Immunological Cross-Reactivity Between Simian Virus 40 Large T Antigen and D2 Hybrid T Antigen

A specific antiserum was raised in rabbits against D2 hybrid T antigen that had been purified from HeLa cells infected with the adenovirus/simian virus 40 hybrid, Ad2(+)D2. The specificity of this serum was compared with that of a conventional hamster antiserum against simian virus 40-induced tumors by immunoprecipitation and by a new radioimmune assay that can detect nanogram quantities of D2 hybrid T antigen.     

Association of Simian Virus 40 T Antigen with Simian Virus 40 Nucleoprotein Complexes

Viral nucleoprotein complexes were extracted from the nuclei of simian virus 40 (SV40)-infected TC7 cells by low-salt treatment in the absence of detergent, followed by sedimentation on neutral sucrose gradients. Two forms of SV40 nucleoprotein complexes, those containing SV40 replicative intermediate DNA and those containing SV40 (I) DNA, were separated from one another and were found to have sedimentation values of 125 and 93S, respectively. [(35)S]methioninelabeled proteins in the nucleoprotein complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to VP1, VP3, and histones, a protein with a molecular weight of 100,000 (100K) is present in the nucleoprotein complexes containing SV40 (I) DNA. The 100K protein was confirmed as SV40 100K T antigen, both by immunoprecipitation with SV40 anti-T serum and by tryptic peptide mapping. The 100K T antigen is predominantly associated with the SV40 (I) DNA-containing complexes. The 17K T antigen, however, is not associated with the SV40 (I) DNA-containing nucleoprotein complexes. The functional significance of the SV40 100K T antigen in the SV40 (I) DNA-containing nucleoprotein complexes was examined by immunoprecipitation of complexes from tsA58-infected TC7 cells. The 100K T antigen is present in nucleoprotein complexes extracted from cells grown at the permissive temperature but is clearly absent from complexes extracted from cells grown at the permissive temperature and shifted up to the nonpermissive temperature for 1 h before extraction, suggesting that the association of the 100K T antigen with the SV40 nucleoprotein complexes is involved in the initiation of SV40 DNA synthesis.     

Replication Stress and Mitotic Dysfunction in Cells Expressing Simian Virus 40 Large T Antigen

We previously demonstrated that simian virus 40 (SV40) large T antigen (LT) binds to the Bub1 kinase, a key regulator of the spindle checkpoint and chromosome segregation. Bub1 mutations or altered expression patterns are linked to chromosome missegregation and are considered to be a driving force in some human cancers. Here we report that LT, dependent on Bub1 binding, causes micronuclei, lagging chromatin, and anaphase bridges, which are hallmarks of chromosomal instability (CIN) and Bub1 insufficiency. Using time-lapse microscopy, we demonstrate that LT imposes a Bub1 binding-dependent delay in the metaphase-to-anaphase transition. Kinetochore fibers reveal that LT, via Bub1 binding, causes aberrant kinetochore (KT)-microtubule (MT) attachments and a shortened interkinetochore distance, consistent with a lack of tension. Previously, we showed that LT also induces the DNA damage response (DDR) via Bub1 binding. Using inducible LT cell lines, we show that an activated DDR was observed before the appearance of anaphase bridges and micronuclei. Furthermore, LT induction in serum-starved cells demonstrated γ-H2AX accumulation in cells that had not yet entered mitosis. Thus, DDR activation can occur independently of chromosome segregation defects. Replication stress pathways may be responsible, because signatures of replication stress were observed, which were attenuated by exogenous supplementation with nucleosides. Our observations allow us to propose a model that explains and integrates the diverse manifestations of genomic instability induced by LT.     

The J Domain of Simian Virus 40 Large T Antigen Is Required To Functionally Inactivate RB Family Proteins

Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellular tumor suppressors. Transformation minimally requires the following three domains: (i) a C-terminal domain that mediates binding to p53; (ii) the LXCXE domain (residues 103 to 107), necessary for binding to the retinoblastoma tumor suppressor protein, pRB, and the related p107 and p130; and (iii) an N-terminal domain that is homologous to the J domain of DnaJ molecular chaperone proteins. We have previously demonstrated that the N-terminal J domain of TAg affects the RB-related proteins by perturbing the phosphorylation status of p107 and p130 and promoting the degradation of p130 and that this domain is required for transformation of cells that express either p107 or p130. In this work, we demonstrate that the J domain of TAg is required to inactivate the ability of each member of the pRB family to induce a G₁ arrest in Saos-2 cells. Furthermore, the J domain is required to override the repression of E2F activity mediated by p130 and pRB and to disrupt p130-E2F DNA binding complexes. These results imply that while the LXCXE domain serves as a binding site for the RB-related proteins, the J domain plays an important role in inactivating their function.     

Simian Virus 40 Large T Antigen Is Phosphorylated at Multiple Sites Clustered in Two Separate Regions

The phosphorylation sites of simian virus 40 large T antigen were determined within the primary structure of the molecule. Exhaustive digestion of ³²P-labeled large T antigen with trypsin generated six major phosphopeptides which could be separated in a newly developed isobutyric acid-containing chromatography system. By partial tryptic digestion, large T antigen was cleaved into an amino-terminal fragment of 17,000 daltons and overlapping fragments from the carboxy-terminal region ranging in size between 71,000 and 13,000 daltons. The location of the phosphopeptides was then determined by fingerprint analyses of individual fragments. Their physical properties were analyzed by sizing on polyacrylamide gels and by sequential digestion and peptide mapping; their amino acid composition was determined by differential labeling with various amino acids. The amino-terminal 17,000-dalton fragment gave rise to only one phosphopeptide (phosphopeptide 3) that contained half of the phosphate label incorporated into large T antigen. It contained phosphoserine and phosphothreonine sites, all of which were clustered within a small segment between Cys₁₀₅ and Lys₁₂₇. This segment contained five serines and two threonines. Among these, Ser₁₀₆, Ser₁₂₃, and Thr₁₂₄ were identified as phosphorylated residues; in addition, either one or both of Ser₁₁₁ and Ser₁₁₂ were phosphorylated. The neighboring residues, Ser₁₂₃ and Thr₁₂₄, were found in three different phosphorylation states in that either Ser₁₂₃ or Thr₁₂₄ or both were phosphorylated. Phosphopeptides 1, 2, 4, 5, and 6 were all derived from a single fragment extending 26,000 daltons upstream from the carboxy terminus of large T antigen. Phosphopeptide 6 was identical with the previously determined phosphothreonine peptide phosphorylated at Thr₇₀₁. Phosphopeptides 1, 2, 4, and 5 contained only serine-bound phosphate. Phosphopeptides 1, 2, and 4 represented overlapping peptides, all of which were phosphorylated at Ser₆₃₉ located next to a cluster of six acidic residues. In phosphopeptide 5, a large peptide ranging from Asn₆₅₃ to Arg₆₉₁, at least two of seven serines were phosphorylated. Thus, large T antigen contains at least eight phosphorylation sites. Their clustering within two separate regions might correlate with structural and functional domains of this protein.     

Archetype JC Virus Efficiently Replicates in COS-7 Cells, Simian Cells Constitutively Expressing Simian Virus 40 T Antigen

JC polyomavirus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), is ubiquitous in humans, infecting children asymptomatically and then persisting in the kidney. Renal JCV is not latent but replicates to excrete progeny in the urine. The renal-urinary JCV DNAs carry the archetype regulatory region that generates various rearranged regulatory regions occurring in JCVs derived from the brains of PML patients. Tissue cultures that support the efficient growth of archetype JCV have not been reported. We studied whether archetype JCV could replicate in COS-7 cells, simian cells transformed with an origin-defective mutant of simian virus 40 (SV40). Efficient JCV replication, as detected by a hemagglutination assay, was observed in cultures transfected with five of the six archetype DNAs. The progeny JCVs could be passaged to fresh COS-7 cells. However, when the parental cells of COS-7 not expressing T antigen were transfected with archetype JCV DNAs, no viral replication was detected, indicating that SV40 T antigen is essential for the growth of JCV in COS-7 cells. The archetype regulatory region was conserved during viral growth in COS-7 cells, although a small proportion of JCV DNAs underwent rearrangements outside the regulatory region. We then attempted to recover archetype JCV from urine by viral culture in COS-7 cells. Efficient JCV production was observed in COS-7 cells infected with five of the six JCV-positive urine samples examined. Thus, COS-7 cells should be of use not only for the production of archetype JCV on a large scale but also for the isolation of archetype JCV from urine.     

Sequence Requirements for the Assembly of Simian Virus 40 T Antigen and the T-Antigen Origin Binding Domain on the Viral Core Origin of Replication

The regions of the simian virus 40 (SV40) core origin that are required for stable assembly of virally encoded T antigen (T-ag) and the T-ag origin binding domain (T-ag-obd(131-260)) have been determined. Binding of the purified T-ag-obd(131-260) is mediated by interactions with the central region of the core origin, site II. In contrast, T-ag binding and hexamer assembly requires a larger region of the core origin that includes both site II and an additional fragment of DNA that may be positioned on either side of site II. These studies indicate that in the context of T-ag, the origin binding domain can engage the pentanucleotides in site II only if a second region of T-ag interacts with one of the flanking sequences. The requirements for T-ag double-hexamer assembly are complex; the nucleotide cofactor present in the reaction modulates the sequence requirements for oligomerization. Nevertheless, these experiments provide additional evidence that only a subset of the SV40 core origin is required for assembly of T-ag double hexamers.     

Cytotoxic T-Lymphocyte Epitope Immunodominance in the Control of Choroid Plexus Tumors in Simian Virus 40 Large T Antigen Transgenic Mice

The simian virus 40 (SV40) large tumor antigen (Tag) is a virus-encoded oncoprotein which is the target of a strong cytotoxic T-lymphocyte (CTL) response. Three immunodominant H-2(b)-restricted epitopes, designated epitopes I, II/III, and IV, have been defined. We investigated whether induction of CTLs directed against these Tag epitopes might control Tag-induced tumors in SV11(+) (H-2(b)) mice. SV11(+) mice develop spontaneous tumors of the choroid plexus due to expression of SV40 Tag as a transgene. We demonstrate that SV11(+) mice are functionally tolerant to the immunodominant Tag CTL epitopes. CTLs specific for the H-2Kb-restricted Tag epitope IV were induced in SV11(+) mice following adoptive transfer with unprimed C57BL/6 spleen cells and immunization with recombinant vaccinia viruses expressing either full-length Tag or the H-2Kb-restricted epitope IV as a minigene. In addition, irradiation of SV11(+) mice prior to adoptive transfer with unprimed C57BL/6 spleen cells led to the priming of epitope IV-specific CTLs by the endogenous Tag. Induction of epitope IV-specific CTLs in SV11(+) mice by either approach correlated with increased life span and control of the choroid plexus tumor progression, indicating that CTLs specific for the immunodominant Tag epitope IV control the progressive growth of spontaneous tumors induced by this DNA virus oncogene in transgenic mice.     

Simian Virus 40 Large T Antigen J Domain and Rb-Binding Motif Are Sufficient To Block Apoptosis Induced by Growth Factor Withdrawal in a Neural Stem Cell Line

Serum-free mouse embryo (SFME) cells are a neural stem cell line that is dependent upon epidermal growth factor (EGF) for survival. Removal of EGF results in the G1 arrest and apoptosis of SFME cells. We have shown that the expression of simian virus 40 large T antigen in SFME cells blocks apoptosis and allows cell survival and division in the absence of EGF. Therefore the presence of T antigen abrogates the EGF requirement. The steady-state levels of p53, p21, and mdm-2 do not increase as SFME cells undergo apoptosis upon EGF withdrawal. Furthermore, the amino-terminal 136 amino acids (N136) of T antigen are sufficient to block death and to promote proliferation in the absence of EGF, while the carboxy-terminal fragment (C251-708), which contains the p53 binding site, is unable to block death. Taken together, these data suggest that SFME cells deprived of EGF undergo p53-independent apoptosis. Mutations that disrupt either the J domain or Rb family binding abolish the ability of T antigen to block SFME cell apoptosis and to promote cell growth. We conclude that T antigen must act on one or more members of the Rb family to inhibit SFME cell apoptosis.     

Origin and Direction of Simian Virus 40 Deoxyribonucleic Acid Replication

Double-branched, circular, replicating deoxyribonucleic acid (DNA) molecules of simian virus 40 (SV40) have been cleaved by the R(1) restriction endonuclease from Escherichia coli. This enzyme introduces one double-strand break in SV40 DNA, at a specific site. The site of cleavage in the replicating molecules was used in this study to position the origin and the two branch points. Radioactively labeled molecules fractionated according to their extent of replication were evaluated after cleavage by sedimentation analysis and electron microscopy. The results demonstrate that the R(1) cleavage site is 33% of the genome length from the origin of replication and that both branch points are growing points. These data indicate that SV40 DNA replication is bidirectional and confirm other reports which have shown a unique origin of replication.     

Simian Virus 40-Induced T and Tumor Antigens

Antigen extracts from simian virus 40 (SV40) transplanted hamster tumors were studied by rate-zonal centrifugation. Three species or molecular forms of antigen were demonstrated. The major antigen component corresponded to a molecular weight of 65,000 to 75,000, and two larger species were detectable in smaller quantities. Similar studies were carried out on SV40 virus-induced T antigen from BSC-1 cells. Three antigen components were again detected. Quantitative differences in the expression of "T" and tumor antigen species were reproducibly found.     

Role of the Innate Immune Response and Tumor Immunity Associated with Simian Virus 40 Large Tumor Antigen

We examined properties of the innate immune response against the tumor-specific antigen simian virus 40 (SV40) large tumor antigen (Tag) following experimental pulmonary metastasis in naive mice. Approximately 14 days after mKSA tumor cell challenge, expression of inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and RANTES was upregulated in splenocytes harvested from mice, as assessed by flow cytometry and antibody array assays. This response was hypothesized to activate and induce tumor-directed NK cell lysis since IL-2-stimulated NK cells mediated tumor cell destruction in vitro. The necessary function of NK cells was further validated in vivo through selected antibody depletion of NK cells, which resulted in an overwhelming lung tumor burden relative to that in animals receiving a control rabbit IgG depletion regimen. Interestingly, mice achieved increased protection from experimental pulmonary metastasis when NK cells were further activated indirectly through in vivo administration of poly(I:C), a Toll-like receptor 3 (TLR3) agonist. In a separate study, mice receiving treatments of poly(I:C) and recombinant SV40 Tag protein immunization mounted effective tumor immunity in an established experimental pulmonary metastasis setting. Initiating broad-based immunity with poly(I:C) was observed to induce a Th1 bias in the SV40 Tag antibody response that led to successful antitumor responses not observed in animals treated only with poly(I:C) or SV40 Tag. These data have direct implications for immunotherapeutic strategies incorporating methods to elicit inflammatory reactions, particularly NK cell-driven lysis, against malignant cell types that express a tumor-specific antigen such as SV40 Tag.Considerable interest has been directed toward the role innate immunity plays in reducing malignant growth and progression. Although the innate system by broad definition is not endowed with the antigen specificity and memory recall of adaptive immunity, natural killer (NK) cells are an innate effector population that shares most properties with the adaptive arm of the immune system, excluding receptor rearrangement (28). Interestingly, NK cells can be employed to directly target and destroy malignant cell types through diverse pathways that include tumor major histocompatibility complex class I (MHC-I) loss and upregulation of stress-inducible protein ligands for the NK cell activating receptor NKG2D (24, 29). Much effort is under way in human clinical trials to manipulate NK cell properties for directed therapies against cancer (13, 29).One strategy in eliciting innate immunity in general involves activating the Toll-like receptor (TLR) family, which are preferentially expressed by innate effectors such as NK cells, macrophages, and dendritic cells (DCs) (26). TLR ligands include a variety of pathogen-associated molecular patterns with differing downstream responses based on the cell type involved and specific TLR activated. In TLR-expressing cells, signal transduction pathways follow a MyD88-independent course to produce type I interferons (IFNs) (e.g., TLR3) or a MyD88-dependent pathway that results in the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-6 and expression of costimulatory molecules such as CD40, CD80, and CD86 (e.g., TLR4 and TLR9) (2, 12, 23, 26). In the case of TLR3, activation by poly(I:C) causes DCs and additional accessory cells to secrete type I interferons and IL-12, activating NK cells and prompting NK cell secretion of IFN-γ among other effects (14, 20). Ultimately, modulation of TLR activation results in the generation of a range of cytokines that promote inflammation, Th1 bias, and NK cell-directed killing that can be utilized in a beneficial manner for tumor treatment strategies.TLR agonist incorporation alongside vaccine strategies has resulted in promising results in mouse models of cancer (12). Indeed, the TLR7 agonist imiquimod is an effective FDA-approved topical compound used to treat superficial basal-cell carcinoma and external genital warts (9). However, to our knowledge, modulating TLR activity while also incorporating recombinant simian virus 40 (SV40) large tumor antigen (Tag) protein immunizations in a therapeutic tumor setting has not been previously reported. SV40 Tag is a clinically relevant tumor-specific antigen that has been shown to be expressed by a number of human malignancies, including malignant pleural mesothelioma (MPM), and represents a potential target for immunotherapeutic strategies.Our laboratory has previously defined a unique role for antibody-dependent cell-mediated cytotoxicity (ADCC) reactions—specific against SV40 Tag—promoting cytotoxic T-lymphocyte (CTL) activity in response to neoantigens through cross-presentation of tumor cell debris in a model of experimental pulmonary metastasis (16, 17). In this report, we analyze the role of innate immunity in mediating tumor cell lysis during the early course of tumorigenesis in the absence of vaccination. Overall, we find that activated NK cells are necessary effector cells in achieving antitumor reactions and providing partial tumor immunity during the onset of tumorigenesis and that these functioning NK cells are likely activated in vivo due to inflammation as a result of tumor growth and progression. The burden of tumor challenge could be further reduced in naive animals with the indirect activation of NK cells using poly(I:C) as a TLR3 agonist prior to and during malignant dissemination. Interestingly, in an established pulmonary tumor setting, therapeutic treatment of mice with poly(I:C) and recombinant SV40 Tag resulted in enhanced protection that was not observed using poly(I:C) or SV40 Tag alone. One effect of instituting poly(I:C) treatment alongside SV40 Tag immunizations was a Th1 skewing of the SV40 Tag IgG antibody response that correlated with therapeutic tumor protection.Our results have direct implications for the prevention and treatment of malignancies, such as MPM, that express the SV40 Tag oncoprotein. Combining specific aspects of innate and adaptive immunity by targeting both NK cells and humoral activity against SV40 Tag, respectively, represents a novel and clinically significant immunotherapeutic strategy for potential use in patients.     

A Structure-Guided Mutational Analysis of Simian Virus 40 Large T Antigen: Identification of Surface Residues Required for Viral Replication and Transformation

Simian virus 40 large T antigen (TAg) transforms cells in culture and induces tumors in rodents. Genetic studies suggest that TAg interaction with the chaperone hsp70 and tumor suppressors pRb and p53 may not be sufficient to elicit complete transformation of cells. In order to identify additional cellular factors important for transformation, we designed mutations on the solvent-exposed surface of TAg. We hypothesized that surface residues would interact directly with cellular targets and that the mutation of these residues might disrupt this interaction without perturbing TAg''s global structure. Using structural data, we identified 61 amino acids on the surface of TAg. Each surface amino acid was changed to an alanine. Furthermore, five patches containing clusters of charged amino acids on the surface of TAg were identified. Within these patches, we selectively mutated three to four charged amino acids and thus generated five mutants (patch mutants 1 to 5). We observed that while patch mutants 3 and 4 induced foci in REF52 cells, patch mutants 1 and 2 were deficient in focus formation. We determined that the patch 1 mutant is defective in p53 binding, thus explaining its defect in transformation. The patch 2 mutant can interact with the Rb family members and p53 like wild-type TAg but is unable to transform cells, suggesting that it is defective for action on an unknown cellular target essential for transformation. Our results suggest that the histone acetyltransferase CBP/p300 is one of the potential targets affected by the mutations in patch 2.Simian virus 40 (SV40) large T antigen is a multifunctional protein that is essential for productive viral infection and for cellular transformation (26). T antigen possesses several biochemical activities, some of which map to discrete domains that can act independently and/or coordinately. To effect replication and transformation, T antigen binds to several cellular targets via different domains/regions. For example, during replication, T antigen associates with components of the cellular replication apparatus such as DNA polymerase α, replication protein A, and topoisomerase I (11, 14, 24, 31, 39). Three regions of T antigen are essential to elicit cellular transformation (1, 2, 36). The LXCXE motif mediates binding to the members of the Rb family (pRb, p107, and p130) and in conjunction with the J domain results in the inactivation of the Rb family function. While these domains reside in the N terminus of T antigen, a third transforming function in the C terminus of T antigen is essential for inactivation of the tumor suppressor p53. Genetic studies suggest that inactivation of pRb and p53 is not always sufficient to induce T-antigen-mediated transformation (7, 30, 38), thus indicating the presence of additional targets of T antigen contributing to transformation. In the past few years, several additional targets of T antigen, including CBP/p300, Bub1, Cul7, Fbw7, and IRS-1, have been discovered (8, 9, 12, 17, 29, 40, 42); however, their roles in T-antigen-mediated transformation are not clear. T antigen also targets the DNA-damage-sensing and -processing complex Mre11-Rad50-Nbs1 and may induce genetic instability that contributes to transformation (10, 42). The issue is complicated further by the observation that T antigen has redundant functions, that is, it can act on critical targets via multiple mechanisms (7, 37).One of the key strategies to delineate functions of T antigen required for transformation is the use of amino acid substitution and truncation mutants. However, a caveat to this approach is the production of mutants that are defective in transformation due to a loss of integrity of the secondary, or even local, structure. In this study, we combined available sequence data with structural information to design mutants. Sequence alignments allow the identification of conserved amino acid residues, while structural data provide information about amino acid residues on the surface of the molecule. This approach allows us to combine structural elements and target binding sites. In addition, identification of residues conserved across species, followed by mutation of these conserved residues, will likely yield better insights into common biological pathways. Using this method, we have generated four mutants, of which two are defective in transformation and, thus, of great interest for the identification of novel cellular pathways regulating cell growth and proliferation.     

Binding of p110 Retinoblastoma Protein Inhibits Nuclear Import of Simian Virus SV40 Large Tumor Antigen

Nuclear import of the simian virus 40 large tumor antigen (T-ag) is dependent on its nuclear localization signal (NLS) within amino acids 126–132 that is recognized by the importin α/β1 heterodimer, as well as a protein kinase CK2 site at serine 112 upstream of the NLS, which enhances the interaction ∼50-fold. Here we show for the first time that T-ag nuclear import is negatively regulated by N-terminal sequences (amino acids 102–110), which represent the binding site (BS) for the retinoblastoma (Rb) tumor suppressor protein (p110^Rb). Quantitative confocal laser scanning microscopic analysis of the transport properties of T-ag constructs with or without Rb binding site mutations in living transfected cells or in a reconstituted nuclear transport system indicates that the presence of the RbBS significantly reduces nuclear accumulation of T-ag. A number of approaches, including the analysis of T-ag nuclear import in an isogenic cell pair with and without functional p110^Rb implicate p110^Rb binding as being responsible for the reduced nuclear accumulation, with the Ser¹⁰⁶ phosphorylation site within the RbBS appearing to enhance the inhibitory effect. Immunoprecipitation experiments confirmed association of T-ag and p110^Rb and dependence thereof on negative charge at Ser¹⁰⁶. The involvement of p110^Rb in modulating T-ag nuclear transport has implications for the regulation of nuclear import of other proteins from viruses of medical significance that interact with p110^Rb, and how this may relate to transformation.     

Nonidentity of Some Simian Virus 40-induced Enzymes with Tumor Antigen

The complement-fixing tumor (T) antigen induced by simian virus 40 (SV40) has been prepared from SV40-infected cell cultures, from infected cell cultures treated at the time of infection with 1-beta-d-arabinofuranosylcytosine (ara-C), and from SV40-transformed cells. Upon partial purification, the T antigen exhibited the following properties: it was tightly adsorbed by calcium phosphate gel, it was precipitated by acetic acid at pH 5 or by ammonium sulfate at about 20 to 32% saturation, and it had a molecular weight greater than 250,000, as estimated by Sephadex G-200 gel chromatography. In contrast, deoxycytidylate (dCMP) deaminase, thymidylate (dTMP) kinase, and thymidine (dT) kinase were less strongly bound to calcium phosphate and were not precipitated at pH 5; these enzymes also had much lower molecular weights than the T antigen, as did dihydrofolic (FH(2)) reductase. Furthermore, higher ammonium sulfate concentrations were required to precipitate dCMP deaminase, dTMP kinase, and FH(2) reductase activities than to precipitate the T antigen. Another difference was that the T antigen was not stabilized, but dCMP deaminase, dTMP kinase, and dT kinase, were stabilized, respectively, by dCTP, dTMP, and dT or dTTP. Deoxyribonucleic acid (DNA) polymerase activity resembled the T antigen in adsorption to calcium phosphate, in precipitation by ammonium sulfate or at pH 5, and in the rate of inactivation when incubated at 38 C. However, the polymerase activity could be partly separated from the T antigen by Sephadex G-200 gel chromatography. The cell fraction containing partially purified T antigen also contained a soluble complement-fixing antigen (presumably a subunit of the viral capsid) which reacted with hyperimmune monkey sera. The latter antigen was present in very low titers or absent from cell extracts prepared from SV40-infected monkey kidney cell cultures which had been treated with ara-C at the time of infection, or from SV40-transformed mouse kidney (mKS) or hamster tumor (H-50) cells. The T antigen, however, was present in usual amounts in SV40-transformed cells or ara-C treated, infected cells.     

Superinfection of Simian Virus 40-Transformed Permissive Cells with Simian Virus 40

Evidence that the resistance of simian virus (SV40)-transformed permissive cells to superinfection with SV40 is due to lack of virus uptake is presented. When virus uptake is enhanced, the events of infection proceed as in normal permissive cells, resulting in production of infectious virus.     

Interaction of Simian Virus 40 chromatin with Simian Virus 40 T-antigen.

We have studied the binding of the tumor antigen (T-antigen) of simian virus 40 to simian virus 40 chromatin (minichromosomes). The minichromosomes isolated from infected cells by a modification of standard techniques were relatively free of contaminating RNA and cellular DNA and had a ratio (by weight) of protein to DNA of approximately 1; their DNA was 50 to 60% digestible to an acid-soluble form by staphylococcal nuclease. Cleavage of this chromatin with restriction endonucleases indicated that the nuclease-resistant regions were randomly distributed in the population of minichromosomes, but were not randomly distributed within minichromosomes. Only 20 to 35% of these minichromosomes adsorbed nonspecifically to nitrocellulose filters, permitting binding studies between simian virus 40 T-antigen and chromatin to be performed. Approximately two to three times as much T-antigen was required to bind chromatin as to bind an equivalent amount of free DNA. When T-antigen was present in excess, both chromatin and free DNA were quantitatively retained on the filters. On the other hand, when DNA or chromatin was present in excess, only one-third as much chromatin as DNA was retained. We suggest that T-antigen-chromatin complexes may be formed by the cooperative binding of T-antigen to chromatin, whereas T-antigen-DNA complexes may be formed by simple bimolecular interactions.