Mutational analysis of simian virus 40 small-t antigen.

Several point mutations in the simian virus 40 (SV40) small-t antigen have been analyzed for their effects on protein stability, transformation, transactivation, and binding of two cellular proteins. All mutations which affected cysteine residues in two cysteine clusters produced highly unstable small-t antigens. Four point mutations outside these clusters and one in-frame deletion mutant, dl890, produced stable proteins but reduced transformation efficiency. These were able to transactivate the EII promoter and bind the cellular proteins, suggesting that these activities are not sufficient for small-t-mediated enhancement of transformation.     

Simian virus 40 small-t antigen binds two zinc ions.

Six cysteine residues of the simian virus 40 small-t antigen (small-t) are important for stability of the protein. Stability has been shown to be related to the ability of small-t to bind zinc ions in vitro. Purified small-t expressed either in bacteria or from baculovirus vectors binds two molecules of zinc per molecule of protein. Thus, small-t may resemble GAL4, which contains a Zn(II)2Cys6 binuclear cluster.     

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.     

An antibody to a synthetic peptide recognizes polyomavirus middle-T antigen and reveals multiple in vitro tyrosine phosphorylation sites.

Antibodies were raised against three synthetic peptides corresponding to sequences surrounding tyrosine 315, a putative in vitro phosphorylation site in polyomavirus middle-T antigen. Only one of the peptides (called C and corresponding to residues 311 to 330) elicited antibodies that recognized middle-T efficiently. Middle-T present in immunoprecipitates formed with purified anti-C serum still accepted phosphate on tyrosine in an in vitro kinase reaction. This implies that tyrosines other than 315 and 322 that lie within the antibody binding region are phosphorylated under these conditions. This conclusion was supported by the altered partial V8 proteolysis fingerprint of the labeled middle-T. Two-dimensional tryptic fingerprint analysis of 32P-labeled middle-T showed that several tryptic peptides identified as including tyrosine 315 and 322 were missing from middle-T labeled in anti-C immunoprecipitates compared with middle-T labeled in immunoprecipitates made by using anti-tumor cell serum. However, one major labeled peptide remained. This peptide was also present in fingerprints of 32P-labeled middle-T coded by M45, dl23, pAS131, and dl1013, but a peptide with altered mobility was present in dl8 middle-T. This identified the peptide as including tyrosine 250. We deduce from these data that (i) the presence of the antibody against peptide C inhibits phosphorylation of tyrosines 315 and 322; (ii) middle-T labeled in the kinase reaction after immunoprecipitation with anti-C serum is phosphorylated on tyrosine 250; and (iii) when anti-tumor cell serum is used in the in vitro kinase reaction, middle-T is phosphorylated at multiple sites, including residues 250, 315, and 322.     

Presence in growth-arrested cells of cellular proteins that interact with simian virus 40 small-t antigen.

Two cellular proteins, 56K and 32K, found in association with simian virus 40 small-t antigen were not induced by viral infection. In addition, the proteins were expressed by cells in the growth arrest period, a time in which small-t function is of importance in infection and transformation.     

SV40 T-antigen is a histocompatibility antigen of SV40-transgenic mice

Although the extensive family of non-H-2 histocompatibility (H) antigens provides a formidable barrier to transplantation, the origin of their encoding genes are unknown. Recent studies have demonstrated both the linkage between H genes and retroviral sequences and the ability of integrated Moloney-murine leukemia virus to encode what is operationally defined as a non-H-2 H antigen. The experiments described in this communication reveal that skin grafts from an SV40 T-antigen transgenic C57BL/6 mouse strain are rejected by coisogenic C57BL/6 recipients with a median survival time of 49 days, which is comparable to those of many previously defined non-H-2 H antigens. The specificity of this response for SV40 T-antigen was demonstrated by the identification of SV40 T-antigen-specific cytolytic T lymphocytes and antibodies in multiply-grafted recipients. Although these cytolytic T lymphocytes could detect SV40 T-antigen on syngeneic SV40-transformed fibroblasts, they neither could be stimulated by splenic lymphocytes from T-antigen transgenics nor could they lyse lymphoblast targets from T-antigen transgenics. These observations suggest a limited tissue distribution of SV40 T-antigen in these transgenics. These results confirm the role of viral genes in the determination of non-H-2 histocompatibility antigenes by the strict criteria that such antigenes stimulate (1) tissue graft rejection and (2) generation of cytolytic T lymphocytes. Furthermore, they suggest that the SV40 enhancer and promoter region can target expression of SV-40 T-antigen to skin cells of transgenic animals.     

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.     

High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda pL promoter

Several plasmids were constructed in which the SV40 small-t antigen gene was inserted in close proximity downstream from the thermoinducible leftward promoter (pL) of bacteriophage lambda. Upon temperature induction the best of our constructions expressed a small-t-related 19 000-dalton polypeptide in an amount corresponding to approx. 2.5% of total de novo protein synthesis. This 19 000-dalton protein was identified as small-t by specific immunoprecipitation with anti-T serum and by two-dimensional fingerprint analysis. In addition to the 19 000-dalton product, representative plasmids expressed fairly large amounts (up to 7% of total de novo protein synthesis) of a protein with an apparent Mr of 14 500. This 14 500-dalton polypeptide was shown to be related to authentic small-t. Presumably the secondary structure of the mRNA starting at pL is such that translation initiation at an internal AUG codon of the small-t gene is favored over initiation at the true initiating codon.     

Simian virus 40 (SV40) small t antigen inhibits SV40 DNA replication in vitro.

We describe a biochemical function of simian virus 40 small t antigen, the inhibition of simian virus 40 large T antigen-mediated viral DNA replication in an in vitro replication system. Our results suggest that in this system, small t antigen prevents protein phosphatase 2A-mediated activation of large T 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.     

SV40 T antigen and the exocytotic pathway.

A chimeric gene consisting of DNA coding for the 15-amino acid signal peptide of influenza virus hemagglutinin and the C-terminal 694 amino acids of SV40 large T antigen was inserted into a bovine papilloma virus (BPV) expression vector and introduced into NIH-3T3 cells. Cell lines were obtained that express high levels (approximately 5 X 10(6) molecules/cell) of the chimeric protein (HA-T antigen). The biochemical properties and intracellular localization of HA-T antigens were compared with those of wild-type T antigen. Wild-type T antigen. Wild-type T antigen is located chiefly in the cell nucleus, although a small fraction is detected on the cell surface. By contrast, HA-T antigen is found exclusively in the endoplasmic reticulum (ER). During biosynthesis, HA-T antigen is co-translationally translocated across the membrane of the ER, the signal peptide is cleaved and a mannose-rich oligosaccharide is attached to the polypeptide (T antigen contains one potential N-linked glycosylation site at Asn154). HA-T antigen does not become terminally glycosylated or acylated and little or none reaches the cell surface. These results suggest that T antigen is incapable of being transported along the exocytotic pathway. To explain the presence of wild-type T antigen on the surface of SV40-transformed cells, an alternative route is proposed involving transport of T antigen from the nucleus to the cell surface.     

Expression of SV40 tumor antigen in SV40 transformed teratocarcinoma-derived cell lines

The relative importance of viral tumor antigen expression and the cellular background in the maintenance of a transformation phenotype was examined in five SV40-transformed teratocarcinoma-derived cell lines. These cell lines show qualitative differences in growth characteristics associated with transformation, and vary in their state of differentiation. Viral T antigen expression was evaluated by two criteria: 1) the amount of immunoprecipitated antigen in growing cells, and 2) the amount and rate of antigen synthesis in density-inhibited cells. There was no direct correlation found between retention, or rate of synthesis, of the viral T antigen and the degree of transformation. These findings imply that the cellular environment has a more important influence on the growth properties of a stably transformed cell than the quantitative levels of viral T antigen expression.     

An N-terminal deletion mutant of simian virus 40 (SV40) large T antigen oligomerizes incorrectly on SV40 DNA but retains the ability to bind to DNA polymerase alpha and replicate SV40 DNA in vitro.

A peptide encompassing the N-terminal 82 amino acids of simian virus 40 (SV40) large T antigen was previously shown to bind to the large subunit of DNA polymerase alpha-primase (I. Dornreiter, A. Höss, A. K. Arthur, and E. Fanning, EMBO J. 9:3329-3336, 1990). We report here that a mutant T antigen, T83-708, lacking residues 2 to 82 retained the ability to bind to DNA polymerase alpha-primase, implying that it carries a second binding site for DNA polymerase alpha-primase. The mutant protein also retained ATPase, helicase, and SV40 origin DNA-binding activity. However, its SV40 DNA replication activity in vitro was reduced compared with that of wild-type protein. The reduction in replication activity was accompanied by a lower DNA-binding affinity to SV40 origin sequences and aberrant oligomerization on viral origin DNA. Thus, the first 82 residues of SV40 T antigen are not strictly required for its interaction with DNA polymerase alpha-primase or for DNA replication function but may play a role in correct hexamer assembly and efficient DNA binding at the origin.     

Topoisomerase activity associated with SV40 large tumor antigen.

Purified preparations of simian virus 40 (SV40) large tumor antigen (LT) from three different sources, including LT expressed from a recombinant baculovirus, were found to relax negatively supercoiled cyclic DNA molecules, whether or not they contained SV40 sequences. Relaxation was stimulated by MgCl2 but not by ATP, and inhibited by camptothecin, suggesting the involvement of an enzymatic activity similar to that of topoisomerase I (topo I). However, the pH requirements for relaxation by respectively LT and topo I are different. Also, antibodies reacting with LT inhibited relaxation by preparations of LT but not topo I, whereas antibodies inhibiting relaxation by topo I had no effect on relaxation by LT. Reconstruction experiments suggested that both procedures used to purify LT, immunoaffinity chromatography and DEAE-Sepharose chromatography, separate topo I from LT. Finally, relaxing activity was found in over 40 preparations of LT, and in the few instances where activity could not be found, it probably had been lost during storage, rather than absent from the start. Whereas these results seem to exclude that the activity being detected is that of a contaminant of LT, they would be consistent with this activity being that of a stable topo-LT complex, or else intrinsic to LT itself.