Wolfgang Deppert

Different structural systems of the nucleus are targets for SV40 large T antigen

To define the interaction of SV40 large T with different structural systems in the nuclei of SV40-transformed cells (BALB/c mKSA), we have employed an in situ cell fractionation procedure leading to the preparation of the nuclear matrix, and giving rise to defined nuclear extracts comprising soluble nuclear proteins (nucleoplasm) and the solubilized chromatin. Large T could be detected in the nucleoplasmic fraction and in the chromatin fraction, as well as in tight association with the nuclear matrix. From the nuclear matrix, large T could be solubilized by treatment with a zwitterionic detergent. Different solubility properties, differences in the amount of the cellular phosphoprotein p53 coprecipitating with large T, and different stabilities in its association with the nuclear structural systems indicate that distinct subclasses of large T were isolated from their in vivo location in SV40-transformed cells.

Intermediate filament systems are collapsed onto the nuclear surface after isolation of nuclei from tissue culture cells.

Abstract Standard procedures for the biochemical isolation and purification of tissue culture cell nuclei are very similar to recently described methods for the preparation of detergent-resistant cytoskeletons. But the latter in addition to extracted nuclei contain cytoplasmic filament systems. We, therefore, investigated the distribution of these filamentous systems during nuclear isolation both by immunofluorescence microscopy and by SDS-gel electrophoresis. The intermediate filaments copurify with each single isolated nucleus. The majority of the filaments is collapsed onto isolated nuclei and still constitutes a filamentous system. This can be shown by partially unfolding the collapsed filament systems.

SV40-transformed cells express SV40 T antigen-related antigens on the cell surface

Abstract SV40 tumor antigen (T-Ag)-related antigens were detected serologically on the surface (surface T) of living SV40-transformed human and mouse monolayer cells by an 125 I-protein A binding assay. In immunofluorescence analysis, these cells were negative for surface T. However, on mKSA, a SV40-transformed mouse cell line grown in suspension or on SV40-transformed human and mouse monolayer cells put into suspension, surface T could be visualized by immunofluorescence microscopy. The antisera used in these experiments were raised in rabbits with purified, SDS-denatured SV40 T-Ag or came from hamsters bearing SV40 tumors. Both types of antisera had in common high titers against SV40 T-Ag (⩾1:1000). All these antisera were negative on normal cells or on polyoma virus-transformed cells. The specificity of both antisera for SV40 T-Ag-related binding sites on the surface of SV40-transformed cells were demonstrated by an 125 I-IgG blocking assay in which preincubation of the cells with rabbit anti-T-Ag serum inhibited the binding of hamster SV40 tumor serum to the cell surface by about 85%. These results demonstrate the expression of T-Ag-related antigens on the surface of living cells and, therefore, support the hypothesis that SV40 T-Ag-related antigens participate in the formation of the SV40-specific tumor transplantation antigen (TSTA).

Acylation: A new post-translational modification specific for plasma membrane-associated simian virus 40 large T-antigen

SV40 transformed mouse cells (mKSA) were labeled in parallel with either [35S]methionine or [3H]palmitate and subfractionated. Nuclear extracts and solubilized plasma membranes were analyzed for the presence of either 35S‐ or 3H‐labeled SV40 large tumor antigen by immunoprecipitation and SDS polyacrylamide gel electrophoresis. The majority of the [35S]methionine labeled large T was recovered from the nuclear fraction, only minor amounts were detected in plasma membranes. In contrast, large T labeled specifically with [3H]palmitate was found only in the plasma membrane fraction. Our results demonstrate a specific acylation of large T associated with plasma membranes, suggesting that the membrane location of this predominantly nuclear protein is specific.

Domains of simian virus 40 large T-antigen exposed on the cell surface

Abstract The orientation of SV40 large T on the surface of SV40-transformed mouse cells and of human cells infected with nondefective adenovirus 2 SV40 hybrid viruses has been studied. Using antibodies against a synthetic peptide corresponding to a region of 11 amino acids at the carboxyterminus of large T, the surface of formaldehyde-fixed SV40-transformed cells could be specifically stained by indirect immune fluorescence. Staining was inhibited by an excess of the peptide. These data suggest that the carboxyterminus of large T is exposed on the surface of formaldehyde-fixed cells. Antibodies against the carboxyterminus of large T also stained the surface of cells infected with the hybrid viruses Ad2 + ND1, Ad2 + ND2, and Ad2 + ND4. Thus, the carboxytermini of the SV40-specific proteins synthesized in hybrid virus-infected cells are also exposed on the cell surface. When analyzed with an antiserum against purified denatured large T, which among many other determinants also recognizes the large T carboxyterminus, surface fluorescence was observed in cells infected by all three hybridviruses. The surface fluorescence of Ad2 + ND1-infected cells, expressing an SV40-specific protein of 28 K, and Ad2 + ND2-infected cells expressing SV40-specific proteins of 42 K and 56 K molecular weight, was completely inhibited by carboxyterminal peptide. However, the surface fluorescence of Ad2 + ND4-infected cells, expressing SV40-specific proteins up to nearly full size large T, was unaffected by carboxyterminal peptide. Our data suggest that a major portion of large T, located between a region near the carboxyterminus and a region corresponding to the aminoterminus of the 56 K protein, is not exposed on the surface of hybridvirus-infected cells. However, some parts of the aminoterminal one-third of large T appear to be exposed again. We conclude that SV40 large T on the surface of SV40-transformed cells is oriented in a specific manner, suggesting that it is specifically associated with the plasma membrane.

A new sensitive target-bound DNA binding assay for SV40 large T antigen.

We have developed a new sensitive target-bound DNA binding assay (TB assay) for SV40 large T antigen (large T). The major advantage of this assay is that in contrast to commonly used DNA binding assays, DNA binding is not performed in large T extracts, but instead is performed with immunopurified target-bound large T. Thereby interference of cellular components present in large T extracts is avoided. Thus the TB assay allows DNA binding analysis of large T from different sources (extracts, cell lines) under standardized conditions. Large T is first immunopurified with an anti-T monoclonal antibody not interfering with DNA binding and protein A-Sepharose. Then SV40 DNA is added to the large T immune complex. For analysis of bound DNA and large T, we developed a two-step elution procedure by which bound DNA and large T in the immune complex can be analyzed separately and which allows the determination of the actual amounts of bound DNA and large T. Binding data obtained with the TB assay allowed us to determine an equilibrium dissociation constant (Kd). As a further application of this assay, we analyzed the ORI binding of SVR9D mutant large T which has been reported to exhibit no ORI binding activity. We found that a small percentage of SVR9D large T binds specifically to the SV40 ORI.

Specific complex of the late nonstructural 100,000-Dalton protein with newly synthesized hexon in adenovirus type 2-infected cells

Analysis of cellular extracts of HeLa cells infected with adenovirus type 2 (Ad2) by immunoprecipitation with antiserum against the late nonstructural 100,000-dalton (100K) protein revealed the presence of a specific complex between the 100K protein and newly synthesized hexon molecules. Serological analysis of the hexon molecule in the 100K/hexon complex with antibodies specific for hexon monomers or trimers showed that only monomeric hexon molecules were associated with the 100K protein. By immunofluorescence microscopy this monomeric hexon was primarily found in the cytoplasm, whereas the trimeric form was mainly confined to the nucleus of infected cells. We conclude that in the cytoplasm of Ad2-infected cells newly synthesized, monomeric hexon molecules can interact with the 100K protein. This suggests that the 100K protein may play some role either in trimerization of newly synthesized, monomeric hexon molecules and/or in its transport from the cytoplasm into the nucleus.

Detection of simian virus 40 T-antigen-related antigens by a 125I-protein A-binding assay and by immunofluorescence microscopy on the surface of SV40-transformed monolayer cells.

Simian virus 40 (SV40)-transformed cells express the SV40-specific tumour transplantation antigen (TSTA) on the cell surface and the SV40-coded tumour antigen in their nuclei. TSTA is defined by SV40-specific transplantation immunity, whereas T-antigen (T-Ag) can be detected serologically by indirect immunofluorescence. Both antigens, however, are derived from the A gene of SV40. We therefore analysed SV40-transformed cells for the presence of serologically detectable T-Ag-related molecules. Such antigens could not be detected on the surface of living SV40-transformed cells in monolayers. However, after a short formaldehyde fixation it was possible to stain the cell surfaces of SV40-transformed cells with sera from rabbits immunized with purified SDS-denatured T-Ag, but not with sera from hamsters bearing SV40-induced tumours. T-Ag-related antigens could be detected with both types of antisera by applying a more sensitive 125I-protein A assay. The T-Ag specificity of the binding of hamster SV40 tumour sera was demonstrated be a 125I-IgG-blocking assay in which preincubation of formaldehyde-fixed SV40-transformed cells with rabbiet anti-SDS-T-Ag serum inhibited the binding of hamster SV40 tumour serum by about 70%. The localization of T-Ag-related antigens on the outside of plasma membranes of formaldehyde-fixed cells was shown by an anti-SDS-T-Ag serum-specific binding of fluorescein isothiocyanate-labelled Staphylococcus aureus to the cell surface. Out results are consistent with the hypothesis that SV40 T-Ag-related antigens are involved in the formation of TSTA.

Analysis of mechanisms controlling the interactions of SV40 large T antigen with the SV40 ORI region

We have characterized the interactions of simian virus 40 (SV40) large tumor antigen (large T) with the control region of the SV40 genome, the SV40 ORI, by analyzing the specific binding of large T antigen to SV40 wild-type origin DNA and to isolated binding sites I and II, respectively. DNA binding affinities of large T antigen were determined under standardized conditions and DNA excess, using a target-bound DNA binding assay (M. Hinzpeter, E. Fanning, and W. Deppert, 1986, Virology 148, 159-167). Our results show that large T antigen exhibits similar affinities for isolated binding sites I and II and for combined sites I and II on wild-type ORI DNA. When the fraction of large T antigen molecules (calculated per large T antigen monomers) able to bind specifically to these sites was determined (DNA binding activity of large T antigen) we found that only 2% of large T antigen molecules present in extracts of lytically infected cells were able to bind to isolated site II, whereas about 50% bound to isolated site I. However, only about 10% of large T antigen molecules bound to the complete wild-type ORI, containing combined binding sites I and II. Thus, a much larger proportion of large T antigen molecules is capable of binding specifically to site I as is suggested by analysis of large T antigen binding to combined sites I and II on the SV40 wild-type ORI. These findings indicate that the interaction of large T antigen with the SV40 wild-type ORI is restricted on one hand by the ability of large T antigen to bind to site II, and on the other hand by the spatial arrangement of binding sites I and II on the SV40 wild-type ORI.

Acylated simian virus 40-specific proteins in the plasma membrane of HeLa cellsinfected with adenovirus 2-simian virus 40 hybrid virus Ad2+ND2

HeLa cells infected with the adenovirus 2-simian virus 40 (Ad2+SV40) hybrid virus Ad2+ND2 were labeled with either [35S]methionine or [3H]palmitate and fractionated into cytoplasmic, nuclear, and plasma membrane fractions. Analysis of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the SV40-specific proteins in the plasma membrane fraction were specificially acylated.