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High-titer antiserum raised in rats against the tumor (T) antigen of polyoma virus was used to purify the T antigen by the Staphylococcus protein A antibody adsorbent technique. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis allowed the identification of a protein with an apparent molecular weight of 100,000-108,000 as a major component induced in lytically infected mouse cells. In cells infected by ts A mutants this component was temperature sensitive. Several minor components were also observed. In pulse and chase experiments there was a slight decrease in electrophoretic mobility of T antigen during the chase period at the permissive temperature, suggesting that the T antigen is a modified protein. In two lines of transformed cells, the amount of T antigen seemed to be considerably less than in lytically infected cells, but the size of the antigen appeared to be equal.
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... Research therefore focused on detecting SV40 in human sera samples and discovering how it functioned due to the potential pandemic that could arise. Early experiments using sera from SV40-infected animals identified two major viral proteins [9,10]; large T antigen (TAg), a 90-100 kDa protein located in the nucleus, and small t antigen (tag), a smaller 17-22 kDa protein. Importantly, these studies also identified a protein of host origin that was found to bind to the large T viral protein [11,12]. ...
... In the second, simpler approach, denatured EBNA is isolated in radiochemically pure form from cell extracts by binding of EBNA/anti-EBNA immune complexes to matrix-bound staphylococcal protein A, followed by elution and gel electrophoresis in sodium dodecyl sulfate (SDS)-containing buffers. The latter method has recently been used to purify the T antigens of polyoma virus and simian virus 40 for structural studies (6,20). It is a considerably more efficient way of isolating immune complexes than indirect immunoprecipitation, which has been inadequate for EBNA purification. ...
The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified from extracts of the human lymphoid cell lines Raji, Namalwa, and B95-8/MLD by two different methods. In the first approach, the apparently native antigen was purified 1,200-fold by a four-step procedure involving DNA-cellulose chromatography, blue dexptran-agarose chromatography, hydroxyapatite chromatography, and gel filtration, employing complement fixation as the assay procedure. Such EBNA preparations specifically inhibited the anticomplement immunofluorescence test for EBNA and bound to methanol/acetic acid-fixed metaphase chromosomes. The purified antigen, which has a molecular weight of 170,000 to 200,000, yielded a single protein band of molecular weight about 48,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These data indicate that native EBNA has a tetrameric structure. In the second purification method, EBNA-containing cell extracts containing radioactively labeled proteins were incubated with anti-EBNA-positive sera, and antigen-antibody complexes were adsorbed to matrix-bound staphylococcal protein A. The bound proteins were then released with an SDS-containing buffer, and denatured EBNA was separated from antibody chains by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. The denatured EBNA obtained in radiochemically pure form by this procedure has a molecular weight of about 48,000, so both methods yield an EBNA monomer of the same size.
... A Dissertation Submitted to the Faculty of the DEPARTMENT Nucleotide sequence analysis showed the coding capacity of this region within one reading frame equivalent to a polypeptide of approximately 100,000 d. This protein classically defined as the T (tumor) antigen was isolated by 1977 for both virus systems (Tegtmeyer, 1974;Ito, Spurr, and Dulbecco, 1977). Other researchers soon discovered a second protein of about 20,000 d., now called small t, which also mapped to the region coding for large T in both viral genomes (Prives li..e.J.., 1977; Sleigh JU..ru..., 1978; Ito and Spur, 1979). ...
We analyzed large and small species of T-antigen by immunoprecipitation and two-dimensional gel electrophoresis. The T-antigen species were subjected to electrophoresis either directly or after reduction and alkylation with N-ethylmaleimide. Treatment with N-ethylmaleimide improved the resolution of large-T by two-dimensional gel electrophoresis and was a requirement for the resolution of small-t antigen on two dimensional gels. Large-T did not form a discrete protein spot, but rather formed a streak from approximately pH 6.5 to 6.9 on isoelectric focusing gels. Small-t formed a sharp protein spot at approximately pH 7.2 when subjected to electrophoresis under non-equilibrium conditions which extended the pH gradient to include proteins with basic isoelectric points. Treatment with N-ethylmaleimide decreased the mobility of the T-antigen species during sodium dodecyl sulfate gel electrophoresis. We suggest that the apparent increase in molecular weight was due to the association of N-ethylmaleimide with cysteine-rich regions of these proteins. Viable deletion mutants of simian virus 40 which do not induce the synthesis of small-t but product small-t-related polypeptides were used to localize the cysteine-rich region of small-t to between 0.54 and 0.59 on the genetic map of simian virus 40.
We have studied the tumor (T) antigens induced by wild-type polyoma virus and several nontransforming mutants using immunoprecipitation with antisera from animals bearing polyomya-induced tumors followed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. In a variety of mouse cells, wild-type virus induces a major T antigen species with apparent molecular weight of 100,000 daltons, and four minor T antigen species with apparent molecular weights of 63,000, 56,000, 36,000 and 22,000 daltons. Hr-t mutants, which have an absolute defect in transformation, induce a normal 100,000 dalton T antigen but are altered in the minor T antigen species. Hr-t deletion mutants induce none of the minor T antigen species seen in wild-type virus. In their place, these mutants induce T antigen species with molecular weights in the range of 6,000--9,000 daltons. The size of the very small T antigen products does not correlate in any simple way with the size or location of the deletions in the viral DNA. Point hr-t mutants induce two of the four minor T antigen species; they make apparently normal amounts of the 56,000 dalton product and reduced amounts of the 22,000 dalton product, but none of the 63,000 or 36,000 dalton species. Ts-a mutants, which have a temperature-sensitive defect in the ability to induce stable transformation, and which complement hr-t mutants, induce T antigens with the same mobility as wild-type; however, the 100,000 dalton T antigen of ts-a mutants is thermolabile compared to wild-type. A double mutant virus carrying both a ts-a mutation and a deletion hr-t mutation induces a thermolabile 100,000 dalton product and none of the minor T antigen species. Cell fractionation studies with productively infected cells have been carried out to localize the T antigen species.
POLYOMA, the small DNA animal tumour virus, like the SV40 virus, has attracted interest because its genome is small enough to be analysed at the molecular level1-6, enabling study of the molecular basis of oncogenic transformation. In polyoma virus there are several deletion mutants, designated hr-t (host-range transformation) mutants, which are unable to transform cells (or cause tumours) and which will grow in certain types of cells only, mainly those which have already been transformed. We report here that we have sequenced the region of the DNA which probably determines the non-transforming phenotype of one such hr-t mutant (NG-18)7 and compared it with the sequence of wild-type polyoma virus DNA. These studies show NG-18 to be a frame-shift mutant, and to have lost 187 of the nucleotides present in wild-type DNA, including several nucleotide triplets which code for cysteine.
Detergent extracts of polyoma virus-infected mouse cells contain three major proteins of approximately 100,000--108,000 (100K), 55,000 (55K) and 21,500 (22K) daltons, which react with sera obtained from rats carrying tumors induced by the virus. A comparison of the 35S-methionine-, 3H-leucine- and 3H-proline-labeled tryptic peptides of each of these proteins by cation-exchange chromatography followed by descending paper chromatography has shown that: at least five peptides are shared by all three T-reactive proteins; at least three peptides are shared by the 55K and 22K proteins, but not by the 100K protein; at least three peptides are found only in the 22K protein; at least six peptides are found only in the 55K protein; and at least sixteen peptides are found only in the 100K protein. The results are consistent with the hypothesis that the polypeptide chains of the 100K, 55K and 22K dalton tumor antigens of polyoma virus share a common virus-coded amino terminal region. The data also suggest that there is a portion of the polypeptide chains (probably immediately adjacent to the common amino terminal region of the molecules) that is shared by the 55K and 22K proteins, but not by the 100K protein (perhaps because this portion of the genetic information is spliced out of the messenger RNA coding for the 100K protein). The facts that all the peptides common to the 100K and 55K proteins are also found in the 22K protein and are thus assigned to the common amino terminal region of the molecules, and that there are several peptides unique to the 100K protein, as well as several peptides unique to the 55K protein, suggest that the presumed carboxy terminal portion of the polypeptide chain of the 100K protein is considerably, if not entirely, different from that of the 55K protein.
Purified simian virus 40 and polyoma DNAs injected into nuclei of Xenopus oocytes were transcribed and subsequently translated into virus-specific tumor antigens and capsid proteins. Simian virus 40 large and small tumor antigens synthesized in the oocytes were indistinguishable, by gel electrophoresis and [35S]methionine-labeled tryptic peptide mapping, from the corresponding polypeptides synthesized in CV-1 African green monkey cells. The synthesis of large simian virus 40 tumor antigen implies the correct splicing of its mRNA, which is complementary to nonadjacent nucleotide sequences in the early region of the viral genome. Polyoma DNA directed synthesis of two polyoma tumor antigen polypeptides, 57,000 Mr and small tumor antigen, and of the main capsid protein.
Polyoma virus-specific RNA isolated from the cytoplasm of lytically infected cells can be translated in vitro to yield three T antigens, of Mrs approximately 90,000, 60,000, and 22,000. The tryptic peptide patterns of the T antigens synthesized in vitro are similar or identical to the patterns of the corresponding proteins in polyoma-infected cells. All three proteins incorporate methionine donated from initiator tRNA in vitro. Polyoma cRNA codes for a protein that is slightly larger than the 22,000 T antigen and that, by other criteria, is similar to the 22,000 T antigen. Translation of cRNA does not yield the 90,000 and 60,000 T antigens, suggesting that the generation of the mRNAs for these T antigens requires the removal of intervening sequences. The mRNA for the 90,000 T antigen is smaller than the mRNAs for the 22,000 and 60,000 proteins. All three proteins share common NH2-terminal sequences, and the 60,000 T antigen may be translated partially in a different reading frame from sequences also coding for the 90,000 T antigen. The demonstration that polyoma virus codes for three different T antigens raises the possibility that all three proteins may be involved in cell transformation.
Early viral polypeptides synthesized in simian virus 40 rat transformants were identified by immunoprecipitation using anti-T (tumor) antigen immune serum. Four polypeptide classes could be identified, which were not detectable in extracts of nontransformed cells and were not precipitated from transformed cell extracts by nonimmune serum. Their apparent M(r) were 92,000, 63,000, 56,000, and 19,000. A similar pattern was observed in extracts from lytically infected cells, but the relative rate of radioactive labeling of the M(r) 63,000 and 56,000 species was in this case significantly lower than in transformed cells. In tsA30 transformants of type A, which maintain the transformed phenotype at high temperature, only minor quantitative variations of this pattern were observed when the cultures were shifted from 33 degrees to 40.5 degrees . In contrast, the rate of labeling of the four virus-specific polypeptides was decreased by 90% or more at high temperature in the temperature-sensitive N transformants. In all cases, a coordinated variation of the radioactivity associated with the different polypeptide classes was observed. These results suggest that the synthesis or processing, or both, of the viral early proteins may be controlled by different mechanisms in various types of simian virus 40 transformants and, furthermore, that it may be under the positive control of a virus-coded protein in transformed cells of type N.
Polyoma virus-transformed rat cell lines were isolated as colonies growing in agar after infection of F2408 cells with low multiplicities of wild-type virus. Viral DNA present in the transformed cells was analyzed by fractionating the cellular DNA on agarose gels before and after digestion with various restriction endonucleases, followed by detection of the DNA fragments containing viral sequences using the procedure described by Southern (E. Southern, J. Mol. Biol., 98:503--515, 1975). Five lines, independently derived, were studied in detail. All five lines, when examined after a minimum number of passages in culture, contained both free and apparently integrated viral DNA. The free polyoma DNA in three of the lines was indistinguishable, by restriction enzyme analysis, from wild-type viral DNA, whereas the two other lines also contained smaller free DNA molecules which lacked parts of the wild-type genome. The integrated DNA in the five lines studies existed as head-to-tail tandem repeats of unit-length polyoma DNA covalently attached to nonviral DNA. The same five polyoma-transformed rat lines were examined after further passage in culture. Free viral DNA was then either undetectable or greatly reduced in amounts, whereas the high-molecular-weight, integrated units persisted after passage of the cells. The subclones, derived from one of the five lines selected for detailed analysis, showed some variations in the quantity and size of the free viral DNA as well as minor alterations in the pattern of the apparently integrated sequences.
The frequency of transformation of rodent fibroblasts by polyomavirus is enhanced by a viral gene product, large T-antigen. However, this effect of large T-antigen cannot be demonstrated with pBR322-cloned viral DNA. Recently, it was discovered that pBR322 contains cis-acting sequences inhibitory to DNA replication in mammalian cells. Because polyomavirus large T-antigen is required for viral DNA replication, we examined the possibility that our inability to demonstrate a requirement for large T-antigen in transformation with pBR322-cloned viral DNA was due to the failure of the chimeric DNA to replicate in the transfected cells. To this end we constructed polyomavirus recombinant molecules with a plasmid (pML-2) that lacks these "poison" sequences and measured their capacity to transform cells. Here we report that recombinant plasmids capable of replicating in the transfected cells transform these cells at frequencies approximately sixfold greater than their replication-defective counterparts.
Hr-t mutants of polyoma virus are restricted in their growth properties (host range) and defective in cell transformation and tumor induction. The present study indicates that these mutants have lost the ability to induce morphological transformation, but have retained a mitogenic function. Thus an early and dramatic difference between wild-type virus and hr-t mutant-infected cultures of rat fibroblasts is the morphological change in individual cells observed by light, fluorescence and scanning electron microscopy. Viruses containing an intact hr-t function (wild-type virus and ts-a mutants) induce a transformed phenotype consisting of stellate cell shape, loss of defined cytoplasmic actin architecture, cellular “under-lapping,” and increased nuclear and nucleolar sizes. These prominent alterations constitute an abortive transformation, peaking 24–48 hr post-infection, and subsequently resolving in most or all of the cells. In contrast, cells infected with hr-t mutants do not develop the above structural changes, but rather retain their preinfection appearance.
The ability of interferon (IF) to inhibit T antigen (Ag) production in simian virus 40 (SV40)-infected or transformed cells was studied primarily through the use of immunoprecipitation followed by gel electrophoresis and autoradiography. Addition of IF to monkey cells prior to or subsequent to inoculation with SV40 resulted in an inhibition in the amount of T Ag that was synthesized late in infection. In contrast when a similar experiment was performed with a is mutant of SV40, tsA58, which does not replicate at the nonpermissive temperature, there was no inhibition in the amount of immunoprecipitable T Ag when IF was added at 30 hr postinfection at 40.5°. The effect of IF on an integrated versus nonintegrated genome within the same cell population was studied in an SV40-transformed mouse cell, H6-15, which is temperature sensitive for the transformed phenotype and for expression of T antigen. In shift-down experiments it was shown that the reappearance of SV40 T Ag was insensitive to the addition of IF whereas superinfection of these same cells with polyoma virus resulted in a dose-dependent inhibition of polyoma T Ag infection. An SV40-transformed mouse cell line (nonpermissive) and two SV40-transformed human cell lines (semipermissive) were passaged in the presence of IF for four generations. Approximately the same amount of labeled T Ag could be immunoprecipitated from IF-treated compared to control mouse cultures whereas, there was a marked decrease in the amount of newly synthesized T Ag in IF-treated human cultures. All these results are compatible with the hypothesis that IF affects differentially the expression of early viral genes whether the viral DNA is integrated or not integrated.
In vitro binding of a Herpesvirus ateles (HVA)-associated soluble antigen to amphibian erythrocyte nuclei was demonstrated by the acid-fixed nuclear binding technique in combination with anticomplement immunofluorescence. Incubation of concentrated salt-extracted soluble antigens derived from HVA-carrying marmoset lines with methanol/acetic acid-fixed erythrocytes of frogs and salamanders resulted in a brilliant nuclear fluorescence after exposure to a live virus-boostered, anti-HVS-positive squirrel monkey serum. Anti-HVS-negative sera did not stain. The activity of the positive serum could be abosrbed completely with extracts of HVA-carrying cells but not with Epstein-Barr virus-carrying or Herpesvirus papio-carrying cells. The HVA-associated antigen was also present in lytically HVA-infected marmoset kidney cells.
At least three distinct forms of polyoma virus tumor antigens were isolated from productively infected and transformed hamster cells by immunoprecipitation with anti-T serum. These proteins had approximate molecular weights of 105,000 (large T antigen), 63,000 (middle T antigen), and 20,000 (small T antigen) as estimated by acrylamide gel electrophoresis. An examination of the appearance of these antigens in polyoma-infected mouse cells showed that all three polypeptides were synthesized maximally at approximately the same time after infection. Analysis of the methionine-containing tryptic peptides of these proteins indicated that the large, middle, and small forms of polyoma T antigens contained five similar or identical peptides. In addition, the 63,000- and 20,000-dalton antigens contained two other methionine peptides absent from the large T-antigen species. Other methionine peptides were found only in the large or middle T-antigen forms. These results and results obtained previously suggested that the three T-antigen species have the same NH2-terminal end regions but different COOH termini. A model is presented describing the synthesis of these polypeptides from different regions of the polyoma virus genome.
The sequence of about one third of the polyoma virus genome is presented. This sequence covers the origin of replication of two large plaque strains (A2 and A3) of polyoma virus. The two strains differ by 11 bp in the origin region. A model for replication is suggested. The sequence probably also covers the entire coding region of two of the polyoma virus early proteins--small and middle T antigens--as well as part of the coding region for large T antigen. Over a small region of the DNA, all three coding frames contain termination codons, which argues a need for spliced early messenger RNAs. In another region of the DNA, two coding frames can be used. Correlation with protein data suggests that one frame codes for part of middle T antigen and the other for part of large T antigen.
We have investigated the DNA sequence alterations in several hr-t mutants of polyoma virus. These mutants are defective in one of the two known viral functions essential for transformation and are altered with respect to several minor T antigen species. The lesions in some of these mutants have been mapped previously by marker rescue experiments to Hpa II fragment 4 (Hpa II-4, 78.4--91.7 map units) in the proximal part of the early region of the viral DNA. Thirteen of sixteen hr-t mutants examined carry deletions 2 to 5 map units (100--250 bp) long in Hpa 11-4. Three mutants carry either point mutations or very small deletions/insertions. Eight of the deletion mutants were mapped closely with restriction enzymes. Seven of them have deletions located entirely within the Hae III subfragment A of Hpa II-4 (the Hae A subfragment, 78.4--85.2 map units), and one extends just beyond this subfragment, ending at 85.5 map units. The complete sequence of the wild-type Hae A subfragment was determined and compared with those of four deletion mutants, NG-18, A-8, 6B5 and B-2. The deletion in each of these mutants is out-of-phase: NG-18, 187 bp; A-8, 127 bp; 6B-5, 179 bp; B-2, 241 bp. All are expected to remove protein sequences in the C terminal part of the small t antigen.
The nucleotide sequence of the early region of the polyoma genome has been determined by the chemical method of Maxam and Gilbert (1977) and the primed synthesis method of Sanger, Nicklen and Coulson (1978). The sequence consists of 3013 nucleotide pairs and contains the regions encoding the three related forms of the tumor antigens, as well as the regions encoding the origin of DNA replication and sequences regulating messenger RNA transcription and splicing. The extent of the open reading frames, together with estimated mRNA splice positions, defines potential coding regions and approximate sizes for the three forms of the tumor (T) antigens. There are uninterrupted open frames corresponding to the probable small t coding region, the 5′ ends of the large T and medium T coding regions, the maximum length for the 3′ end of the large T coding region, and a region potentially available for the 3′ end of the medium T antigen. The 5′ untranslated region contains sequences that are capable of interacting in several different ways with the 3′ end of the 18S ribosomal RNA, thereby representing a possible ribosomal binding site. There are two other 5′ untranslated sequences similar to sequences found at the 5′ ends of other eucaryotic genes that might represent capping signals and/or promoter signals. The 3′ untranslated region contains the sequence 5′-AA-TAAA-3′ just 3′ to the TGA termination triplet, a sequence that may constitute an eucaryotic polyadenylation signal. Codon utilization in the polyoma early region shows deviation from random, especially in its deficiency of triplets ending in TC and in its paucity of CG. There is extensive nucleotide, and deduced amino acid, homology with the early region of SV40 (Fiers et al., 1978; Reddy et al., 1978) throughout regions thought to encode the respective small t and large T antigens. Furthermore, a portion of the possible medium T antigen coding region shows homology with the 3′ end of the SV40 large T antigen coding region, suggesting that the function of the polyoma medium T antigen is performed by the SV40 large T antigen.
Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the "curing" of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for "curing" by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo "curing" with about the same frequency at 33 degrees or 39 degrees C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 degrees than at 39 degrees C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable.
The Epstein-Barr virus-producing cell lines P3HR-1 and B95-8 and the nonproducer cell lines Raji clone No. 7 and NC37 were induced to viral antigen synthesis by the tumor promoter TPA and then analyzed by immunoprecipitation with human sera for early and late virus-associated polypeptides. After labeling of producer cells for a 4-day period with [35S]methionine, two polypeptides with molecular weights of 140,000 and 150,000 were identified reacting predominately with virus capsid antigen (VCA+) sera. Analysis of purified Epstein-Barr virus demonstrated that the 140,000 polypeptide presumably represents an envelope protein while the 150,000 polypeptide is a nucleocapsid protein. In 4-hr radioactively labeled producer cells an additional polypeptide with a molecular weight of 130,000 was found to be immunoreactive with VCA+ sera. Immunoprecipitation of [35S]methionine-labeled cell extracts from nonproducer cells resulted in the specific precipitation of two polypeptides with molecular weights of 85,000 and 35,000 which most likely represent early EBV-associated proteins. Producer cells exhibit three additional apparently early EBV-associated polypeptides with molecular weights of 120,000, 18,000, and 16,000. None of these polypeptides could be detected in EBV genome-negative Ramos cells after TPA treatment.
Evidence is presented that the 55,000-dalton polyoma virus-specific protein in the plasma membrane fraction of productively infected mouse cells is a virus-coded early protein. A non-transforming mutant does not express this protein.
Polyoma-infected 3T6 cells contain a number of proteins precipitable by serum from rats carrying polyoma-induced tumors. The virus codes for three species having apparent molecular weights of 90,000, 60,000 and 22,000 daltons, as determined by polyacrylamide gel electrophoresis (90K, 60K and 22k). The 90K and 22K species produced by a large plaque and a small plaque wild-type polyoma have similar mobilities, but the 60K species produced by the large plaque wild-type. In cells infected by each of seven polyoma tsA mutants, the 90K species is unstable at the nonpermissive temperature, while the 60K and 22K species are stable. In cells infected by a mutant carrying a deletion between roughly 98 and 3 map units in the early region of the viral genome, the 22K species is present, but the 90K and 60K species are absent. Tryptic peptide analysis of the isolated 90K, 60K and 22K species shows that the three species have common N terminal regions. The 60K and 22K species contain amino acid sequences not found in the 90K species , and the 60K species has several unique, methionine-containing peptides not found in either the 22K or 90K species. Two polyoma-transformed BHK cell lines do not have detectable amounts of the 90K protein.
The tumor antigens directed by human papovaviruses BK and JC and the monkey papovavirus simian virus 40 have two methionine-containing tryptic peptides in common. These peptides are constituents of the small forms of papovavirus tumor antigen (17,000 daltons) which are present in lytically infected and transformed cells and which are believed to share some amino acid sequences with the amino-terminal portion of the larger tumor antigen species (97,000 daltons). In addition to the two peptides, which are present in all three papovavirus tumor antigens, the larger forms of the tumor antigens specified by simian virus 40 and BK virus share four other methionine-containing tryptic peptides, two of which are also present in the smaller (17,000 daltons) species of antigen. The occurrence of common peptides at the amino-terminal portion of tumor antigens of primate papovaviruses suggests that these conserved regions may play a fundamental role in the function of these proteins and in the propagation of these viruses in nature. The tryptic peptides of the small forms of papovavirus tumor antigen were examined and compared to those present in the large species. Out of a total of nine and ten methionine-containing peptides in the 17,000-dalton tumor antigens of simian virus 40 and BK virus, seven and nine peptides, respectively, are constituents of the corresponding larger (97,000 daltons) forms of the antigen.
Antisera used previously to assay SV40 T antigen have been produced in tumor-bearing hamsters. This report describes the production and characterization of antiserum produced by injection of rabbits with denatured large-T antigen (Ag) purified by immunoprecipitation and electrophoresis. The resulting antiserum reacts with native large-T Ag as determined by indirect immunofluorescence, complement fixation, and indirect immunoprecipitation assays and binds to species such as “small-T Ag” and previously described proteolytic fragments of large-T Ag.
We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events.
We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.
As indicated by its name, polyoma virus induces a variety of histologically different tumors (Stewart, 1960). Like its close relative SV40, the virus is in fact able to transform a large spectrum of differenciated rodent cells. Most commonly used target cells are rodent (rat, mouse, hamster) fibroblasts: two types of cultures are available, the most normal primary cultures derived from 12–15 day rodent embryos (REF cells) and the established (3T3) lines, which, although in principle non tumorigenic, have acquired one of the characters of a transformed cell, the ability to grow in culture for an indefinite number of generations (Todaro & Green, 1963). Transformation of REF or 3T3 cells leads to the same “highly transformed” phenotype: as originally shown by the pioneer work of Vogt & Dulbecco (1962,1963), these cells grow in all cases indefinitely in culture, grow in culture past confluency, in suspension, in the absence of serum factors, exhibit a completely disorganized cytoskeleton, etc. These cell lines are highly tumorigenic in the syngeneic animal.
The natural occurrence of herpes viruses has been demonstrated within a broad range of animal species. All herpes viruses share typical properties of the group, such as common morphology and the ability to establish persistent infections. Nevertheless, the different herpes viruses of man (HSV 1 and 2, VZV, CMV and EBV) as well as those of animals are quite distinct from each other. Immunological cross-reactivity and molecular DNA studies have shown that there is more affinity between biologically similar herpes viruses of different host species than between different herpes viruses of one individual species (except HSV 1 and HSV 2). All members of the herpes virus group studied so far display an extensive heterogeneity of strains. Sixty-three corneal HSV 1 isolates were compared for intratypic strain differences. Studies of biological properties (neurovirulence in mice) and biochemical analysis (DNA cleavage with restriction endonucleases) revealed considerable differences between individual strains. Attempts to correlate biological and biochemical strain characteristics with clinical features failed. One explanation for this outcome may be that many other pathophysiological factors are involved in the pathogenesis of herpetic diseases. Nevertheless, further studies on the heterogeneity of viral strains are considered necessary for elucidation of the pathogenesis of herpetic diseases.
In recent years, considerable progress has been made in the identification of cellular genes implicated in the genesis of human neoplasia. Among the first evidence for dominant transforming genes were transfection experiments, in which DNA-mediated gene transfer was used to pass from tumor cells a malignant phenotype onto normal fibroblasts. Some of these genes were found to be cellular homologues of viral oncogenes, which confer the ability to cause cancer to acutely transforming retroviruses. With the advent of banding techniques, cytogenetic studies have revealed characteristic abnormalities in several forms of human cancer. To date, the correlation between these non-random karyotypic abnormalities and the chromosomal localization of cellular oncogenes is of limited value. Further, only in very few cases, a cellular oncogene can be directly implicated in the genesis of human tumors. Among the notable advances, however, are findings relating the function of cellular oncogene products to cell proliferation control, since cellular oncogenes code for growth factors, growth factor receptors, and mediators of intracellular message systems. In addition, it was possible to show that the cooperation of oncogenes might by a fundamental principle in oncogenesis, and the genetic dissection of tumorigenesis in at least two parts, immortalization and transformation, provides a frame-work for further studies. Of the alterations of cellular oncogenes in human tumors by amplification, base-pair mutation, and rearrangement, respectively, neither mechanism appears to be sufficient to trigger oncogenic transformation. However, these alterations can serve as definite markers for the classification of human tumors. This concept is elaborated for T- and B-cell leukemia/lymphoma, chronic myelogenous leukemia, Burkitt lymphoma, and retinoblastoma.
Simian virus 40 and polyoma virus chromosomes have long been used as models for the replication and structure of chromatin in mammalian nuclei. This article highlights recent developments and provides simple working models for events common to both viral chromosomes. RNA-primed DNA synthesis by DNA primase-DNA polymerase-α or -δ is initiated repeatedly only on the retrograde arm of replication forks where one of many initiation sites is selected stochastically within a single-stranded DNA region (“initiation zone”) that is defined by chromatin structure rather than DNA sequence. Old histone octamers are distributed to both arms of a fork and newly replicated DNA is rapidly assembled into nucleosomes with little regard for sequence specificity or strand preference. RNA primed-DNA synthesis is first initiated on the early mRNA template strand of the origin of replication (ori) by the same mechanism used to initiate Okazaki fragments at replication forks. Bidirectional DNA replication begins at a unique site at one end of the required ori sequence (ori-core) following binding of a T-antigen/permissive cell-factor initiation complex; binding to ori-core is facilitated by either promoter or enhancer elements adjacent to the late-gene side of ori-core. These transcriptional elements can determine cell-type, but not species, specificity for ori activation. Replication terminates at whatever sequence is 180° from ori, but the termination site strongly affects the way in which sibling chromosomes are separated. Topoisomerase II appears to be required specifically for termination of replication although formation of catenated intertwines is not an obligatory pathway in the separation of sibling chromosomes, suggesting that topoisomerase II acts behind replication forks rather than in front of them.
Striking changes in actin cytoarchitecture occur when cultured fibroblasts become oncogenically transformed by tumor viruses. These changes were first observed by electron and immunofluorescence miscroscopy as a loss of large bundles of actin microfilaments called “stress fibers.” Recent technical innovations for visualizing the cytoskeleton have revealed that actin structure in normal fibroblasts is much more complex than was originally supposed and that the changes in actin structure that occur on transformation, collectively referred to as “the actin transformation,” are manifold. In cells transformed by certain DNA- and RNA-containing tumor viruses, these actin alterations have been shown to be the consequence of specific viral genes that simultaneously set in motion a variety of pleiotypic changes in the infected cell and that are also required for tumorigenicity. Several of these transforming genes code for proteins that may directly induce transformation and tumorigenesis. Considerable progress has been made in recent years toward the identification of these proteins. Although the transforming proteins encoded by different viruses are distinct, they share certain properties that may be important in bringing about the actin transformation. Furthermore, since transformation induced by these distinct tumor viruses results in a more or less common actin phenotype in the infected cell, it is likely that the actin transformation contributes to the common features of transformation and tumorigenicity. Indeed, a wealth of studies have accumulated that show correlations among the actin transformation, tumorigenicity, and individual transformation parameters, some of which are related to growth control and others of which are not obviously growth-related. Nonetheless, a cause-and-effect relationship has not been demonstrated between the actin transformation and any of these parameters.
In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression.
Plasmids encoding the amino terminal portion of an influenza virus hemagglutinin (HA) fused to polyoma virus middle T (mT) or large T (lT) sequences have been constructed. Stable expression of the chimeric proteins was obtained in established rat embryo fibroblasts following plasmid co-transfection and selection for G418 resistance. The synthesis and localization of the proteins was followed by metabolic labeling with [35S]methionine and [3H]mannose, cell fractionation, and immunoprecipitation with anti-polyoma T antibody. The HA leader and amino terminal peptide direct the synthesis of the lT and mT proteins into the endoplasmic reticulum where they undergo glycosylation, but this occurs with a very low efficiency. Most of the HA-mT and HA-lT fusion protein molecules do not enter completely into the endoplasmic reticulum, but rather achieve their normal locations in the cell as slightly higher molecular weight proteins, presumably due to the extra sequences derived from HA at their amino termini. HA-mT fusion protein is found to have associated tyrosine-specific protein kinase activity precipitable with anti-src as well as anti-T antibody, and cells expressing this fusion protein have a transformed phenotype.
Polyoma is a small, double stranded DNA, mouse virus that normally propagates by a lytic infectious cycle. Under some circumstances, however, polyoma virus can induce the formation of a wide variety of tumors. The early proteins, or T-antigens, exert this oncogenic effect, which is paralleled by an ability to transform cells in culture. The three T-antigens co-operate to induce tumorigenesis, but the main transforming role is contained within one polypeptide, the middle T-antigen (MT). MT interacts with, and alters the regulation of, a number of host proteins that control cell proliferation. MT binds the A and C subunits of protein phosphatase 2A, and this complex then associates with a member of the src-family of non-receptor tyrosine protein kinases. As a consequence, the kinase is activated, and MT itself becomes tyrosine phosphorylated on at least three residues. These act as binding sites for the SH2 domains of phosphatidylinositol (3′) kinase and phospholipase Cγ-1, and the phosphotyrosine binding domain of Shc. Each of these polypeptides is in turn phosphorylated on tyrosine residues, which initiates a series of intracellular events culminating in transformation. These pathways are the same as those stimulated by tyrosine kinase associated growth factor receptors, leading to the conclusion that MT acts as a functional homologue of such a receptor. MT, therefore, not only supplies us with information about the mechanisms behind tumor induction in vivo and transformation in cell culture, but can also be used to examine the molecular details of signal transduction induced by growth factor receptors. MT also interacts with the constitutively synthesised heat shock protein 70, and some members of the 14-3-3 family of polypeptides, but the role of these interactions is less clear.
We have compared ts-a mutants of polyoma virus with ts-A mutants of SV-40, and hr-t mutants of polyoma virus with the viable deletion mutants of SV-40 mapping between 0.54 and 0.59 map unit (referred to as dl). All four groups of mutants are either totally or partially defective in inducing stable transformation as assayed by anchorage-independent growth. In each virus system, two classes of mutants—hr-t and ts-a of polyoma virus and dl and tsA of SV-40—complement to induce stable transformation. Two distinct functions essential for transformation are therefore encoded within the early regions of these papova viruses. Two approaches have been taken in attempts to define the roles of these early viral genes in cell transformation. In the first approach, a clonal analysis was made of cells transformed at the permissive temperature by ts-a/A mutants. Selections were carried out either for anchorage-independent growth or for focus formation. Although the variation in expression of the selected parameter of transformation among multiple clones derived with the same virus and cell line is often high, the majority of clones show no temperature dependence of either selected or unselected properties when compared to wild-type virus-transformed clones. In some instances, temperature-sensitive clones are observed. No correlation is seen between the appearance of a temperature-sensitive phenotype in individual clones and the expression of T-antigen species at permissive and nonpermissive temperatures determined by immunofluorescence or immunoprecipitation of [35Slmethio-nine-labeled proteins. In the second approach, mutants of all four groups were tested for their ability to induce abortive transformation measured as the transient loss of anchorage-dependent growth. This assay circumvents the problem of clonal variation and gives a clearcut result. ts-a/A mutants retain the ability to induce abortive transformation, behaving like wild-type virus at the nonpermissive temperature. hr-t mutants are virtually negative, while the dl mutants show a reduced ability to induce abortive transformation. The simplest explanation which is adequate for the majority of the results is that the ts-a/A function is required only transiently to carry out an initiation event which stabilizes transformation, while the hr-t/dl function acts to induce parameters of the transformed phenotype in the manner of a maintenance function. Additional interpretations are put forward to explain the results with temperature-sensitive clones transformed by ts-a/A mutants.
Formalin-fixed, heat-inactivated Staphylococcus aureus Cowen Type I (SAC) (Kessler, S. W. (1975) J. Immunol.115, 1617–1624) has been used to isolate antibody-bound tyrosine aminotransferase (EC 18.104.22.168) from radioactively labeled cell-free extracts and translation reactions of polysomes and poly(A)-containing messenger RNA. Under our conditions, SAC immunoprecipitation of cell-free extracts generated a radioactive background in control serum precipitations of 0.1%, an average of fivefold lower than backgrounds obtained by direct and indirect antibody precipitations. It is shown that the major source of the background, which limits sensitivity of the assay, comes from the amounts of antiserum and bacterial adsorbent in the assay. Procedures are presented to provide low backgrounds in more general applications of the precipitation method. From these experiments, SAC immunoprecipitation is shown to be a rapid, economical method to assay for specific protein synthesis under a variety of experimental conditions with noise levels much lower than with other standard methods.
Antisera, raised in rats, containing specificities directed against tumor antigen of polyoma virus also react with several proteins present in the plasma membrane of mouse cells infected with the virus. The main component has an apparent molecular weight of 55,000. The appearance of this protein after infection with early temperature-sensitive A mutants was temperature-dependent like tumor antigen itself. Pulse and chase isotope experiments suggest that this protein originates from a precursor, perhaps by cleavage; its production appears to be facilitated by the A mutation. Two other components with apparent molecular weights of 61,000 and 28,000 were also present but were more variable from experiment to experiment. All proteins were absent from the plasma membranes of cells infected with a transformation-defective mutant, NG-18. Up to four virus-specific proteins could be isolated from the plasma membranes of rat, hamster, and mouse cells transformed by the virus. The possible role of the plasma membrane proteins in cell transformation is discussed.
Large quantities of a species of T antigen with an apparent molecular weight of 84,000 have been isolated from monkey kidney cells infected with SV40 by using the protein A Antibody Adsorbent (P.A.A.) technique and preparative SDS-polyacrylamide gel electrophoresis. The purified polypeptide was found to be immunogenic, inducing a specific antibody response in a rabbit. The resulting antiserum was 10 times as potent as a hamster anti-tumor serum and reacted with native as well as SDS- and DTT-treated T antigens from SV40-transformed or lytically infected cells. It failed to show any reaction with T antigen from polyoma-infected cells and showed similar specificity to antitumor serum obtained from hamsters which had been inoculated with cells of an SV40-transformed, virus-free cell line. In both cases two distinct polypeptides, large T (84,000 and 94,000) and small t (19,000) were precipitated from extracts of SV40-transformed or lytically infected cells. The rabbit antiserum was shown to be capable of specifically precipitating small-t antigen in the absence of large-T antigen and therefore these two polypeptides must share common antigenic determinants. A radioimmunoassay showed large-T antigen to be very heat stable in direct contrast to earlier results obtained using the complement fixation test. The reasons for this discrepancy and its functional significance are discussed.
The monomer form of BK virus (BKV) tumor antigen (T Ag) was immunoprecipitated from extracts of BKV-transformed cells and had a molecular weight of approximately 113,000. This compared with 97,000 for the molecular weight of either BKV or simian virus 40 (SV40) T Ag from lytically infected cells. The SV40 and BKV T Ag's from productively infected cells were compared by examining their methionine-labeled tryptic peptides. Out of a total of 20 SV40-and 21 BKV-specific peptides, there were seven pairs of similar peptides on the basis of ion-exchange chromatography, These coeluting peptides contained approximately 25 to 30% of the total methionine radioactivity. Similar results were obtained when the tryptic peptides of SV40 T Ag from lytically infected cells were compared with those of BKV T Ag from virally transformed cells.
When isolated by means of an anti-polyoma tumor (T) antiserum, the major product from mouse cells productively infected by wild-type polyoma virus is a polypeptide of 100,000 apparent molecular weight as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In cells infected by NG-18, an hr-t mutant carrying a deletion of about 150 base pairs in the early region of the viral DNA, a T antigen species appears that comigrates with that of the wild-type virus. Comparisons of peptides after partial proteolysis reveal no differences between mutant and wild-type products. Both wild-type and mutant 100,000 products can be labeled in vivo with [³²P]orthophosphate. An independent and more reliable estimate of the molecular weight of this protein using guanidine/Sepharose chromatography yields a value of 81,000 for both mutant and wild-type species. The apparent identity of wild-type and mutant products indicates that the deletion in NG-18 lies outside of the region encoding this major T antigen species.
Immunoprecipitates from wild-type infected cells shows four bands in addition to the “100,000” band; these have apparent molecular weights of 63,000, 56,000, 36,000, and 22,000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis; the 56,000 and 36,000 species are phosphorylated. All four of these lower molecular weight bands are absent or drastically reduced in the immunoprecipitates from NG-18-infected cells.
The accumulation of virus-specific early RNA in mouse 3T6 cells infected by wild type polyoma virus or by a tsA mutant, tsA25E, was measured by hybridization of cytoplasmic RNA to radiolabeled "early" strand polyoma DNA. Cells infected by the tsA25E mutant accumulated approximately 20 times more virus-specific early RNA during the early phase of lytic infection than did wild type-infected cells at both the permissive and the nonpermissive temperature under identical conditions of infection and hybridization. Cells infected by the tsA25E mutant at the permissive temperature continued to accumulate virus-specific early RNA during the late phase of infection after being shifted to the nonpermissive temperature to block further viral DNA replication. A mixed infection of cells by wild-type polyoma and tsA25E showed that the overproduction of early RNA by the tsA mutant alone could be suppressed by coinfection with the wild type. The results suggest that the A gene product of polyoma regulates transcription of early RNA, as has been suggested for SV40 (Reed et al., 1976) and that the wild-type A-gene product overcomes the effect of the temperature-sensitive A-gene product.
Two techniques were used to search for the polypeptide encoded by the avian sarcoma virus (ASV) src gene. First, antiserum from rabbits bearing ASV-induced fibrosarcomas was used to immunoprecipitate a transformation-specific antigen from ASV-transformed chick embryo fibroblasts. This antigen has an apparent molecular weight (Mr) of 60,000. Second, the 3' one-third of the ASV genome, selected by oligo(dT)-cellulose chromatography and sucrose gradient sedimentation, was translated in a mRNA-dependent reticulocyte cell-free lysate. This RNA species programmed the synthesis of a polypeptide that comigrated with the transformation-specific antigen of Mr 60,000 immunoprecipitated from transformed cells. The methionine-containing tryptic peptides from the polypeptides of Mr 60,000 obtained from translation in vitro and from immunoprecipitation were found to be identical upon two-dimensional fractionation.
The DNA sequence of the early coding region of polyoma virus is presented. It consists of 2739 nucleotides. The sequence predicts
that more than one reading frame can be used to code for the three known polyoma virus early proteins (designated small, middle
and large T-antigens). From the DNA sequence, the ‘splicing’ signals used in the processing of viral RNA to functional messenger
RNAs can be predicted, as well as the sizes and sequences of the three proteins. Other unusual aspects of the DNA sequence
are noted. Comparisons are made between the DNA sequences and the predicted amino acid sequences of the respective large T-antigens
of polyoma virus and the related virus Simian Virus (SV) 40.
In mouse cells transformed with the ts-a mutant of polyoma virus (ts-a-3T3), only low amounts of the virus-specific T antigen were synthesized at high temperature (39 C). After a shift-down to the permissive temperature (31 C), these cells exhibited the same level of T-antigen production as wild-type polyoma transformants. The T antigen produced by ts-a-transformed cells was inactivated at 39 C in vitro at a faster rate than that produced by wild-type-transformed cells. These observations indicate that T antigen is, or includes, a virus-coded peptide.
Cell lines were established from cultures derived from Fischer rat embryos according to the transfer schedule described by Todaro and Green (1963) for mouse 3T3 cells where cell crowding and serum exhaustion were kept to a minimum. Cell growth rate did not decline greatly during the course of successive 3-day transfers. Like 3T3 cells, the rat cell lines possess very low saturation densities under standard culture conditions. A clonal cell line with a relatively high plating efficiency was obtained from one of the cell lines, 3Y1. In these cloned cultures, virus growth was not detectable upon infection with SV40, while a small amount of virus was produced upon infection with polyoma virus. Morphological transformation of the cloned 3Y1 cells by SV40 and polyoma virus could be assayed with single-hit kinetics and with efficiencies comparable to those of the previously available transformation systems for each virus. Independent cell lines transformed by SV40 were consistently virus-free and all the lines tested produced SV40 upon fusion with permissive monkey cells. Most of the independent transformed cell lines isolated after polyoma infection appeared to be virus-free, although the cultures of some lines produced a small amount of polyoma virus spontaneously after a prolonged cultivation. Most of the virus-free polyoma-transformed lines produced virus upon fusion with permissive mouse cells.
T antigen induced in African green monkey kidney cells by a temperature-sensitive mutant of simian virus 40, defective in a function required for cell transformation, was characterized. The number of T antigen-positive cells estimated by an immunofluorescent techniques was almost equal at permissive (32.5 C) and restrictive (38.5 C) temperatures, but was slightly reduced when the infected cells were incubated at a higher temperature (40.5 C). However, a complement fixation test indicated that the amount of T antigen induced by the mutant is not significantly different from that induced by wild-type virus at 40.5 C. These results suggest that the T antigen-inducing ability of the mutant is not defective. Two distinct molecular species of T antigen were induced by the mutant at the permissive temperature, whereas only one form was observed at the restrictive temperature. The larger molecular form (14 to 15S) induced by the mutant at the permissive temperature was more heat labile than that induced by wild-type virus, suggesting that the mutated gene product is a component of the larger molecular form.
SV40 may cause either productive infection of permissive cells or transforming infection of restrictive cells. The viral gene(s) expressed early in productive infection are the only gene(s) expressed in transforming infection (Khoury et al. 1972; Sambrook et al. 1972). Although several virus-induced early antigens have been identified in infected cells (Black et al. 1963; Lewis and Rowe 1971), only one early gene (gene A) has been detected by genetic techniques (Tegtmeyer and Ozer 1971; Chou and Martin 1974). That gene may be directly or indirectly responsible for the induction of each of the early antigens. Gene A codes for a diffusible gene product to be referred to as the A protein. Its function is directly and continuously required to initiate, but not to propagate or complete, each round of viral DNA replication (Tegtmeyer 1972). Expression of the same gene is only transiently required to initiate the continuous transcription of late...
Stimulus for the study of small DNA tumor viruses comes from the fact that not only do they have the ability to induce neoplastic transformation, but also they interact with the host cell and affect its function and control. Recently restriction enzymes have become available as powerful tools for the analysis of these viral genomes. These enzymes cleave double-stranded DNA at specific nucleotide sequences and have been used to produce large and homogeneous quantities of limited portions of the viral genome. The ordering of restriction enzyme fragments has led to the formation of physical maps for polyoma (Griffin et al. 1974) and SV40 (Danna et al. 1973) DNA.
The following report deals with a further analysis of restriction enzyme fragments of the polyoma wild-type strain A-2 and the restriction enzyme cleavage patterns of different temperature-sensitive mutants and purified defective DNA isolates...
Characterization of Polyoma Strain A-2 DNA
Polymon DNA is
Simian-virus-40-specific T-antigen was isolated by immunoprecipitation. From other studies we have proof that the T-antigen described in this work is coded by the viral DNA. The molecular weight estimated from electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels of T-antigen isolated from nonpermissive mouse cells in abortive infection is 86,000 and from permissive monkey cells in lytic infection is 82,000. The 86 kilodalton T-antigen is readily converted in vitro into an 82 kilodalton form by incubation with extracts from permissive monkey cells but not with extracts from nonpermissive mouse or hamster cells. This and the results of fingerprinting analysis of tryptic peptides suggest that T-antigen may be processed in permissive cells.
T-antigens from simian virus 40 (SV 40)-transformed and lytically infected cells have been isolated by immunoprecipitation and their molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T-antigen from SV40-transformed mouse and hamster cells has an apparent molecular weight of 94,000 whereas that from several lines of SV40-infected monkey cells is 84,000. In a wheat germ cell-free system, mRNA from either transformed or productively infected cells is translated into a 94,000 species. Experiments with the protease inhibitors L-l-(tosylamide-2-phenyl)ethylchloromethyl ketone HCl and N-alpha-p-tosyl-L-lysylchloromethyl ketone HCl suggest that the 84,000 species of T-antigen found in infected cells is derived from the larger species by proteolytic cleavage. Further, the cleavage pathway probably involves a two-step reaction with an 89,000 intermediate. The biological significance of the two molecular weight forms of T-antigen is unknown, but the possibility that they have different physiological activities is discussed.
The major stimulus for the studies on two papovaviruses—polyoma virus and simian virus 40 (SV40)—has been their oncogenic potential. Both viruses cause tumors in a variety of rodents in vivo. In tissue-culture systems, polyoma virus transforms number of different types of rodent cells, whereas SV40 can transform, in addition to rodent cells, monkey and human cells. These viruses do not appear to be important tumor-causing agents in the wild. However, large numbers of transformed cells are produced in vitro in a simple and reproducible manner after viral infection. Thus, these viruses appear to offer a good model system for elucidating in vitro some of the events that lead to in vivo tumor formation. The genetic information of both polyoma virus and SV40 resides in their DNAs, which have molecular weights of about 3.4 x l06 daltons. The DNAs exist in “native” form as covalently closed circular superhelical molecules with a coding capacity for about 200,000 daltons of protein. Three distinct genes have so far been identified. One can be correlated with an early function concerned with viral DNA synthesis and the other two with proteins found in the viral capsid.
The T (tumor) antigen from transformed mouse cells has been purified 400-fold and prominent polypeptides of molecular weights 85–90,000 and 70–75,000 resolved. Polypeptides exhibiting same molecular weights are adsorbed specifically on insolubilized anti-T antibodies.
The Cowan I strain of the bacterium Staphylococcus aureus has been used as an adsorbent for antibodies complexed with radiolabeled antigens from cell lysates. This application is advanced as a superior alternative to other methods of immune precipitation for the isolation of antigens. It exploits the high adsorption capacity for IgG molecules by protein A molecules on the cell walls of certain strains of staphylococci, along with the advantageous sedimentation properties of the bacteria. The interaction of immune complexes with the adsorbent was defined initially using a model system of bovine serum albumin with a high excess of rabbit anti-bovine serum albumin antibodies (IgG). The uptake of immune complexes under these conditions was extremely rapid, occurring within seconds, whereas maximum binding of free IgG was much slower. In addition, once bound the complexed antigen could not be displaced from the adsorbent either by large amounts of normal IgG or by extra free antibody. Antigen could be eluted almost completely from the inert adsorbent for analytic or preparative purposes with a variety of solvent systems, such as the detergent SDS in combination with urea and high temperature, and neutral salts with strong lyotropic salting in properties. The efficacy of the protein A-antibody adsorption technique was tested in direct comparisons with a conventional double antibody precipitation method for the isolation of mouse lymphocyte IgM. The bacterial adsorbent not only had a distinct advantage in speed of antigen isolation, but analyses by polyacrylamide gel electrophoresis in SDS also revealed consistently higher antigen recoveries, lower levels of background radioactivity, and an absence of other cell components which may nonspecifically bind to and complicate analyses using conventional immune precipitates.
Polyoma virus-induced complement-fixing antigen (PV-ICFA) has been demonstrated by immunofluorescence in polyoma primary rat kidney and hamster subcutaneous fibrosarcomas, in one polyoma trans-plantable mouse tumor and in one polyoma-transformed in vitro hamster cell line. The antigen has a typical nuclear localization appearing either in a granular or in a diffused form throughout the whole nucleus except for the nucleoli. A lower concentration of ICFA was found in the hamster tumors than in the rat and mouse tumors. The PV-ICFA was also demonstrated in polyoma-infected mouse, hamster, and rat embryonic tissue cultures. In primary infected cultures of all three species, the synthesis of PV-ICFA preceded that of viral antigen (PV-A). Many more cells synthesized PV-ICFA than PV-A, The difference was especially high in the hamster and the rat cultures in which the ratio of PV-ICFA to PV-A positive cells averaged about forty.
A line of polyoma-transformed mouse cells has been isolated which is fully susceptible to lytic infection by polyoma virus. This line has been used to select virus mutants which have lost most or all of their ability to grow in the untransformed parental line while retaining the ability to grow in the transformed derivative. These virus mutants are also defective in their ability to transform cells of rat or hamster origin. Since the DNA extracted from the mutants has the same host range as the whole virus, the mutants appear to be blocked at some intracellular step which is required both for the completion of virus development in mouse cells and for transformation in rat or hamster cells.
Temperature-sensitive mutants of polyoma virus have been tested for their ability to produce polyoma T antigen in mouse embryo cells at permissive and nonpermissive temperatures. One “late” mutant, which is not defective in viral DNA synthesis, exhibits normal T antigen production at the nonpermissive temperature. Four “early” mutants, which are defective in viral DNA synthesis, are also defective in T antigen production at the nonpermissive temperature. However, the inhibition of viral DNA synthesis by cytosine arabinoside or 5-fluorodeoxyuridine does not prevent T antigen production by wild-type polyoma virus, or by “early” mutants at the permissive temperature. These observations suggest that the failure of these “early” mutants to form T antigen at the nonpermissive temperature is not a consequence of defective viral DNA replication per se, but reflects an alteration in either the gene which codes for T antigen, or in a gene the expression of which is required for both T antigen formation and viral DNA replication.
The ts-a mutant of polyoma virus, and other mutants of the ts-a type, are defective in viral DNA synthesis, T-antigen synthesis and stable transformation at the restrictive temperature. Viral DNA synthesis ceases in mutant-infected cells following a shift from the permissive to the restrictive temperature. Inhibition of RNA or protein synthesis during a shift up in temperature has no effect on the cessation of viral DNA synthesis. Pulse-chase experiments suggest that initiation of new rounds of viral DNA synthesis is blocked at the restrictive temperature. Completion of rounds of viral DNA synthesis at the restrictive temperature occurs at the same rate in mutant-infected cells as in wild-type infected cells.
Two of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrids induce SV40 transplantation resistance in immunized hamsters. These two hybrids, Ad2(+)ND(2) and Ad2(+)ND(4), contain 32 and 43% of the SV40 genome, respectively. The pattern of induction of SV40 transplantation antigen (TSTA) by the various hybrids differentiates TSTA from both SV40 U and T antigens. Since the SV40 RNA induced by both these hybrids is early SV40 RNA, these findings confirm that TSTA is an early SV40 function. By combining available data on SV40 antigen induction by these hybrids with electron microscopy heteroduplex mapping studies, the DNA segment responsible for the induction of SV40 TSTA can be inferred to lie in the region between 0.17 and 0.43 SV40 units from the site on the SV40 chromosome cleaved by E. coli R(1) restriction endonuclease.
The RNAs and proteins specified by five early genes of bacteriophage T7 have been identified by electrophoresis on sodium dodecyl sulfate, polyacrylamide gels. Extracts of cells infected by different deletion strains and point mutants of T7 are analyzed on a slab gel system in which 25 samples can be run simultaneously and then dried for autoradiography. The high capacity of this system makes it possible to do many types of experiment that would be extremely tedious by other means.The five early genes are designated 0.3,0.7, 1, 1.1 and 1.3, in order from left to right on the T7 genetic map. The stop signal that prevents host RNA polymerase from transcribing into the late region of T7 DNA is located to the right of gene 1.3 (ligase). Most deletions that affect gene 1.3 also delete the stop signal, and some of them affect at least one late protein, the 1.7 protein. Several small, early RNAs can be resolved that are not affected by any of the deletions. These small RNAs could not come from between the five early genes or from the right end of the early region, and other work (Dunn & Studier, 1973) indicates that at least some of them come from the region to the left of gene 0.3.Deletions have been found that enter either end of the gene 1 RNA or the right ends of the 0.3 or 1.1 RNAs without seeming to affect the proteins specified by these RNAs. Perhaps all of the early messenger RNAs of T7 have untranslated regions at both ends. Some deletions that enter the left end of the gene 1 RNA reduce the amount of gene 1 protein that is synthesized, presumably by interfering with initiation of protein synthesis.
A simple method is described for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X-ray film. The gel is dehydrated in dimethyl sulphoxide, soaked in a solution of 2,5-diphenyloxazole (PPO) in dimethylsulphoxide, dried and exposed to RP Royal “X-Omat” film at -70 °C. Optimal conditions for each step are described. β-particles from 3H interact with the 2,5-diphenyloxazole emitting light which causes local blackening of an X-ray film. The image produced resembles that obtained by conventional autoradiography of isotopes with higher emission energies such as 14C. 3000 dis. 3H/min in a band in a gel can be detected in a 24-h exposure. Similarly 500 dis./min can be detected in one week.
When applied to the detection of 35S and 14C in polyacrylamide gels, this method is ten times more sensitive than conventional autoradiography. 130 dis. 35S or 14C/min in a band in a gel can be detected in 24 h.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
Disaggregated mouse embryo cells, grown in monolayers, underwent a progressive decline in growth rate upon successive transfer, the rapidity of the decline depending, among other things, on the inoculation density. Nevertheless, nearly all cultures developed into established lines within 3 months of culture. The first sign of the emergence of an established line was the ability of the cells to maintain a constant or rising potential growth rate. This occurred while the cultures were morphologically unchanged. The growth rate continued to increase until it equaled or exceeded that of the original culture. The early established cells showed an increasing metabolic autonomy, as indicated by decreasing dependence on cell-to-cell feeding. It is suggested that the process of establishment involves an alteration in cell permeability properties. Chromosome studies indicated that the cells responsible for the upturn in growth rate were diploid, but later the population shifted to the tetraploid range, often very rapidly. Still later, marker chromosomes appeared. Different lines acquired different properties, depending on the culture conditions employed; one line developed which is extremely sensitive to contact inhibition.
One or more new specific antigens are present in “virus-free” polyoma tumors, as demonstrated by their complement fixation reaction with serum from animals carrying transplantable tumors. These antigens cannot be found in normal animal tissues or in tumors induced by other viruses or by methylcholanthrene. Mouse and hamster cell tissue cultures after exposure to polyoma virus are positive for the antigen and corresponding control cultures are negative. The presence of the antigen in these tissue culture cells is not related to the presence or absence of infectious virus in a carrier culture relationship or to the ability of the culture cells to produce tumors in isologous animals. Sera from tumor-bearing animals having high titers of CF antibody against tumor antigen are negative for antiviral antibody activity. Some sera from virus immunized animals are negative against the tumor antigen but have high anti-viral antibody titers. The presence of demonstrable anti-tumor CF antibodies is not necessary for virus-immunized animals to resist challenge with an isologous polyoma tumor.
The effect of DNA antagonists and various antibiotics on steps in the synthesis of SV40 virus in green monkey kidney cells was investigated. Both the early forming tumor (T) antigen, as well as the later synthesized virus (V) antigen, were synthesized in the presence of fluorouracil and iododeoxyuridine. Cytosine arabinoside (and fluorodeoxyuridine in starved cells) prevented synthesis of V antigen but not T antigen. The synthesis of T antigen therefore does not require synthesis of virus DNA. Virus particles formed only in the presence of the iododeoxyuridine and they were non-infectious. Actinomycin D inhibited synthesis of both tumor and virus antigens, suggesting that the synthesis of these antigens involves DNA-dependent RNA. Puromycin allowed synthesis of the T antigen which remained localized at the nucleolar membrane. This finding with puromycin suggests that the T antigen is a protein of low molecular weight. Virus antigen forming in the presence of mitomycin C, p-fluorophenylalanine, iododeoxyuridine, or fluorouracil was distributed atypically. These inhibitors caused the V antigen to be diffusely spread throughout the nucleus, or to be concentrated at the nuclear membrane.