SV40 T antigen acts as a minor histocompatibility antigen of SV40 T antigen tolerant transgenic mice

The ability of normal mice to mount an SV40 T antigen-specific cytolytic T lymphocytes response when immunized in vivo with splenocytes from the SV40 T antigen transgenic 427-line mice and restimulated in vitro with SV40-transformed fibroblasts, or when immunized with SV40 and restimulated with 427-line splenocytes, was analyzed. Both immunization schemes resulted in an SV40 T antigen-specific immune response, indicating the presence of SV40 T antigen-positive cells in the spleens of these transgenic mice. Normal mice engrafted with skin from 427 donors showed no rejection of the graft. Thus, SV40 T antigen in transgenic 427-line mice is expressed on an undefined cell type in the spleen and acts as a tissue-specific minor histocompatibility antigen.     

c-myc protein can be substituted for SV40 T antigen in SV40 DNA replication.

Replicating activity of SV40 origin-containing plasmid was tested in human cells as well as in monkey CosI cells. All the plasmids possessing SV40 ori sequences could replicate, even in the absence of SV40 T antigen, in human HL-60 and Raji cells which are expressing c-myc gene at high level. The copy numbers of the replicated plasmids in these human cells were 1/100 as high as in monkey CosI cells which express SV40 T antigen constitutively. Exactly the same plasmids as the transfected original ones were recovered from the Hirt supernatant of the transfected HL-60 cells. Furthermore, replication of the SV40 ori-containing plasmids in HL-60 cells was inhibited by anti-c-myc antibody co-transfected into the cells. These results indicate that the c-myc protein can be substituted for SV40 T antigen in SV40 DNA replication.     

An immune complex assay for SV40 T antigen.

The two forms of ferredoxin from $\text{Desulfovibrio gigas}$, Fd I and Fd II, are studied by differential pulse polarography 1. Fd I and Fd II give one well defined peak corresponding to $\text{E}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= ?0.33}\text{and}\text{?0.35}$ V (vs. the hydrogen electrode) respectively, at c > 5 μM. The influence of the concentration on the peak potentials Ep and the peak heights i_p is examined. The denaturation of the two forms of ferredoxin is studied by polarography in conjunction with UV spectrophotometry. Two new peaks at negative potentials before the reduction of the solvent are observed in denaturated proteins.     

Partial purification and characterization of the SV40 T antigen

Multiple SV40-infected cell lines and induced tumors have been assayed for their content of T antigen. SV80, a human cell line, contains superior levels of this antigen. The SV80 T antigen has been partially purified by salt fractionation and adsorbent chromatography; it exists in the form of a high molecular weight aggregate in nondenaturing solvents. The antigen binds to phosphocellulose but not carboxymethylcellulose at pH 6.2, suggesting that it may have specific affinity for phosphate ligands but is not a basic protein. This property may reflect an underlying function of this protein in the cell.     

Topoisomerase activity is associated with purified SV40 T antigen.

Purified SV40 T antigen has been assayed for topoisomerase activity. The ability to relax negatively-supercoiled SV40 DNA was found in preparations of T antigen purified either from human 293 cells infected with Ad5-SVR111 virus or from insect Sf9 cells infected with recombinant baculovirus 941T. The T antigen-associated relaxing activity was stimulated by MgCl2 and was not dependent on ATP, suggesting that it is not due to cellular topoisomerase II. The topoisomerase activity was immunoprecipitated by a monoclonal antibody specific for T antigen, but not by a control monoclonal antibody. In addition, immunoblotting of purified T antigen from human 293 cells with antihuman topoisomerase I and anti-human topoisomerase II antibodies failed to detect cellular topoisomerases I or II. Sedimentation analysis of purified T antigen revealed that the topoisomerase activity co-sedimented with the hexameric form of T antigen at 23S. The topoisomerase activity is, therefore, either inherent to T antigen or due to a cellular topoisomerase I tightly bound to, and co-purifying with, T antigen.     

Interactions between SV40 T antigen and DNA polymerase alpha

Simian virus 40 large T antigen is the only viral protein required for SV40 DNA synthesis in vivo and in vitro. This complex protein recruits the cellular DNA replication apparatus to the SV40 origin and provides a good model for the initiation of cellular DNA replication. The interaction between SV40 large T antigen (TAg) and DNA polymerase alpha has been shown previously to be inhibited by murine p53, the nuclear protein product of a cellular anti-oncogene. The murine p53 protein will inhibit SV40 replication both in vivo and in vitro. Using monoclonal antibodies to TAg, p53, and polymerase alpha, we developed immunoassays to measure the complexes formed between TAg and polymerase alpha and between TAg and p53. The assays allowed us to detect the TAg-polymerase alpha and TAg-p53 complexes in lytically infected and transformed cells. The amount of TAg complexed to p53 was far lower in infected cells than in transformed cells. We used a large range of monoclonal antibodies to different sites on T antigen and found that antibodies that inhibited the formation of the TAg-polymerase alpha complex also inhibited the formation of the TAg-p53 complex. Finally, we found that the tsA58 and 5080 point mutations in TAg, previously shown to inhibit the binding of TAg to p53, also inhibit its binding to polymerase alpha. Together these results emphasize the specificity and functional importance of the TAg-polymerase alpha complex. The disruption of this interaction by the cellular anti-oncogene p53 provides an interesting model for the normal action of p53 and the effects of its removal on the regulation of cellular DNA synthesis.     

RNA unwinding activity of SV40 large T antigen

Large T antigen, the regulatory protein encoded by simian virus 40, has DNA helicase activity and unwinds double-stranded DNA at the expense of ATP. T antigen also functions as an RNA helicase separating duplex regions in partially double-stranded RNA substrates. Surprisingly, T antigen RNA helicase activity requires UTP, CTP, or GTP as a cofactor, whereas ATP is an inefficient energy source for the RNA unwinding reaction. Accordingly, T antigen has both an intrinsic non-ATP NTPase activity that is stimulated by single-stranded RNA and an ATPase activity stimulated by single-stranded DNA. Thus, it appears that the bound nucleotide determines whether T antigen acts as an RNA helicase or as a DNA helicase.     

SV40 large tumor antigen (T antigen): database of mutants.

The SV40 T antigen database (http://www.pitt.edu/pipaslab/) lists viruses and plasmids expressing mutant forms of large T antigen. Each entry contains information regarding the mutant designation, mutant type, virus strain, nucleotide change, amino acid change and pertinent references. The database is now available as an internet searchable index.     

Activation of the human epsilon- and beta-globin promoters by SV40 T antigen.

We have studied the effect of the SV40 T antigen on expression from human globin promoters fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and compared its effect with the SV40 enhancer and the adenovirus E1A protein. We have observed that expression of p epsilon GLCAT and p beta GLCAT (the epsilon-globin or beta-globin promoter linked to the CAT gene) was significantly stimulated when cotransfected with a cloned T antigen plasmid into CV-1 cells, indicating that trans-activation of the globin promoters was mediated by SV40 T antigen. Transfection of the p beta GLCAT-SV (p beta GLCAT containing the SV40 enhancer element) into CV-1 cells resulted in a 50-60-fold increase in CAT activity as compared to p beta GLCAT (no enhancer). However, cotransfection of the p beta GLCAT-SV with the cloned T antigen resulted in an additional increase of CAT expression, which suggests that T antigen and the SV40 enhancer activate globin gene expression independently. We found that T antigen but not E1A could further stimulate the expression of an enhancer-containing plasmid in CV-1 cells; whereas E1A but not T antigen could further stimulate p epsilon GLCAT expression in COS-1 cells which constitutively express the SV40 T antigen. These results suggest that T antigen and E1A also act independently. Deletion analysis showed that the minimum sequence required for a detectable level of stimulation of the epsilon-globin promoter by T antigen is 177 bp 5' to the cap site, suggesting that the target sequences for response to T antigen do not reside in the canonical 100 bp promoter region, but rather reside in sequences further upstream, and therefore the cellular factors interacting with T antigen are not the TATA or CAT box binding proteins, but the proteins interacting with upstream regulatory sequences.     

Insulin-induced mitogenesis associated with transformation by the SV40 large T antigen.

Simian virus 40 (SV40) large T antigen-transformed cells typically show a markedly reduced serum requirement for growth and the inability to growth arrest and differentiate. An SV40 large T antigen-transformed 3T3 T cell line, CSV3-1, that can growth arrest and differentiate into adipocytes with high efficiency has, however, recently been described (Scott et al: Proc. Natl. Acad. Sci. U.S.A. 86:1652-1656, 1989; Estervig et al: J. Virol. 63:2718-2725, 1989; J. Cell. Physiol. 142:552-558, 1990). The results of the current studies using these cells show that whereas quiescent 3T3 T cells show no mitogenic response to insulin, quiescent CSV3-1 cells show a highly significant insulin-induced mitogenic responsiveness in the absence of other added growth factors. Maximum mitogenesis was observed at an insulin concentration of 1 microgram/ml, which induced 40-70% of the cells to undergo DNA synthesis within 48 hours. The half maximum response was achieved with 1-10 ng/ml of insulin. Insulin's mitogenic effect on CSV3-1 cells was evident under several different culture conditions that induce quiescence and was not mediated by any detectable autocrine growth factors that might make CSV3-1 cells competent to respond to insulin. In CSV3-1 cells insulin appears to act on its own receptor rather than on the IGF-1 receptor, because at comparable dosages IGF-1 is 10- to 100-fold less effective than insulin. Insulin also is shown to be a mitogen for another SV40-transformed cell line, CSV3-35, which can be growth arrested; in contrast insulin has no mitogenic effect on two control cell lines that are stably transfected with pSV2neo, a plasmid containing SV40 early promoter/enhancer but lacking large T antigen gene: These results suggest a significant relationship between SV40 T antigen-associated transformation and the expression of mitogenic responsiveness to insulin.     

T antigen and template requirements for SV40 DNA replication in vitro.

A cell-free system for replication of SV40 DNA was used to assess the effect of mutations altering either the SV40 origin of DNA replication or the virus-encoded large tumor (T) antigen. Plasmid DNAs containing various portions of the SV40 genome that surround the origin of DNA replication support efficient DNA synthesis in vitro and in vivo. Deletion of DNA sequences adjacent to the binding sites for T antigen either reduce or prevent DNA synthesis. This analysis shows that sequences that had been previously defined by studies in vivo to constitute the minimal core origin sequences are also necessary for DNA synthesis in vitro. Five mutant T antigens containing amino acid substitutions that affect SV40 replication have been purified and their in vitro properties compared with the purified wild-type protein. One protein is completely defective in the ATPase activity of T antigen, but still binds to the origin sequences. Three altered proteins are defective in their ability to bind to origin DNA, but retain ATPase activity. Finally, one of the altered T antigens binds to origin sequences and contains ATPase activity and thus appears like wild-type for these functions. All five proteins fail to support SV40 DNA replication in vitro. Interestingly, in mixing experiments, all five proteins efficiently compete with the wild-type protein and reduce the amount of DNA replication. These data suggest that an additional function of T antigen other than origin binding or ATPase activity, is required for initiation of DNA replication.     

Purification of the simian virus 40 (SV40) T antigen DNA-binding domain and characterization of its interactions with the SV40 origin.

To better define protein-DNA interactions at a eukaryotic origin, the domain of simian virus 40 (SV40) large T antigen that specifically interacts with the SV40 origin has been purified and its binding to DNA has been characterized. Evidence is presented that the affinity of the purified T antigen DNA-binding domain for the SV40 origin is comparable to that of the full-length T antigen. Furthermore, stable binding of the T antigen DNA-binding domain to the SV40 origin requires pairs of pentanucleotide recognition sites separated by approximately one turn of a DNA double helix and positioned in a head-to-head orientation. Although two pairs of pentanucleotides are present in the SV40 origin, footprinting and band shift experiments indicate that binding is limited to dimer formation on a single pair of pentanucleotides. Finally, it is demonstrated that the T antigen DNA-binding domain interacts poorly with single-stranded DNA.     

Regulation of SV40 DNA replication by phosphorylation of T antigen.

The role of phosphorylation in regulating the biochemical properties of SV40 large T antigen has been examined. Treatment of purified T antigen with calf intestinal alkaline phosphatase resulted in the removal of 80% of the 32P label. This partially dephosphorylated T antigen displayed an increase in its ability to support DNA replication in vitro. This increase in replication activity was paralleled by an activation of specific DNA binding to site II, a necessary element within the origin of SV40 DNA replication. In contrast, the ATPase activity of dephosphorylated T antigen remained unchanged. These results demonstrate that DNA replication is regulated by phosphorylation of an origin specific DNA binding protein.     

Phenotypic complementation of the SV40 tsA mutant defect in viral DNA synthesis following microinjection of SV40 T antigen.

African green monkey cells (CV-1P) were microinjected with highly purified SV40 T antigen using protein-loaded red cell ghosts and polyethylene glycol as fusagen. The microinjected cells were infected with a temperature-sensitive mutant of SV40 (tsA209) which is defective in the initiation of viral DNA synthesis. Using in situ hybridization as an assay method, we found that PEG-microinjection of both partially and highly purified T antigen resulted in an increase in the amount of viral DNA sequences in the monolayer. Moreover, 3H-thymidine-labeled and unlabeled Hirt supernatant from microinjected, tsA209-injected cells contained significantly more SV40 DNA than comparable extracts from sham-injected, tsA209-infected or uninfected cells, which were tested in parallel. Thus the introduction of highly purified, "large" SV40 T antigen led to phenotypic complementation of the tsA defect in viral DNA synthesis.