types 2, 5, and 6. RNA isolated from virus-free rat embryo cells transformed by adenovirus 2, 5, and 6 hybridized with all group C adenovirus DNA's (adenovirus 1, 2, 5, and 6) equally well, but not appreciably with group A and B adenovirus DNA's. Most likely no viral genes common to group A, B, and C adenoviruses are transcribed in adenovirus-transformed cells.Group C adenoviruses are closely related since they share 83 to 93 per cent of their base sequences as shown by DNA-DNA homology measurements. Group C DNA's share only 10 to 26 per cent of their base sequences with group A and B DNA's. Moreover, the shared sequences are not transcribed detectably in adenovirus transformed cells.Virus-specific RNA isolated from group C transformed cells contains 49 to 51 per cent G + C, but viral DNA's possess a 7 to 9 per cent higher G + C content. These differences suggest that only a portion of the viral genome with an average G + C content of 49 to 51 per cent is transcribed in group C adenovirus transformed cells.     

Polypeptides of cells transformed by RNA or DNA tumor viruses.

Proteins solubilized from normal BALB/3T3 cells and BALB/3T3 transformed by simian virus 40 or Kirsten sarcoma virus have been analyzed by two-dimensional gel electrophoresis and tryptic peptide mapping. A large fraction of the polypeptides of the virus-transformed cells, about 30%, were different from normal cells. In contrast to the marked differences between normal and transformed cells, the polypeptides of the DNA and RNA virus-transformed cells were almost identical. These findings were observed with polypeptides stained by Coomassie Blue, or labeled with [14C]glucosamine or [35S]methionine. Pulse-chase analysis showed that most of the polypeptides were stable during 20 hr of incubation. The identity of several polypeptides was confirmed by tryptic peptide mapping.