Rearrangement of integrated viral DNA sequences in mouse cells transformed by simian virus 40.

The organization of viral DNA sequences in several cell lines derived from a primary colony of simian virus 40 (SV40)-transformed mouse cells was analyzed to examine the origin of the various distinctive patterns of SV40 sequence arrangement present in transformed cells. This analysis revealed a complex arrangement of viral sequences in the uncloned transformed cells but simplified arrangements in cloned derivatives of the primary transformant. The cell lines studied had certain SV40 sequence arrangements in common, but the cloned lines had lost some parental arrangements and acquired new arrangements. These results indicate that the arrangement of viral sequences in some SV40-transformed cells is not fixed but that alterations occur after integration, creating a heterogeneous population of transformants. In the process, expression of viral genes may be altered. Possible causes for and implications of this genetic instability are discussed.     

Simian virus 40-specific proteins in HeLa cells infected with nondefective adenovirus 2-simian virus 40 hybrid viruses.

The synthesis of simian virus 40 (SV40)-specific proteins in HeLa cells infected with the nondefective adenovirus 2 (Ad2)-SV40 hybrid viruses, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5, was investigated. Infected-cell proteins were labeled with radioactive amino acids late after infection, when host protein synthesis was shut off, and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All polypeptides normally seen in Ad2-infected cells were found in cells infected by the hybrid viruses. In addition to the Ad2-specific proteins, cells infected with Ad2+ND2 contain two SV40-specific proteins with apparent molecular weights of 42,000 and 56,000, cells infected with Ad2+ND4 contain one protein with an apparent molecular weight of 56,000, and cells infected with Ad2+ND5 contain one protein with an apparent molecular weight of 42,000. Cells infected with Ad2+ND3 do not contain detectable amounts of proteins not seen during Ad2 infection. Pulse-chase experiments demonstrate that the SV40-specific proteins induced by Ad2+ND2, Ad2+ND4, and Ad2+ND5 are metabolically unstable. These proteins are not present in purified virions. Two nonstructural Ad2-specific proteins have been demonstrated in Ad2 and hybrid virus-infected cells which have a smaller apparent molecular weight after a short pulse than after a pulse followed by a chase. The molecular weight increase during the chase may be caused by the addition of carbohydrate to a polypeptide backbone.     

Simian virus 40 tumor-specific proteins: subcellular distribution and metabolic stability in HeLa cells infected with nondefective adenovirus type 2-simian virus 40 hybrid viruses.

HeLa cells infected with adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses produce several SV40-specific proteins. These include the previously reported 28,000-dalton protein of Ad2+ND1, and 42,000- and 56,000-dalton proteins of Ad2+ND2, the 56,000-dalton protein of Ad2+ND4, and the 42,000-dalton protein of Ad2+ND5. In this report, we extend the list of SV40-specific proteins induced by Ad2+ND4 to include proteins of apparent molecular weights of 28,000 42,000, 60,000, 64,000, 72,000, 74,000, and a doublet of 95,000. Cell fractionation studies demonstrate that the SV40-specific proteins are detectable in the nuclear, cytoplasmic, and plasma membrane fractions. By pulse-chase and cell fractionation experiments, three classes of SV40-specific proteins can be distinguished with regard to metabolic stability: (i) unstable in the cytoplasmic but stable in the nuclear and plasma membrane fractions; (ii) stable in the nuclear, cytoplasmic, and plasma membrane fractions; and (iii) unstable in all subcellular fractions. Immunoprecipitation of infected cell extracts demonstrates that most of the above proteins share antigenic determinants with proteins expressed in hamsters bearing SV40-induced tumors. Only the 42,000-dalton protein of Ad2+ND5 is not immunoprecipitable.     

Structure of integrated simian virus 40 DNA in transformed mouse cells

The structure of integrated viral DNA sequences in four lines of simian virus 40 (SV40)-transformed Balb/c 3T3 cells has been probed using restriction endonucleases and the Southern (1975) transfer method. By considering data from a large number of restriction digests of DNA from each line, and by using a novel method of handling the data, we have constructed fairly detailed physical maps of the integrated DNA in each line. The most striking of the features of the maps described here is that none is easily explained by the integration of a single SV40 genome into the DNA of the host cell. Three of the lines contain at least two distinct integrated segments and the fourth contains a single segment longer than the viral DNA. Considered individually, only two of the seven segments that we have mapped might be unit length. Of the remaining five, two are longer and three are shorter than the viral genome. It seems likely, therefore, that at least in SV40-transformed Balb/c 3T3 cells simple, single integrations are rare.The endpoints of these seven segments of integrated DNA fall at many positions distributed over the entire genome, confirming earlier studies (Ketner &; Kelly, 1976; Botchan et al., 1976), which indicated that SV40 integration is not absolutely site-specific.Finally, one of the lines mapped here (SVB209) does not possess an intact SV40 early region, an observation that suggests the possibility that a normal viral large T polypeptide is not synthesized by this line.     

Simian virus 40-specific ribosome-binding proteins induced by a nondefective adenovirus 2-simian virus 40 hybrid.

We have studied the intracellular distribution of the two simian virus 40-specific proteins, with apparent molecular weights of 56,000 and 42,000, detectable in human KB cells infected by a nondefective adenovirus 2-simian virus 40 hybrid, Ad2+ND2. After a 20-min pulse of [35S]methionine, about two-thirds of the newly synthesized 56K protein and one-third of the 42K protein were found localized on the plasma membrane. The remainder of each protein was found in the cytoplasm, whereas the nuclear fraction was virtually free of either component. A significant portion of both proteins present in the cytoplasmic fraction was complexed to the 40S ribosomal subunits and was not removed by treatment with 0.5 M KCl. Moreover, the portion that was found free in the cytoplasm could bind preferentially and quantitatively to purified 40S ribosomes in vitro, leading us to propose that these simian virus 40 proteins may act as translational control elements in cells.     

Human glioblastoma cells persistently infected with simian virus 40 carry nondefective episomal viral DNA and acquire the transformed phenotype and numerous chromosomal abnormalities.

A stable, persistent infection of A172 human glioblastoma cells with simian virus 40 (SV40) was readily established after infection at an input of 450 PFU per cell. Only 11% of the cells were initially susceptible to SV40, as shown by indirect immunofluorescent staining for the SV40 T antigen at 48 h. However, all cells produced T antigen by week 11. In contrast, viral capsid proteins were made in only about 1% of the cells in the established carrier system. Weekly viral yields ranged between 10(4) and 10(6) PFU/ml. Most of the capsid protein-producing cells contained enormous aberrant (lobulated or multiple) nuclei. Persistent viral DNA appeared in an episomal or "free" state exclusively in Southern blots and was indistinguishable from standard SV40 DNA by restriction analysis. Viral autointerference activity was not detected, and yield reduction assays did not indicate defective interfering particle activity, further implying that variant viruses were not a factor in this carrier system. Interferon was also not a factor in the system, as shown by direct challenge with vesicular stomatitis virus. Persistent infection resulted in cellular growth changes (enhanced saturation density and plating efficiency) characteristic of SV40 transformation. Persistent infection also led to an increased frequency of cytogenetic effects. These included sister chromatid exchanges, a variety of chromosomal abnormalities (ring chromosomes, acentric fragments, breaks, and gaps), and an increase in the chromosome number. Nevertheless, the persistently infected cells continued to display a bipolar glial cell-like morphology with extensive process extension and intercellular contacts.     

Viral DNA sequences and gene products in hamster cells transformed by adenovirus type 2.

Complementary strand-specific adenovirus DNA of full length or from endonuclease BamHI fragments was used as a probe to estimate the fractional representation and abundance of viral sequences in five hamster cell lines (Ad2HE1-5) transformed with UV-inactivated adenovirus type 2. The fraction of the viral genome present in the five transformed cell lines varied from 44% in the Ad2HE5 cell line to 84% in the Ad2HE3 cell line. The number of viral DNA copies per diploid cell equivalent ranged from 1.8 in the Ad2HE1 line to 7.1 in the Ad2HE4 line. In vivo labeling with [35S]methionine followed by immunoprecipitation with an antiserum against adenovirus type 2 early proteins revealed virus-specific polypeptides with molecular weights of 42,000 to 58,000 in extracts from all five hamster cell lines. Several other early viral polypeptides were detected in some of the adenovirus type 2-transformed hamster cell lines.     

Structural relationship between the 100,000- and 17,000- molecular-weight T antigens of simian virus 40 (SV40) as deduced by comparison with the SV40-specific proteins coded by the nondefective adenovirus type 2-SV40 hybrid viruses.

The two-dimensional peptide maps of the methionine-containing tryptic peptides of the 100,000-molecular-weight (100K) and 17K T antigens of simian virus 40 (SV40) have been compared. The two proteins share nine methionine-containing tryptic peptides in common. The 17K T antigen has two peptides not found in the 100K T antigen, and the 100K T antigen has 14 unique peptides. The peptide maps of the 100 K and 17K T antigens were also compared with those of the SV40-specific proteins found in cells infected by the nondefective adenovirus type 2-SV40 hybrid viruses, which we have previously shown are encoded by defined sequences within the early region of SV40 (K. Mann, T. Hunter, G. Walter, and H.K. Linke, J. Virol. 24:151-169, 1977). This comparison shows that the 100K and 17K T antigens share common N-terminal sequences coded for between 0.65 and 0.59 map units on the SV40 genome. Furthermore, none of the sequences in the 17K T antigen arises from the region between 0.54 and 0.18 map units. We deduce that the sequences unique to the 17K T antigen originate between 0.59 and 0.54 map units. This type of structural relationship between the 100K and 17K T antigens fits well with the proposed model (L.V. Crawford, C.N. Cole, A. E. Smith, E. Paucha, P. Tegtmeyer, K. Rundell, and P. Berg, Proc. Natl. Acad. Sci. U.S.A. 75:117-121, 1978) for the expression of the early region of SV40.     

Arrangement of integrated viral DNA sequences in cells transformed by adenovirus types 2 and 5.

The organization of the viral DNA sequences in 15 adenovirus-transformed cell lines was analyzed by the Southern blotting procedure. The site of adenovirus integration in the cellular genome was found not to be unique, and the viral DNA sequences involved in integration were not confined to a specific region of the adenovirus genome. Several cell lines showed simple integration patterns that demonstrated the presence of large continuous stretches of viral DNA. In four cell lines, containing sequences from both molecular ends of the viral genome, the left- and right-hand-terminal sequences appeared to be linked to each other.     

Interference with simian virus 40 DNA replication by adenovirus type 2 during mixed infection of monkey cells.

Infection of monkey cells with human adenovirus (Ad) is abortive, but the infection can be enhanced by coinfecting with simian virus 40 (SV40). However, in the coinfected monkey cells, Ad interferes strongly with SV40 DNA biosynthesis. This interference was found to be a reproducible, delicately controlled phenomenon that was proportional to the multiplicity of infection of Ad and dependent on the active expression of the Ad genome. Newly synthesized SV40 DNA was not broken down in cells after delayed superinfection with Ad, and several early events of SV40 infection such as adsorption, penetration, uncoating, induction of cellular DNA synthesis, and enhancement of Ad infection were not markedly influenced by Ad-mediated interference. It is unlikely that interference is simply due to competition between SV40 and Ad for metabolites, enzymes, or replication sites. The interference effect could be partially neutralized by an increase in the multiplicity of coinfecting SV40 or by an increase in the time interval between SV40 infection and Ad coinfection. Interference was shown to be due to the activity of an Ad early gene product. However, the detailed mechanism of this Ad interference is still unclear.     

Amount of viral DNA in the genome of cells transformed by adenovirus type 2

The number of copies of viral DNA in DNA of cells transformed by adenovirus type 2 has been determined by following the kinetics of reassociation of ³²P-labeled viral DNA in the presence of unlabeled DNA extracted from transformed and control cells. There is close to one copy of adenovirus 2 DNA for every diploid quantity of cell DNA.     

Integration and expression of viral DNA in cells transformed by host range mutants of adenovirus type 5.

Group I host range (hr) mutants of adenovirus type 5 are unable to transform rat embryo or rat embryo brain cells but induce an abnormal transformation of baby rat kidney cells. We established several transformed rat kidney cell lines and characterized them with respect to the transformed phenotype and the structure of the integrated viral DNA. The hr mutant-transformed cells, unlike wild-type virus transformants, were fibroblastic rather than epithelial, failed to grow in soft agar, and were also less tumorigenic in nude mice. Studies on the structure of the integrated viral DNA sequences showed that hr-transformed cells always contained the left end of the adenovirus DNA, but the size of the integrated DNA fragment varied among different lines, and a high percentage of the lines contained the entire viral genome colinearly integrated. The patterns of integration were maintained after prolonged growth in culture and after subcloning. Attempts to rescue infectious virus from lines which contained the entire genome were unsuccessful. Using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we analyzed the viral proteins expressed in hr-transformed cells. Results of these studies indicated that, like wild type-transformed cells, hr transformants expressed E1B proteins of molecular weight 58,000 and 19,000.     

Monoclonal antibodies against simian virus 40 tumor antigens: analysis of antigenic binding sites, using adenovirus type 2-simian virus 40 hybrid viruses.

The antigenic binding sites of two monoclonal antibodies are located in the COOH-terminal region (clone 412) and probably in an internal region (clone 7) of simian virus 40 large T antigen. A third monoclonal antibody (clone 122), which has been shown to bind nonviral T antigen, does not react with HeLa cells infected with nondefective adenovirus type 2 (Ad2)-simian virus 40 hybrid viruses Ad2+ND1, Ad2+ND2, or Ad2+ND4.     

Rat cells transformed by simian virus 40 give rise to tumor cells which contain no viral proteins and often no viral DNA.

Rat 3T3 cells transformed by simian virus 40 were injected into rats to examine their capacity to develop into tumors. Both large T-dependent (N) transformants and large T-independent (A) transformants were used. All the transformed cell lines contained large T and small t and could multiply efficiently in agar. Only some transformants could develop into tumors. All tumor cells examined had lost both large T and small t. Tumor cells in which the viral genome could still be detected were found together with tumor cells in which the simian virus 40 DNA could no longer be detected. N transformants which displayed the transformed phenotype in a temperature-sensitive manner became temperature insensitive during tumor formation.     

Replication of Chinese hamster embryo cells transformed by temperature-sensitive T-antigen mutants of simian virus 40.

Chinese hamster embryo cells transformed by simian virus 40 temperature-sensitive T-antigen mutants replicated when confluent at 40.5 degrees C, regardless of the selection method, selection temperature, or virus strain used.     

Excision of viral DNA from host cell DNA after induction of simian virus 40-transformed hamster cells.

Simian virus 40-transformed hamster cells were induced to produce infectious virus by treatment with mitomycin C or gamma-irradiation. A portion of the simian virus-40 DNA, which is integrated into the host cell genome in uninduced cells, was recovered in a pool of relatively low-molecular-weight DNA early after induction treatment in the absence of DNA replication. The data indicte that excision of the viral genome occurs subsequent to the induction stimulus.     

Spontaneous rearrangement of integrated simian virus 40 DNA in nine transformed rodent cell lines.

Frequencies of spontaneous DNA rearrangement within or near integrated simian virus 40 (SV40) DNA were measured in four transformed mouse and rat cell lines of independent origin and in five clones of the SV40-transformed mouse line SVT2. Rearrangements were detected as polymorphisms of restriction enzyme fragment length in subclones of the lines. At least 17% of the subclones of each line had detectable rearrangements. The rate of rearrangement was calculated to be at least 5 x 10(-3) events per cell per division. No rearrangements were detected in sequences of an immunoglobulin gene, part of the coding region of the mouse protein p53, and five proto-oncogenes. The possible role of recombination between duplicated segments of integrated SV40 DNA in generating rearrangements was studied in the five SVT2 clones, which differed in the number of duplications within a single SV40 DNA segment. The SVT2 clone that had no duplications, M3, became rearranged further at least as frequently as did closely related lines with one, two, or three duplications. Another line in this group that had one small duplication, X1, had a much higher frequency of rearrangement than did the others; integrated SV40 DNA of X1 became mostly rearranged within 100 cell divisions. The examples of M3 and X1 suggested that the high rate of rearrangement characteristic of integrated SV40 DNA was influenced more by the presence of particular sequences within or near integrated SV40 DNA than by the number or extent of duplicated sequences.