Distinct transformation phenotypes induced by polyoma virus and simian virus 40 in rat fibroblasts and their control by an early viral gene function.

Several transformed cell lines established from Fisher rat cells (FR 3T3) infected with wild-type polyoma virus or simian virus 40 or early temperature-sensitive mutants (polyoma tsa and simian virus 40 tsA30) were studied for their transformation phenotypes. The distinct patterns which were obtained for polyoma and simian virus 40 transformants led to the conclusion that these two viruses express different transforming abilities in rat cells. The results obtained with temperature-sensitive mutant-derived transformants indicate that all of the transformation characteristics studied so far may be under the control of a viral function in polyoma tsa-transformed cells.     

Oncogenes: Growth regulation and the papovaviruses polyoma and SV40

Cellular oncogenes and their activated and retrovirus-coded counterparts play an important role in cellular regulation. Here the relationship between such oncogenes and the genes coding for the transforming proteins of the papovaviruses, polyoma viruses, and simian virus 40 (SV40) is discussed. It is concluded that polyoma virus may transform established cells by a mechanism involving activation of a cellular oncogene product, whereas SV40 may transform by a mechanism involving a previously little studied cytoplasmic form of the transforming protein.     

Serological relationship of woodchuck hepatitis virus to human hepatitis B virus.

Two antigenic systems of the woodchuck hepatitis virus have been identified. The relationship between viral antigens of the woodchuck hepatitis virus and the human hepatitis B virus was determined by using immunoprecipitation, hemagglutination, and immune electron microscopy techniques. Antigens found on the cores of the two viruses were cross-reactive. Lack of cross-reactivity between the surface antigens of the two viruses in immunodiffusion experiments suggested that the major antigenic determinants of the viral surfaces are different; however, results of passive hemagglutination tests indicated that there are common minor determinants. Nucleic acid homology, as measured by liquid hybridization, was found to be 3 to 5% of the viral genomes. The results of this study provide further evidence that woodchuck hepatitis virus is the second member of a new class of viruses represented by human hepatitis B virus. Since virus-infected woodchucks may acquire chronic hepatitis and hepatocellular carcinoma, these antigens and their respective antibodies will be useful markers for following the course of virus infection in investigations of the oncogenic potential of this class of viruses. The nucleocapsid antigen described may be a class-specific antigen of these viruses and, thus, may be useful in discovering new members of the group.     

Common structural antigen of papovaviruses of the simian virus 40-polyoma subgroup.

An antigenic determinant common to the major capsid polypeptide (VP1) of simian virus 40 (SV40) and polyoma virus is described. Antisera prepared against intact viral particles reacted only with cells infected with the homologous virus by immunofluorescence tests (IF). However, antisera prepared against disrupted SV40 particles reacted in IF with both polyoma- and SV40-infected permissive cells. The cross-reaction with polyoma was localized to VP1 by the following evidence. (i) The IF cross-reaction was inhibited by preincubation of the antiserum with purified SV40 VP1; (ii) purified radiolabeled polyoma VP1 was precipitated by the cross-reactive serum, and this reaction was inhibited by unlabeled SV40 VP1; (iii) other antisera prepared against purified SV40 VP1 or polyoma VP1 reacted in IF with both SV40- and polyma-infected permissive cells. These cross-reacting antisera also reacted in IF with permissive cells infected with BK virus, rabbit kidney vacuolating virus, and the stumptailed macaque virus, suggesting that all members of the polyoma-SV40 subgroup share a common antigenic determinant located in their major capsid polypeptides.     

Comparison of JC and BK human papovaviruses with simian virus 40: DNA homology studies.

Studies were performed to ascertain the relationship of human papovavirus JC to BK virus and to simian virus 40 (SV40) by further restriction endonuclease analysis and by DNA-DNA competition hybridization on membrane filters. Form I DNA extracted from two new isolates from cases of progressive multifocal leukoencephalopathy of human papovaviruses that were JC-like in their antigenic properties were found to yield restriction endonuclease fragmentation patterns similar to those of prototypic JC virus DNA and different from those of BK or SV40. Form I DNA preparations of JC and BK viruses were found to be related to each other and to SV40 DNA to a similar extent, with JC and BK virus DNAs containing sequences homologous to both early and late regions of the SV40 genome. The relatedness in each comparison was less than 50%, and heterologous hybrids between either JC or BK and SV40 DNAs were found to be less stable than homologous SV40-SV40 hybrids in high concentrations of formamide, suggesting substantial mismatch within homologous regions, to the extent of 15 to 30%. The new JC-like isolates were also studied in competition hybridization reactions with SV40 DNA and yielded results similar to those obtained with JC virus.     

Cross-reactivity of antibodies against synthetic peptides

Antiserum against the synthetic peptide Lys-Arg-Ser-Arg-His-Phe, corresponding to the carboxy terminus of polyoma virus medium tumor antigen (medium T antigen), immunoprecipitates a protein of 36,000 daltons from polyoma virus-infected and uninfected cell extracts treated with the sulfhydryl group reagent N-ethyl-maleimide. This protein appears to share an antigenic determinant with medium T antigen that is normally buried inside the protein or covered up by another protein or cellular structure. The two-dimensional tryptic fingerprints of the 36K protein and of medium T antigen are apparently unrelated to each other. Antiserum against the octapeptide Ac-Met-Asp-Lys-Val-Leu-Asn-Arg-Tyr, including the amino-terminal heptapeptide sequence of the simian virus 40 (SV40) large tumor (T) and small T antigens, cross-reacts with polyoma virus large T antigen, which has an identical amino-terminal heptapeptide sequence except that Lys is replaced by Arg and Asn by Ser. The problem of cross-reactivities of antipeptide sera is discussed.     

Characterization of tau antigens isolated from uninfected and simian virus 40-infected monkey cells and papovavirus-transformed cells.

Tau antigens (also known as cellular or nonviral tumor antigens) were detected in uninfected and simian virus 40-infected monkey cells after immunoprecipitation with serum from hamsters bearing simian virus 40-induced tumours (anti-T serum). These two proteins (56,000 daltons) were digested to similarly sized peptides with various amounts of Staphylococcus aureus V8 protease. The Tau antigen isolated from infected monkey cells was closely related but was not identical to the corresponding protein from human cells transformed by simian virus 40, as determined by two-dimensional mapping of their methionine-labeled tryptic peptides. Hamster cells transformed by various primate papovaviruses (simian virus 40, BK virus, and JC virus) synthesized indistinguishable Tau antigens, as determined by two-dimensional peptide mapping. When tested by the same procedure, these proteins and the ones made in monkey and human cells were found to be related to the Tau antigens isolated from simian virus 40-transformed mouse and rat cells. Based on these results, an "evolutionary tree" was constructed to show the relationship among the methionine-containing tryptic peptides of all of these proteins.     

Further characterization of the polyoma virus Y8e from a Rauscher leukaemia virus producing mouse cell line and detection of partial sequence homology between polyoma virus Y8eDNA and hamster papovavirus DNA.

A DNA virus of the papovavirus group spontaneously appeared in RLV-infected spleen and thymus cells of mice in vitro was further characterized as polyoma virus Y8e by haemagglutination test, banding in density gradients, sedimentation coefficients of DNA and molecular hybridization of its DNA. The latter technique showed nearly complete sequence homology to polyoma virus strain SE DNA, partial sequence homology to hamster papovavirus DNA and mouse host DNA and little or no sequence homology to SV 40 DNA. The relationship between rodent papovaviruses and primate papovaviruses is discussed.     

An electron microscopic method for studying and mapping the region of weak sequence homology between simian virus 40 and polyoma DNAs.

A procedure for investigating the possibility of small amounts of partial DNA sequence homology between two defined DNA molecules has been developed and used to test for sequence homology between simian virus 40 and polyoma DNAs. This procedure, which does not necessitate the use of separated viral DNA strands, involves the construction of hybrid DNA molecules containing a simian virus 40 DNA molecule covalently joined to a polyoma DNA molecule, using the sequential action of EcoRI restriction endonuclease and Escherichia coli DNA ligase. Denaturation of such hybrid DNA molecules then makes it possible to examine intramolecularly rather than intermolecularly renatured molecules. Visualization of these intramolecularly renatured “snapback” molecules with duplex regions of homology by electron microscopy reveals a 15% region of weak sequence homology. This region is denatured at about 35 °C below the melting temperature of simian virus 40 DNA and therefore corresponds to about 75% homology. This region was mapped on both the simian virus 40 and polyoma genomes by the use of Hemophilus parainfluenzae II restriction endonuclease cleavage of the simian virus 40 DNA prior to EcoRI cleavage and construction of the hybrid molecule. The 15% region of weak homology maps immediately to the left of the EcoRI restriction endonuclease cleavage site in the simian virus 40 genome and halfway around from the EcoRI restriction endonuclease cleavage site in the polyoma genome.     

Biological properties and viral surface antigens of Burkitt lymphoma- and mononucleosis- derived strains of Epstein-Barr virus released from transformed marmoset cells.

Three strains of Epstein-Barr virus (EBV), two from Burkitt lymphoma (BL) and one from infectious mononucleosis (IM) were used to transform separate cultures of the same batch of primary marmoset leukocytes, and the viruses released from the transformants were compared. The three viruses shared properties of the transforming biotype of EBV, namely, stimulation of DNA synthesis and immortalization of cord blood leukocytes, and failure to induce "early antigen" in lymphoblast lines. All viruses produced more virus in transformed marmoset cells than in transformed human cells, as measured by the number of EBV genomes detected by complementary RNA/DNA hybridization, by virus capsid antigen expression, or by released virions and biologically active virus. Reference human sera and sera from primary EBV infections were used to compare the three virus strains in a virus neutralization test based on inhibition of stimulation of DNA synthesis. Specimens taken late in convalescence from patients with mononucleosis and sera from marmosets experimentally infected with virus from a patient with mononucleosis neutralized the homologous virus, as well as the two virus strains isolated from patients with BL. This finding indicates that viral antigens that elicit neutralizing antibodies are shared among the strains. However, in certain sera the neutralizing-antibody titer against one strain was consistently higher than against another strain. Furthermore, sera taken early after onset of IM contained low levels of neutralizing antibody against IM-derived virus, but failed to neutralize BL-derived virus. These latter findings suggest the existence of heterogeneity among surface antigens of EBVs. The results emphasize the biological and antigenic similarity of EBV isolates from BL and IM and do not suggest major subtype variations. It remains to be determined whether antigenic diversity such as described or virus genome variation detectable by other means is epidemiologically significant.     

Specific neutralization of defective spleen focus-forming virus in Friend virus complex by rat antiserum.

The spleen focus-forming virus (SFFV), a rapidly transforming, replication-defective virus in Friend virus (FV) complex that is readily neutralized by antisera directed against its helper virus, was examined for the presence of SFFV-specific antigens. Antisera prepared in Fisher rats against an SFFV-infected Fisher rat embryo fibroblast line (SFFV-FRE) neutralized SFFV effectively, but not Friend-associated murine leukemia virus (F-MuLV) whether the latter was tested alone or was mixed with SFFV in the FV complex. In contrast, serum from mice immunized with SFFV-infected nonproducer mouse cells had little or no neutralizing activity against SFFV. Both absorption and immunoprecipitation studies indicate that the SFFV-specific antigen is immunologically related to xenotropic murine leukemia virus antigens. The role of both SFFV- and F-MuLV-specific antigens in the neutralization of SFFV suggests that this defective virus could be an antigenic mosaic and that viruses in the FV complex may participate in a undirectional form of phenotypic mixing.     

Demonstration of T antigens on the surface of cells transformed with primate polyoma viruses

Primate polyoma virus-transformed hamster, mouse, and rat cell lines were examined by indirect immunofluorescence staining for cell surface-associated T antigens, by using a rabbit antiserum prepared against sodium dodecyl sulfate-denatured large T antigen of simian virus 40 (anti-SV40-SDS-T serum). Positive surface staining was shown not only on SV40-transformed cells, but also on BK and JC virus-transformed cells. In contrast, normal cells and cells transformed with mouse polyoma-, human adeno-, and murine sarcoma viruses were negative. The data on SV40-transformed cells confirmed the reports of others demonstrating the cell surface location of SV40 large T antigen, and the data on BK and JC virus-transformed cells proved that these cells have cell-surface T antigens that cross-react with anti-SV40-SDS-T serum.     

Subcellular localisation of the middle and large T-antigens of polyoma virus.

The distribution of two of the polyoma virus early proteins (the large and middle T-antigens) in lytically infected mouse cells and transformed rat cells has been investigated by indirect immunofluorescence and immuno-electron microscopy using well-characterised monoclonal antibodies. By these techniques, the viral large T-antigen was found almost exclusively in the nucleus, sometimes in association with nuclear pores, but never in the nucleolus. In lytically infected, but not transformed cells, fluorescence was detected in discrete areas ('hot spots') within the nucleus and, in a minor population of lytically infected cells, cytoplasmic immunoreactive material was observed. The viral middle T-antigen was found in association with most cytoplasmic membranes and in the majority of cells mainly in the endoplasmic reticulum. Only a fraction of the staining was observed in the plasma membrane and no staining in the nucleoplasm was observed. The data suggest that the site of action of the major transforming activity of polyoma virus need not be at the plasma membrane. Functions associated with the viral antigens are discussed in terms of their subcellular distributions within cells.     

Similar cell surface antigens on hamster cells transformed by different papovaviruses.

Transformed baby hamster kidney (BHK) cells were tested for surface antigens by an immunocytoadhesion method. The cells were sensitized with rabbit antisera to cell clones transformed by polyoma or by BK virus and then rosetted with erythrocytes coated with antibody to rabbit immunoglobulin. These antisera detected common antigens on BHK cells transformed by either of three papovaviruses, polyoma, BK, or SV40, but apparently not on normal BHK cells.     

Lack of Sequence Homology Between the 70S RNA of Various RNA Tumor Viruses and the DNA of Simian Virus 40 or Polyoma Virus

No significant hybridization was detected of DNA from simian virus 40 or polyoma virus, and of 70S RNA from avian myeloblastosis virus, murine leukemia virus (Rauscher), murine sarcoma virus (Kirsten), RD-114B, simian sarcoma virus-1, or Mason-Pfizer virus.     

The effect of antiserum quality on strain specificity assessment of foot and mouth disease virus by the neutralization reaction

The factors affecting the virus strain specificity of antibody to foot an mouth disease virus prepared by a variety of protocols in several species were evaluated by neutralization tests. The time at which the serum was taken, the antigen dose given, whether or not revaccination had occurred and the animal species in which the sera were prepared, did not appear to affect the strain specificity of serum prepared to inactivated antigens when measured in neutralization tests, probably because of the restricted nature of the antigenic site involved. However, variation was observed with convalescent animal sera or sera from animals which had received trypsin cleaved virus were used. For these reasons banks of reference antisera are prepared as pooled sera using one or two inoculations of inactivated antigen.     

Expression of oncornavirus antigens in peripheral leucocytes of patients with chronic myeloid leukaemia.

Peripheral leucocytes from 16 patients with chronic myeloid leukaemia (CML) were examined for the presence of oncornavirus p30 antigens by indirect cytoplasmic immunofluorescence. The leucocytes of 12 patients who could be kept in balance by chemotherapy proved to be negative or contained the p30 antigen of mammalian endogenous oncornaviruses as the only viral antigen. In the leucocytes of four patients being in blastoid crisis, an antigen related to the p30 antigen of mammalian leukaemia-sarcoma viruses was detected. In five of six patients decrease in sensitivity to chemotherapy, or blastoid crisis, was preceded by expression of leukaemia-sarcoma virus p30 antigen(s). Leucocytes from 15 CML patients kept in balance by chemotherapy and those from seven being in blastoid crisis, were examined by indirect membrane immunofluorescence for the presence of antigen(s) related to the gp70 antigen of the simian and murine leukaemia-sarcoma virus. All tests proved to be negative.     

Diagnosis of murine infections in relation to test methods employed

Comparative investigations of Sendai virus, pneumonia virus of mice (PVM), mouse encephalomyelitis virus (mouse polio), minute virus of mice (MVM), and reovirus type 3 (Reo 3) infected murine colonies revealed a 30% higher incidence of positive sera when enzyme-linked immunosorbent assay (ELISA) was employed instead of hemagglutination inhibition (HI) tests. Equivalent sensitivity as in the ELISA was obtained when the same sera were investigated by indirect immunofluorescence (IF) tests. The virus purification techniques described resulted in highly suitable antigens for all indirect ELISA established. Since IIF requires no purified antigens, this test is recommended as an alternative to ELISA as well as to HI and complement fixation (CF) tests for laboratories lacking the necessary equipment for high speed centrifugation. A high incidence of false positive HI reactions was found particularly in Reo 3 routine serology. An updated survey of seromonitoring showed that European murine colonies appeared to be infected far less with Reo 3 if ELISA or IIF tests were employed. During 1982-1984, only 13% of the mouse colonies screened possessed Reo 3 positive sera whereas no natural Reo 3 infection was found in rat colonies. Mouse hepatitis virus (MHV) and the coronaviruses of rats exhibited the highest incidence in murine colonies. A total of 60% of mouse and 41% of rat colonies were found to be infected by these viruses. In comparison with earlier serological surveys, the relative incidence of other murine infections was similar. Antibodies against Bacillus piliformis (Tyzzer's disease) were detected by the IIF test in 41% of the rat colonies screened.     

Independence of H-2 and viral antigens on the cell surface and absence of H-2 antigens on murine leukemia virus and mouse mammary tumor virus particles

By indirect immunoelectron microscopy we tested for the presence of H-2 antigens on murine mammary tumor virus (MMTV) and murine leukemia virus (MuLV) particles. The association of H-2 antigens and viral antigens on the virus-infected cell surface was investigated with antibody-induced redistribution. Mammary tumor cells and leukemia cell lines with different H-2 genotypes and carrying different MuMTV or MuLV were used. No H-2 antigens could be demonstrated on the envelope of MMTV and MuLV particles, even after the permeabilization of their envelopes with saponin. On the surface of virus-infected cells antibody-induced patching or capping of the viral antigens did not result in copatching or cocapping of the H-2 antigens. In the reciprocal tests no co-redistribution of viral antigens with H-2 antigens was seen. Our experiments failed to show any physical association between H-2 antigens and MMTV or MuLV antigens on the cell surface.Abbreviations used in this paper MMTV mammary tumor virus - MuLV murine leukemia virus - MHC major histocompatibility complex - IEM immunelectron microscopy