Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis.

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T antigen lacks those residues known to be required for adenovirus helper function, and the block to productive infection by dlA2459 occurs at the same stage of infection as the block to productive adenovirus infection of CV-1 cells. These results suggest that the adenovirus helper function is required for productive infection by simian virus 40. Mutant dlA2459 was able to grow on the Vero and BSC-1 lines of African green monkey kidney cells. Additional mutants affecting the carboxyl-terminal portion of large T were prepared. Mutant inv2408 contains an inversion of the DNA between the BamHI and BclI sites (0.144 to 0.189 map units). This inversion causes transposition of the carboxyl-terminal 26 amino acids of large T antigen and the carboxyl-terminal 18 amino acids of VP1. This mutant was viable, even though the essential information absent from dlA2459 large T antigen has been transferred to the carboxyl terminus of VP1 of inv2408. The VP1 polypeptide carrying this carboxyl-terminal portion of large T could overcome the defect of dlA2459. This indicates that the carboxyl terminus of large T antigen is a separate and separable functional domain.     

Different regions of primase subunit p48 control mouse polyomavirus and simian virus 40 DNA replication in vitro

DNA polymerase alpha-primase (pol-prim), a complex consisting of four subunits, is the major species-specific factor for mouse polyomavirus (PyV) and simian virus 40 (SV40) DNA replication. Although p48 is the most conserved subunit of pol-prim, it is required for in vitro PyV DNA replication but can inhibit cell-free SV40 DNA replication. Production of chimeric human-mouse p48 revealed that different regions of p48 are involved in supporting PyV DNA replication and inhibiting SV40 DNA replication. The N and C-terminal parts of p48 do not have species-specific functions in cell-free PyV DNA replication, but the central part (amino acids [aa] 129 to 320) controls PyV DNA replication in vitro. However, PyV T antigen physically binds to mouse, human, and chimeric pol-prim complexes independently, whether they support PyV DNA replication or not. In contrast to the PyV system, the inhibitory effects of mouse p48 on SV40 DNA replication are mediated by N- and C-terminal regions of p48. Thus, a chimeric p48 containing human aa 1 to 128, mouse aa 129 to 320, and human aa 321 to 418 is active in both PyV and SV40 DNA replication in vitro.     

Discrete regions of simian virus 40 large T antigen are required for nonspecific and viral origin-specific DNA binding.

The nondefective adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses, Ad2+ND2 and Ad2+ND4, have been used to determine which regions of the SV40 genome coding for the large tumor (T) antigen are involved in specific and nonspecific DNA binding. Ad2+ND2 encodes 45,000 M4 (45K) and 56,000 Mr (56K) T antigen-related polypeptides. The 45K polypeptide did not bind to DNA, but the 56K polypeptide bound nonspecifically to calf thymus DNA, Ad2+ND4 encodes 50,000 Mr (60K), 66,000 Mr (66K), 70,000 Mr (70K), 74,000 Mr (74K), and 90,000 Mr (90K) T antigen-related polypeptides, all of which bound nonspecifically to calf thymus DNA. However, in more stringent assays, where tight binding to viral origin sequences was tested, only the 90K protein specified by Ad2A+ND4 showed specific high affinity for sequences at the viral origin of replication. From these results and previously published experiments describing the SV40 DNA integrated into these hybrid viruses, it was concluded that SV40 early gene sequences located between 0.39 and 0.44 SV40 map units contribute to nonspecific DNA binding, whereas sequences located between 0.50 and 0.63 SV40 map units are necessary for specific binding to the viral origin of replication.     

Identification of a region of simian virus 40 large T antigen required for cell transformation.

A series of replication-competent simian virus 40 (SV40) large T antigens with point and deletion mutations in the amino acid sequence between residues 105 and 115 were examined for the ability to immortalize primary cultures of mouse and rat cells. The results show that certain mutants, including one that deletes the entire region, are able to immortalize. However, consistent with previous data, the immortalized cells are not fully transformed, as judged by doubling time, sensitivity to concentrations of serum, and anchorage-independent growth. The region from 106 to 114 has structural features in common with a region involved in transformation by adenovirus E1a protein (J. Figge, T. Webster, T.F. Smith, and E. Paucha, J. Virol. 62:1814-1818, 1988) and influences the binding of the retinoblastoma gene product to large T (J.A. DeCaprio, J.W. Ludlow, J. Figge, J.-Y. Shew, C.-M. Huang, W.-H. Lee, E. Marsilio, E. Paucha, and D.M. Livingston, Cell 54:275-283, 1988). Together, these results imply that the sequence from 106 to 114 forms part of a domain that is essential for transformation of established cells, is dispensable for immortalization, and is not required for SV40 replication. The results also indicate that the ability of SV40 large T to immortalize primary cells is independent of its ability to bind to the retinoblastoma gene product.     

Salt-resistant association of simian virus 40 T antigen with simian virus 40 DNA in nucleoprotein complexes.

Treatment of nucleoprotein complexes (NPCs) from simian virus 40 (SV40)-infected TC7 cells with NaCl (1 or 2 M) or guanidine-hydrochloride (1 or 2 M) resulted in a significant fraction of T antigen still associated with SV40 (I) DNA. Immunoprecipitation of the salt-treated NPCs with SV40 anti-T serum indicated that T antigen is preferentially associated with SV40 (I) DNA rather than with SV40 (II) DNA. Treatment of the NPCs with 4 M guanidine-hydrochloride, however, resulted in a substantial decrease in the amount of SV40 (I) and (II) DNA associated with T antigen. As the temperature was increased to 37 degrees C during incubation of NPCs with NaCl or guanidine-hydrochloride, there was a decrease in the amount of SV40 (I) and (II) DNA immunoprecipitated with SV40 anti-T serum. In the absence of salt, temperature had no effect on the association of T antigen with the SV40 DNA in the NPCs. Treatment of NPCs from SV40 wildtype or tsA58-infected cells grown at the permissive temperature with 1 or 2 M NaCl indicated that tsA T antigen has the same sensitivities as wild-type T antigen to high salt treatment when bound to DNA in NPCs. Characterization of the proteins associated with SV40 (I) DNA after high salt treatment revealed that, in addition to T antigen, a certain amount of viral capsid proteins VP1 and VP3 remained associated with the DNA. Complexes containing SV40 (I) DNA had a sedimentation value of 53S after 1 M NaCl treatment and 43S after 2 M NaCl treatment.     

Stable T-p53 complexes are not required for replication of simian virus 40 in culture or for enhanced phosphorylation of T antigen and p53.

We generated a number of simian virus 40 (SV40) mutants with single amino acid substitutions in T antigen between residues 388 and 411. All but one mutant (398LV) replicated like wild-type SV40 and gave rise to normal-size plaques. Three different mutations at residue 402 (Asp to Glu, Asn, or His) totally prevented the formation of stable complexes with the cellular protein p53 in monkey cells but had no effect on virus replication. Only one other mutation in this region, involving residue 401 (Met to Thr), slightly inhibited the formation of T-monkey p53 complexes. The three mutant T antigens with substitutions at residue 402 also formed no stable complexes with human p53 but generated low levels of complexes with mouse p53. These results indicate that residue 402 is critical for binding to monkey and human p53 proteins and is important for binding to mouse p53. We suggest that it is one of several points of contact. In cells infected with any one of the three residue 402 mutant viruses. T antigen and p53 became increasingly phosphorylated, as they were in cells infected with wild-type virus. Our data therefore show that stable T-p53 complexes are not required for replication of SV40 in culture or for enhanced phosphorylation of either protein.     

Dimers and complexes with p53 are the prevalent oligomeric forms of a transforming nonkaryophilic T antigen of simian virus 40.

The oligomers formed by a mutant nonkaryophilic large T antigen of simian virus 40, which lacks residues 110 through 152 of normal large T antigen and transforms only established cells (L. Fischer-Fantuzzi and C. Vesco, Proc. Natl. Acad. Sci. USA 82:1891-1895, 1985), were found to consist predominantly of dimers. Anti-p53 antibodies precipitated 14 to 16S complexes containing the mutant nonkaryophilic large T antigen and p53 from extracts of transformed cells, and anti-p53 indirect immunofluorescence stained these cells in the cytoplasm.     

Complex formation of simian virus 40 large T antigen with cellular protein p53

We investigated the formation of native complexes between simian virus 40 large T antigen and the cellular protein p53 (T-p53) by using simian virus 40 tsA58-transformed mouse fibroblasts (tsA58 F2b). We observed that newly synthesized p53 bound to all structural subclasses of large T antigen detectable on sucrose density gradients. This led to various intermediates of T-p53 complexes which converted within 2 h into typical mature aggregates. The final levels of stable T-p53 complexes seemed to be determined by p53 rather than by large T antigen.     

The amino-terminal functions of the simian virus 40 large T antigen are required to overcome wild-type p53-mediated growth arrest of cells.

High levels of the p53 tumor suppressor protein can block progression through the cell cycle. A model system for the study of the mechanism of action of wild-type p53 is a cell line (T64-7B) derived from rat embryo fibroblasts transformed by activated ras and a temperature-sensitive murine p53 gene. At 37 to 39 degrees C, the murine p53 protein is in a mutant conformation and the cells actively divide, whereas at 32 degrees C, the protein has a wild-type conformation and the cells arrest in the G1 phase of the cell cycle. Wild-type simian virus 40 large T antigen and a variety of T-antigen mutants were assayed for the ability to bypass the cell cycle block effected by the wild-type p53 protein to induce colony formation at 32 degrees C. The results indicate that two functions within the amino terminus of T antigen are essential to induce cell growth: (i) the ability to bind to the retinoblastoma protein, Rb, and (ii) the presence of a domain in the first exon that appears to interact with the cellular protein, p300. Thus, the cell cycle arrest triggered by wild-type p53 may be overcome by formation of a T-antigen complex with Rb, p300, or both that could then function to either remove p53-mediated negative growth regulatory signals or promote a positive cell growth signal. Surprisingly, T antigen-p53 complexes are not required to overcome the temperature-sensitive p53 block to the cell cycle in these cells. These data suggest that simian virus 40 T antigen associated with Rb, p300, or both proteins can communicate in a cell with the functions of the wild-type p53 protein.     

Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.

An in vitro nuclear translocation system is described in which isolated rat liver nuclei were incubated in a defined buffered medium containing radiolabeled or fluorescently labeled exogenous proteins. The nuclei were rapidly recovered, extracted, and analyzed for the presence of associated radiolabeled or fluorescently labeled proteins. The isolated nuclei exhibited the same specificity for protein uptake as seen previously in vivo, accumulating simian virus 40 wild-type large-T antigen and p53 while excluding a cytoplasmic variant of large-T antigen (d10) and bovine serum albumin. The rapid nuclear accumulation of wild-type large-T antigen was shown to be selective and dependent upon the recognition of a wild-type nuclear location signal, ATP and temperature dependent, and unidirectional. Taken together, the data suggest that in our in vitro system the nuclear translocation of wild-type large-T antigen exhibits some of the characteristics of an active transport process.     

Rapid and sensitive quantitative immunoassay for the large simian virus 40 T antigen

A quantitative, enzyme-linked immunoadsorbent assay has been developed for the simian virus 40 large T antigen. When hamster anti-simian virus 40 tumor serum was used, this method permitted specific identification of large T antigen and its analog, the D2 hybrid protein, a molecule with the same C-terminal approximately 600 amino acids as large T antigen. The sensitivity limit of this test was 0.63 ng of protein. The slopes of the regression lines of the enzyme-linked immunosorbent assay titrations performed with highly purified D2 or simian virus 40 large T antigen and with crude extracts of simian virus 40-infected monkey and transformed human cells were identical. Thus, the curve generated with a purified protein, such as D2, can serve as a quantitative standard for the measurement of large T antigen in a wide variety of extracts. Furthermore, solutions containing high salt concentrations and buffers containing up to 0.1% Nonidet P-40 did not interfere with the assay, making it applicable to the measurement of large T antigen in a variety of chromatographic fractions. The enzyme-linked immunosorbent assay was three times more sensitive, was significantly faster to perform, and was quantitatively valid over a much broader large-T-antigen concentration range than the complement fixation test. As such, it should be useful in future studies of the structure and function of this protein.     

Thermally inactivated simian virus 40 tsA58 mutant T antigen cannot initiate viral DNA replication in vitro.

The mutation in the temperature-sensitive tsA58 mutant T antigen (Ala-438----Val) lies within the presumptive ATP-binding fold. We have constructed a recombinant baculovirus that expresses large quantities of the tsA58 T antigen in infected insect cells. The mutant T antigen mediated simian virus 40 origin-containing DNA (ori-DNA) synthesis in vitro to nearly the same extent as similar quantities of wild-type T antigen at 33 degrees C. However, if wild-type and tsA58 T antigens were heated at 41 degrees C in replication extracts prior to addition of template DNA, the tsA58 T antigen but not the wild type was completely inactivated. The mutant protein displayed greater thermosensitivity for many of the DNA replication activities of T antigen than did the wild-type protein. Some of the replication functions of tsA58 T antigen were differentially affected depending on the presence or absence of ATP during the preheating period. When tsA58 T antigen was preheated in the presence of ATP at 41 degrees C for a time sufficient to completely inactivate its ability to replicate ori-DNA in vitro, it displayed substantial ATPase and normal DNA helicase activities. Conversely, when preheated in the absence of nucleotide, it completely lost both ATPase and helicase activities. Preheating tsA58 T antigen, even in the presence of ATP, led to drastic reductions in its ability to bind to and unwind DNA containing the replication origin. The mutant T antigen also displayed thermosensitivity for binding to and unwinding nonspecific double-stranded DNA in the presence of ATP. Our results suggest that the interactions of T antigen with ATP that are involved in T-antigen DNA binding and DNA helicase activities are different. Moreover, we conclude, consistent with its phenotype in vivo, that the tsA58 T antigen is defective in the initiation but not in the putative elongation functions of T antigen in vitro.     

Homologous cellular proteins associated with simian virus 40 small T antigen and polyomavirus medium T antigen.

The simian virus 40 small T-associated 56,000-Mr (56K) and 32K cellular proteins were shown to be closely related to the polyomavirus medium T-associated 61K and 37K cellular proteins as demonstrated by two-dimensional polyacrylamide gel electrophoresis and V8 protease peptide mapping.     

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.     

The carboxy terminus of simian virus 40 large T antigen is required to disrupt the yeast cell cycle

Wild-type and J domain mutant simian virus 40 large T antigens alter the cell cycle and bud morphology of Saccharomyces cerevisiae. In contrast, yeast cells expressing mutant T antigen lacking the carboxy-terminal 150 aa exhibit normal morphology, indicating that this region of T antigen is required for cell cycle disruption.