The N-terminal side of the origin-binding domain of simian virus 40 large T antigen is involved in A/T untwisting

We investigated the role of the N-terminal side of simian virus 40 (SV40) large T antigen's origin-binding domain in the initiation of virus DNA replication by analyzing the biochemical activities of mutants containing single point substitutions or deletions in this region. Four mutants with substitutions at residues between 121 and 135 were partially defective in untwisting the A/T-rich track on the late side of the origin but were normal in melting the imperfect palindrome (IP) region on the early side. Deletion of the N-terminal 109 amino acids had no effect on either activity, whereas a longer deletion, up to residue 123, greatly reduced A/T untwisting but not IP melting. These results indicate that the region from residue 121 to 135 is important for A/T untwisting but not for IP melting and demonstrate that these activities are separable. Two point substitution mutants (126PS and 135PL) were characterized further by testing them for origin DNA binding, origin unwinding, oligomerization, and helicase activity. These two mutants were completely defective in origin (form U(R)) unwinding but normal in the other activities. Our results demonstrate that a failure to normally untwist the A/T track is correlated with a defect in origin unwinding. Further, they indicate that some mutants with substitutions in the region from residue 121 to 135 interact with origin DNA incorrectly, perhaps by failing to make appropriate contacts with the A/T-rich DNA.     

The J domain of simian virus 40 large T antigen is required to functionally inactivate RB family proteins

Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellular tumor suppressors. Transformation minimally requires the following three domains: (i) a C-terminal domain that mediates binding to p53; (ii) the LXCXE domain (residues 103 to 107), necessary for binding to the retinoblastoma tumor suppressor protein, pRB, and the related p107 and p130; and (iii) an N-terminal domain that is homologous to the J domain of DnaJ molecular chaperone proteins. We have previously demonstrated that the N-terminal J domain of TAg affects the RB-related proteins by perturbing the phosphorylation status of p107 and p130 and promoting the degradation of p130 and that this domain is required for transformation of cells that express either p107 or p130. In this work, we demonstrate that the J domain of TAg is required to inactivate the ability of each member of the pRB family to induce a G1 arrest in Saos-2 cells. Furthermore, the J domain is required to override the repression of E2F activity mediated by p130 and pRB and to disrupt p130-E2F DNA binding complexes. These results imply that while the LXCXE domain serves as a binding site for the RB-related proteins, the J domain plays an important role in inactivating their function.     

T cells are responsive to the simian virus 40 large tumor antigen transgenically expressed in pancreatic islets

The qualities of a peripheral Ag that determine whether T cells will be tolerant of or responsive to it are poorly understood. To approach this problem, we studied the T cell response in a line of transgenic mice selectively expressing an oncoprotein in the islets of Langerhans. The SV40 large tumor Ag (SV40-T) is directed to islet beta-cells in Rip1-Tag3 (RT3) mice by a hybrid insulin promoter-SV40-T construct. Ag is first detected on these cells between 10 and 12 wk after birth. RT3 mice were bred with mice expressing a transgenic rearranged TCR recognizing SV40-T in the context of the class I MHC molecule, H-2Kk. T cell response in the resultant RT3/TCR-double transgenic mice was then analyzed. T cells are fully responsive to SV40-T in RT3/TCR-transgenic mice, and T cells infiltrate the islets of both RT3 and RT3/TCR-transgenic mice. This work demonstrates that T cells may remain responsive to self-Ag expressed outside the thymus, and that this responsiveness may result in autoimmunity. The developmentally delayed expression or the oncogenic nature of SV40-T in the RT3-transgenic mice may be important in determining this T cell response.     

Context-dependent immunogenicity of an S206G-substituted H-2Db-restricted simian virus 40 large T antigen epitope I variant

SV40 large tumor Ag (Tag) contains four H-2b-restricted (I, II/III, IV, and V) CTL epitopes. A hierarchy exists among these CTL epitopes. CTL directed against epitopes I, II/III, and IV are readily detected following immunization of H-2b mice with SV40, Tag-transformed syngeneic cells, or a vaccinia recombinant that expresses full-length Tag, while epitope V-specific CTL are not. The mechanisms that define this hierarchy remain unknown. Initial studies have shown that the locations of epitopes I and V within SV40 Tag do not determine the immunological potencies of these epitopes. Like the wild-type Tag, derivatives in which the locations of epitopes I and V were precisely reversed within Tag failed to induce epitope V-specific CTL, but did induce epitope I-specific CTL. The use of an S206G-substituted epitope I variant (GAINNYAQKL) revealed that the S206G variant sequence induced CTL when located within the native epitope I context, but failed to do so when located within the epitope V context of Tag. Mutagenesis of residues adjacent to the S206G-substituted epitope I variant revealed that the identity of the residue flanking the amino terminus of the S206G variant was critical when it resided within the epitope V location, but not within the epitope I location. These results demonstrate that effects imposed by both regional context and adjacent residues can modulate immunogenicity, but that the relative importance of such effects varies in an epitope-dependent manner.     

Replication of simian virus 40 origin-containing DNA during infection with a recombinant Autographa californica multiple nuclear polyhedrosis virus expressing large T antigen

Autographica californica multiple nuclear polyhedrosis virus (AcMNPV) has been shown to encode many of the enzymes involved in the replication of its own DNA. Although the AcMNPV genome contains multiple sets of reiterated sequences that are thought to function as origins of DNA replication, no initiator protein has yet been identified in the set of viral replication enzymes. In this study, the ability of a heterologous origin initiator system to promote DNA replication in AcMNPV-infected cells was examined. A recombinant AcMNPV that expressed the simian virus 40 (SV40) large T antigen was surprisingly found to induce the efficient replication of a transfected plasmid containing an SV40 origin. This replication was subsequently found to involve three essential components: (i) T antigen, since replication of SV40 origin-containing plasmids was not induced by wild-type AcMNPV which did not express this protein; (ii) an intact SV40 core origin, since deletion of specific functional motifs within the origin resulted in a loss of replicative abilities; and (iii) one or more AcMNPV-encoded proteins, since viral superinfection was required for plasmid amplification. Characterization of the replicated DNA revealed that it existed as a high-molecular-weight concatemer and underwent significant levels of homologous recombination between inverted repeat sequences. These properties were consistent with an AcMNPV-directed mode of DNA synthesis rather than that of SV40 and suggested that T antigen-SV40 origin complexes may be capable of initiating DNA replication reactions that can be completed by AcMNPV-encoded enzymes.     

Transcriptional activation of the human c-myc gene by simian virus 40 large T antigen without binding to p53 and RB proteins in the transient expression system

Male adult Wistar rats were randomly divided into four groups in a 2 x 2 factorial design and were fed diets containing cooked-stored polished rice (CSPR), with and without 0.7 g/100 g of Nebacitin [bacitracinneomycin sulfate (2:1, wt/wt)] and with and without 1 g cholesterol/100 g diet. The CSPR diet contained 1.87 g resistant starch/100 g. After 4 wk, arterial blood and liver were collected. Feces were collected during the last 7 d. Rats fed the diet with Nebacitin and cholesterol had higher serum total cholesterol than the rats fed diets without cholesterol. Serum triglyceride concentration was greater in rats fed Nebacitin, regardless of dietary cholesterol concentration. Rats fed the diet with Nebacitin and cholesterol had higher serum LDL cholesterol concentration and liver total cholesterol concentration than rats fed the other three diets. Rats fed the CSPR diet with Nebacitin both with and without cholesterol had a higher fecal resistant starch concentration and excretion and lower serum short-chain fatty acid concentration than rats fed the diets without Nabacitin. Hepatic cholesterol concentration was greater in rats fed Nebacitin only when the diet also contained cholesterol. Therefore, dietary Nebacitin alters lipid metabolism in rats, and some effects are most pronounced in those also fed cholesterol.     

A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays

At least three regions of the simian virus 40 small-t antigen (small-t) contribute to the protein's ability to enhance cellular transformation. As we showed previously for rat F111 cells, one region includes sequences from residues 97 to 103 that are involved in the binding and inhibition of protein phosphatase 2A. In the present study, the role of the protein phosphatase 2A binding region was confirmed in two additional small-t-dependent transformation systems. Second, small-t was found to provide a function previously identified as a large-T transformation domain. Mutations in residues 19 to 28 of large-T affected its transforming ability, but these mutations were complemented by a wild-type small-t. A third region of small-t was also required for efficient transformation. This region, the 42-47 region, is shared by large-T and small-t and contains a conserved HPDKGG hexapeptide. The 42-47 region function could be provided by either small-t or large-T in small-t-dependent systems. Mutations in the 42-47 region reduced the ability of small-t to transactivate the cyclin A promoter, of interest because small-t increased endogenous cyclin A mRNA levels in both human and monkey cells, as well as transactivating the promoter in transient assays.     

The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase alpha-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.     

The activity of topoisomerase I is modulated by large T antigen during unwinding of the SV40 origin

When simian virus 40 (SV40) large T antigen binds to the virus origin of replication, it forms a double hexamer that functions as a helicase to unwind the DNA bidirectionally. We demonstrate in this report that T antigen can unwind and release an origin DNA single strand of less than full length in the presence of purified human topoisomerase I. The sites nicked by topoisomerase I in the strands released by T antigen during DNA unwinding were localized primarily to the "late" side of the origin, and the template for lagging strand synthesis was preferred significantly over the one for leading strand synthesis. Importantly, these sites were, for the most part, different from the sites nicked by topoisomerase I in the absence of T antigen. These data indicate that T antigen activates topoisomerase I nicking at discrete sites and releases these nicked strands during unwinding. We hypothesize that a single molecule of topoisomerase I can form a functional complex with a double hexamer of T antigen to simultaneously relax and unwind double-stranded origin-containing DNA.     

Identification of structural elements involved in the interaction of simian virus 40 small tumor antigen with protein phosphatase 2A

SV40 small tumor antigen (small-t) was used as a model to identify structural elements involved in the interactions between regulatory proteins and protein phosphatase 2A (PP2A). Using mutant proteins and synthetic peptides, we identified a small domain within small-t that is a major site for interaction with the dimeric form of PP2A. A series of small-t truncation mutants identified a region surrounding the first of two conserved cysteine clusters that was critical for interaction with PP2A. These mutants also identified additional regions of small-t that contribute to high affinity interaction. Deletion of residues 110-119, which encompass the first cysteine cluster, resulted in a protein that failed to bind to PP2A. Synthetic peptides that contained residues 105-122 of small-t blocked binding of small-t to PP2A. These peptides also inhibited the phosphatase activity of PP2A in a manner analogous to full-length small-t. The active small-t peptides adopt a beta-strand structure that was essential for high affinity interaction with the PP2A dimer. Based on circular dichroism measurements, the same cysteine cluster-containing peptides that bind to PP2A also interact with zinc. Interaction with zinc required the conserved cysteines but was not required for interaction with PP2A.     

Stabilization of the tumor suppressor p53 during cellular transformation by simian virus 40: influence of viral and cellular factors and biological consequences

To understand the process and biological significance of metabolic stabilization of p53 during simian virus 40 (SV40)-induced cellular transformation, we analyzed cellular and viral parameters involved in this process. We demonstrate that neither large T expression as such nor the cellular phenotype (normal versus transformed) markedly influence the stability of p53 complexed to large T in SV40 abortively infected BALB/c mouse fibroblasts. In contrast, metabolic stabilization of p53 is an active cellular event, specifically induced by SV40. The ability of SV40 to induce a cellular response leading to stabilization of p53 complexed to large T is independent from the cellular phenotype and greatly varies between different cells. However, metabolic stability was conferred only to p53 in complex with large T, whereas the free p53 in these cells remained metabolically unstable. Comparative analyses of cellular transformation in various cells differing in stability of p53 complexed to large T upon abortive infection with SV40 revealed a strong correlation between the ability of SV40 to induce metabolic stabilization and its transformation efficiency. Our data suggest that metabolic stabilization and the ensuing enhanced levels of p53 are important for initiation and/or maintenance of SV40 transformation.     

Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses the Int-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.     

Transactivation of a ribosomal gene by simian virus 40 large-T antigen requires at least three activities of the protein

Immunoaffinity-purified paired helical filaments (PHFs) from Alzheimer's disease (AD) brain homogenates contain an associated protein kinase activity that is able to induce the phosphorylation of PHF proteins on addition of exogenous MgCl2 and ATP. PHF kinase activity is shown to be present in immunoaffinity-purified PHFs from both sporadic and familial AD, Down's syndrome, and Pick's disease but not from normal brain homogenates. Although initial studies failed to show that the kinase was able to induce the phosphorylation of tau, additional studies presented in this article show that only cyclic AMP-dependent protein kinase-pretreated recombinant tau is a substrate for the PHF kinase activity. Deletional mutagenesis, phosphopeptide mapping, and site-directed mutagenesis have identified the PHF kinase phosphorylation sites as amino acids Thr361 and Ser412 in htau40. In addition, the cyclic AMP-dependent protein kinase phosphorylation sites that direct the PHF kinase have been mapped to amino acids Ser356 and Ser409 in htau40. Additional data demonstrate that these hierarchical phosphorylations in the extreme C terminus of tau allow for the incorporation of recombinant tau into exogenously added AD-derived PHFs, providing evidence that certain unique phosphorylations of tau may play a role in the pathogenesis of neurofibrillary pathology in AD.     

The effect on the insulin-like growth factor system in human prostate epithelial cells of immortalization and transformation by simian virus-40 T antigen

The insulin-like growth factor (IGF) system has been demonstrated to be important for proliferation and differentiation in tissues. This system has also been demonstrated to be an important regulator of the growth of normal prostate epithelium and has been implicated in the process of transformation to human epithelial prostate cancer. This study examined the function of the various components of the IGF system in benign prostate epithelium (BPE), simian virus-40 (SV40)-T antigen-immortalized prostate epithelial cells, P69SV40-T (P69), and two sublines generated from the parental line by serial passage through athymic mice: one tumorigenic (M2182) and one metastatic (M12). IGF-II messenger ribonucleic acid (mRNA) and protein were detected in BPE cells, and each of the three P69 cell lines. IGF-II protein levels were significantly higher in medium collected from the P69, M2182, and M12 cells than in BPE. Proliferation in response to IGF was P69 > BPE > M2182 > M12. The proliferative responses in the four cell types were paralleled by an increase in c-jun. In addition, as the cells became progressively more tumorigenic, the basal level of c-jun mRNA increased. IGF-binding protein-2 (IGFBP-2), -3, -4, -5, and -6 could be detected in the primary epithelial cell medium; however, as the cells became progressively more tumorigenic, there was a decrease in IGFBP-2, -3, -5, and -6 in the medium. The type 1 IGF receptor (IGFr) also decreased as the cells became more tumorigenic. The M12 cells had 80% fewer receptors than the P69 cells and 70% fewer than M2182 cells. There was no change in the Kd for IGF between the cell lines. Based on these data it would appear that the difference in proliferation between the BPE cells and P69s may be due to an increased concentration of inhibitory IGFBPs in the P69 medium. The decrease in proliferation seen in response to IGF in M2182 and M12 cells compared to the P69s would appear at least in part to be due to a decreased IGFr number. IGFr mRNA is represented by 11.0- and 7.0-kilobase bands in the BPE and P69 cells, but only by an 11.0-kilobase band in M2182 and M12 cells. These data indicate that there are significant changes that occur in the IGF system during the process of malignant transformation of the prostate epithelium. The changes described in the P69 cell system are similar to those seen in vivo and suggest that an intact IGF system may be important in maintaining a differentiated epithelial cell.     

Fusions between Epstein-Barr viral nuclear antigen-1 of Epstein-Barr virus and the large T-antigen of simian virus 40 replicate their cognate origins

Epstein-Barr viral nuclear antigen-1 (EBNA-1) is required for the stable replication of plasmids that contain oriP, the origin of DNA synthesis used during the latent phase of the Epstein-Barr virus life cycle. EBNA-1 acts post-synthetically through unknown mechanisms to facilitate the continued synthesis of oriP plasmids in ensuing S phases. In contrast to viral replicons such as that of SV40, DNA synthesis of oriP is restricted to a single round during each cell cycle. Large T-antigen of SV40 is a DNA helicase and activates the synthesis of SV40 DNA by recruiting cellular proteins required for DNA synthesis to the origin of SV40. Using fusion proteins of EBNA-1 and large T-antigen, we tested whether tethering large T-antigen to oriP is sufficient to initiate multiple rounds of DNA synthesis from oriP during each cell cycle. We report here that, although these fusion proteins retain the biological activities of both EBNA-1 and large T-antigen, their constituent proteins do not confer the properties of one on the other. Thus, it is not possible to subvert the cellular controls that restrict DNA synthesis from oriP to a single round per cell cycle. These results also provide insights into architectural constraints at oriP and at the SV40 ori.