An SV40 mutant T antigen does not bind the SV40 viral origin

F8dl is an SV40 deletion mutant that lacks over 60% of the coding sequences for large T antigen and yet is able to immortalize early passage rat cells, to transform established cell lines, and to cause tumors in animals. We report here on the further characterization of this mutant and show that (a) transformation by F8dl is protein mediated but does not require the action of the SV40 small t antigen; (b) the F8dl T antigens have, or are associated with, an ATPase activity; (c) the 34-kDa mutant T antigen of F8dl is localized in nuclei and cell membranes of F8dl transformants and binds to double-stranded DNA; (d) the 20-25 kDa forms of the mutant T antigen are cytoplasmic; and (e) the F8dl T antigens do not bind with high affinity to the SV40 origin of viral DNA replication.     

Inhibition of nuclear migration of wild-type SV40 tumor antigen by a transport-defective mutant of SV40-adenovirus 7 hybrid virus

A mutant of the defective SV40-adenovirus 7 hybrid, PARA, induces the synthesis of SV40 large T-antigen (T-ag) that is not transported to the nucleus and accumulates in the cytoplasm of infected or transformed cells. The effect of this cT mutation on the transport of wild-type (WT) T-ag was examined by coinfection experiments. Immunofluorescence staining for T-ag revealed that when SV40-transformed green monkey kidney cells were infected with the cT mutant of PARA [PARA(2cT)] there was a total loss of nuclear T-ag (nT-ag) reactivity concomitant with the appearance of cytoplasmic T-ag (CT-ag) reactivity. The dominance of the cT-ag phenotype was also manifest in cells coinfected with PARA(2cT) and either SV40 or WT PARA, both of which induce nT-ag during single infection. Two lines of evidence indicate that the absence of nuclear T-ag reactivity in coinfected cells is due to a failure of migration of WT T-ag into the nucleus, rather than an inhibition of its synthesis. First, coinfection of cells with SV40 and either helper adenovirus or PARA-adenovirus populations, at the multiplicities of infection employed in these studies, did not reduce the yields of infectious SV40. Second, cells coinfected with PARA(2cT) and deletion mutants of SV40 which encode T-ag polypeptides of reduced molecular weight expressed the cT-ag phenotype, while the presence of the deleted forms of T-ag was confirmed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. The observation that the replication of infectious SV40 is not inhibited in cells coinfected with PARA(2cT) and expressing the cT phenotype indicates that levels of nuclear T-ag below the limits of detection by immunofluorescence are sufficient to promote SV40 DNA synthesis. The dominant effect of the cT mutation was specific for transport of SV40 T-ag, since normal migration of adenovirus tumor and virion antigens as well as SV40 virion antigen occurred. Several possible mechanisms for the dominance of the CT-ag phenotype are presented.     

SV40 T-antigen-related surface antigen: correlated expression with nuclear T-antigen in cells transformed by an SV40 A-gene mutant

Rat embryo cells transformed by the SV40 A-gene mutant tsA 28.3 were analyzed at permissive and nonpermissive temperatures for the expression of SV40 nuclear T-antigen (T-Ag) and of SV40 T-Ag-related surface antigen (“surface T”) by indirect immunofluorescence microscopy. At permissive temperature both antigens were detected with rabbit antiserum prepared against purified SDS-denatured T-Ag. Upon shift of tsA 28.3 cells to the nonpermissive temperature, expression of both nuclear T-Ag and of surface T decreased simultaneously. When the cells were returned to the permissive temperature, both nuclear T-Ag and surface T reappeared. Analysis of extracts from tsA 28.3 cells grown and labeled with [³⁵S]methionine either at permissive or at nonpermissive temperature by immunoprecipitation and SDS-polyacrylamide gel electrophoresis demonstrated that tsA 28.3 cells synthesized the T-Ag polypeptide only at the permissive temperature. The T-Ag polypeptide was not detected in extracts of cells grown and labeled at nonpermissive temperature. These results demonstrate a strong correlation between the expression of nuclear T-Ag, surface T, and the synthesis of the T-Ag polypeptide, suggesting that both nuclear T-Ag and surface T are products of the SV40 A-gene.     

An SV40 mutant defective in VP4 expression exhibits a temperature-sensitive growth defect

On reexamination of temperature-sensitive D-type (tsD) mutants of simian virus 40 (SV40), we found that the tsD222 mutant is identical to the VP2 M228I mutant, which is defective in VP4 expression, at the nucleotide level. Although a previous study reported that lack of VP4 caused defects in viral dissemination in BSC-1 cells, this mutant showed a temperature-sensitive growth defect in CV-1 cells. tsD101:VP3 Q113K and tsD202:VP3 P108S exhibited a growth phenotype similar to that of tsD222, and they retained the VP4 open reading frame (ORF). These three mutants did not complement each other, suggesting that their defects were functionally indistinguishable. Transduction of the SV40 vector expressing wild-type VP4 in tsD222-infected cells did not ameliorate the growth defect at the non-permissive temperature. The results indicate that tsD mutation in minor capsid proteins has a more profound impact on viral propagation, and that lack of VP4 ORF seems to have little influence on viral growth.     

Molecular dissection of nuclear entry-competent SV40 during infection

To establish viral infection, SV40 must expose nuclear localization signals (NLSs) that are internal in the virion architecture in order to enter the nucleus via interaction with the host's nuclear import machinery, which includes importin alpha and importin beta. The time course for SV40 association with the importins in infected cells was examined. The viral DNA associated with importin alpha by 1.5h post infection, before associating with the importin beta nuclear import receptor, by 3h post infection. Only a small fraction of cell-internalized SV40 that contained viral DNA was bound by the two importins. This fraction, termed "nuclear entry-competent SV40," was slightly smaller than the virion but, importantly, was larger than the viral chromatin and contained both Vp1 and Vp3. Furthermore, the internalized viral DNA in either anti-importin or anti-Vp3 immune complexes was sensitive to DNase I, whereas the viral DNA in mature virions was resistant. All these results suggest that once SV40 enters the cytoplasm, it undergoes an architectural modification that exposes the virion's NLSs for nuclear entry.     

Tumorigenesis in transgenic mice by a nuclear transport-defective SV40 large T-antigen gene

The SV40(cT) mutant encodes a large tumor antigen (cT-ag) that is defective for transport from the cell cytoplasm into the nucleus. This mutant is able to transform established cell lines at near wild-type virus efficiencies, but has a markedly decreased ability to transform primary cells and to induce tumors in newborn hamsters (R. E. Lanford, C. Wong, and J. S. Butel, 1985, Mol. Cell. Biol. 5, 1043-1050). To explore the biology of transport-defective T-ag in vivo, transgenic mice carrying the cT-ag gene were produced. Five of eight founder animals died early in life of choroid plexus tumors (mean age +/- SE, 52 +/- 11.0 days); renal and thymic lesions were also observed. Mice of an SV40(cT) transgenic line regularly succumb to brain tumors (mean age, 81 +/- 1.2 days). SV40 T-ag is expressed in the tumor cells and is retained in the cytoplasm. The observation that SV40(cT) is equivalent to wild-type virus at tumor induction in transgenic mice emphasizes the probable importance of extranuclear forms of SV40 T-ag in brain tumor formation. This study also indicates that in vitro cell transformation assays may not always be accurate reflections of the oncogenic potential of a transforming gene in vivo, because of the different cell types involved.     

Replicative functions of the SV40(cT)-3 mutant defective for nuclear transport of T antigen

The SV40(cT)-3 mutant is defective in transport of SV40 large tumor antigen (T-ag) to the nucleus. Several properties of T-ag associated with SV40 lytic infection and attributed to its nuclear localization were examined to determine whether biologically significant levels of the mutant T-ag (cT-ag) that were immunologically undetectable were transported to the nucleus in SV40(cT)-3-infected TC-7 cells. SV40(cT)-3 was defective in regulation of T-ag synthesis and initiation of viral DNA synthesis. These defects were presumably due to the lack of nuclear transport of cT-ag, since cT-ag was capable of interacting with the SV40 origin of viral DNA synthesis in a solution binding assay. The level of fatty acid acylation, a modification specific for the cell surface associated T-ag, was not affected by the cT mutation. The cT mutation sufficiently suppressed the nuclear transport of wild-type (WT) T-ag in SV40(cT)-3-infected COS-1 cells to result in the cessation of WT-T-ag-stimulated SV40(cT)-3 viral DNA synthesis. These results are discussed with respect to the recent findings that SV40(cT)-3 is fully competent for the transformation of established cell lines and the induction of cellular DNA synthesis in quiescent cells.     

Replication of a temperature sensitive mutant of simian virus 40 in normal and SV40-transformed monkey cells

An SV40 temperature sensitive mutant, ts¹-101, blocked at an early step of productive infection, was assayed at both permissive and nonpermissive temperatures in normal and Adeno-SV40-hybrid-transformed permissive monkey cells. Synthesis of viral antigen was not restricted at high temperature in the transformed cells. It is suggested that complementation occurred in these cells between the superinfecting virus and the integrated SV40 genome.     

Characterization of SV40 T antigen associated with the nuclear matrix

Simian virus 40 (SV40) T antigen associated with the nuclear matrix of SV40-infected TC7 cells has been characterized. Pulse-chase studies on the turnover of T antigen in the different subcellular fractions show that T antigen turns over most rapidly in its association with the purified SV40 nucleoprotein complexes (NPCs) and undergoes a slower rate of turnover in its association with the nuclear matrix. In contrast, turnover of SV40 T antigen in its association with the other subcellular fractions is not detected during the same period of time. Tryptic peptide maps establish that NPC-associated T antigen and nuclear matrix-associated T antigen are chemically related, in that they have two additional methionine-containing peptides that are not found in the majority of T antigen molecules. The association of T antigen with the nuclear matrix is independent of SV40 DNA replication since T antigen is still present in the nuclear matrix after a 1-hr shift-up of tsA58-infected cells to the nonpermissive temperature. In addition, T antigen is associated with the nuclear matrices of both C6 and Cos7 transformed cells, indicating that the association of T antigen with the nuclear matrix is independent of its ability to initiate and support SV40 DNA replication.     

Rapid small-scale isolation of SV40 virions and SV40 DNA

A rapid method for the small-scale isolation of SV40 virions and SV40 DNA is presented. CV-1 monkey epithelial cells are transfected with linear SV40 DNA. After the onset of transfection, cells are lysed by several freeze/thaw cycles and virions are isolated using polyethylene glycol (PEG) precipitation of DNase I treated lysates. Viral DNA is released by proteinase K and dithiothreitol treatment of the isolated virions followed by phenol/chloroform extraction and ethanol precipitation. This method yields on average 7.5×10⁴ plaque forming units (PFUs) and DNA of adequate purity and concentration to be used for restriction analysis on ethidium bromide agarose gels from a single 35-mm tissue culture dish.     

Crystal structure of SV40 large T-antigen bound to p53: interplay between a viral oncoprotein and a cellular tumor suppressor

The transformation potential of Simian Virus 40 depends on the activities of large T-antigen (LTag), which interacts with several cellular tumor suppressors including the important "guardian" of the genome, p53. Inhibition of p53 function by LTag is necessary for both efficient viral replication and cellular transformation. We determined the crystal structure of LTag in complex with p53. The structure reveals an unexpected hexameric complex of LTag binding six p53 monomers. Structure-guided mutagenesis of LTag and p53 residues supported the p53-LTag interface defined by the complex structure. The structure also shows that LTag binding induces dramatic conformational changes at the DNA-binding area of p53, which is achieved partially through an unusual "methionine switch" within p53. In the complex structure, LTag occupies the whole p53 DNA-binding surface and likely interferes with formation of a functional p53 tetramer. In addition, we showed that p53 inhibited LTag helicase function through direct complex formation.     

Phosphorylation of SV40 large T antigen in SV40 nucleoprotein complexes

Plaques produced by our P^? mutants of vesicular stomatitis virus (VSV), which are defective in the inhibition of total protein synthesis in infected cells, stop increasing in size after several days of incubation under conditions where those produced by P⁺ mutants increase linearly in size. The basis for the arrest in size of P^? plaques has been shown to be due to the induction of interferon, and the phenotype is termed PIF⁺ for “plaque interferon positive.” Thus P^? plaques can inhibit the increase in size of adjacent P⁺ plaques and the factor responsible has the biological and physical properties of interferon. Also P^? mutants, when plaqued on VERO cells which cannot be induced to produce interferon, produce plaques which increase linearly in size like P⁺ plaques. Finally, the inclusion of anti-interferon antibody in the overlay medium also causes P^? mutants to produce plaques like P⁺ mutants. VERO cells were found to be useful to separate the effects of is mutations on plaque size from the interferon effect. Using other cell types the latter effect (PIF assay) can be used as an assay for the ability of viruses to induce interferon, for the isolation of PIF⁺ mutants from PIF^? virus, and as a test for the ability of cells to respond to interferon induction by PIF⁺ viruses. The assay can be increased in sensitivity through the use of specific cell types and of cell cultures preincubated for several days in the stationary phase of growth. In its most sensitive form, the assay could detect PIF⁺ behavior in certain ts mutants of VSV at permissive temperatures and in VSV mutants emerging from persistent infection. The assay has also been used to isolate novel mutants of VSV which show alterations in the viral P function.     

Characterization of defective SV40 isolated from SV40-transformed cells.

Defective SV40 viruses were isolated from SV40-transformed monkey, human and hamster cells after Sendai virus-mediated fusion of the transformed cells with TC7 cells, a stable line of African green monkey kidney cells. Viral isolates were concentrated and purified and the defective viruses examined by electron microscopy. The buoyant densities in CsCl of the defective viruses ranged between 1.32 and 1.33 g/cc. DNA isolated from defective viruses was characterized by dye-buoyant density centrifugation and by velocity sedimentation in neutral CsCl. The DNA was heterogeneous in size and contained some covalently closed double-stranded circular molecules.     

Integration of SV40 DNA and induction of cellular DNA synthesis after a ts SV40 infection

The early temperature-sensitive mutant of simian virus 40 (SV40), ts1101, was characterized in nonpermissive Chinese hamster (ChH) and permissive monkey CV-1 cells for integration of viral DNA and induction of cellular DNA synthesis. At 42° in CV-1 cells, this mutant did not induce the synthesis of T antigen or cellular and viral DNA. The viral DNA did not integrate into the CV-1 DNA, although nuclear penetration occurred. In ChH cells the mutant induced T antigen at 40° and 42°, and the viral DNA integrated into ChH DNA, but ChH DNA synthesis was not induced. These findings suggest that the integration of SV40 DNA does not depend on the replication of cellular DNA and that, at least functionally, integration precedes the induction of cellular DNA synthesis.     

SV40 reporter viruses

Three simian virus 40 (SV40) reporter viruses were constructed in this study. One expresses the green fluorescent protein (GFP) as a fusion protein with the first exon of large-T (LT) antigen and is useful for live-cell imaging. A second reporter virus has a FLAG epitope tag at the C-terminus of large-T antigen (vC-LT^FLAG), and a third has the FLAG tag at the N-terminus of LT (vN-LT^FLAG). The vC-LT^FLAG construct grows to titers near those of wild-type (WT) virus and functions well as a reporter virus for SV40 infection. The vN-LT^FLAG construct, while viable, has a defect in the production and spread of infectious particles. All three viruses are useful in detecting superinfecting virus in cells in which nuclear LT is already present, such as persistently infected human mesothelial cells.     

An immunoaffinity purification procedure for SV40 large T antigen

A rapid purification procedure for SV40 large T antigen has been developed which combines the use of an adenovirus-SV40 hybrid virus which overproduces large T antigen, and immunoaffinity chromatography on an anti-large T monoclonal antibody coupled to protein A Sepharose. The protein exhibits the p53-binding, ATPase, and sequence-specific DNA-binding activities of T antigen. The purification procedure can be completed in 1 day and allows the isolation of milligram amounts of large T in excellent yield. The pure protein is extremely antigenic and is tolerant of iodination to high specific activity, permitting the development of a competition radioimmunoassay for large T that reliably detects nanogram amounts of the protein.