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Pipas, J.M. (2009) SV40: Cell transformation and tumorigenesis. Virology 384:294-303 The story of SV40-induced tumorigenesis and cellular transformation is intimately entwined with the development of modern molecular biology. Because SV40 and other viruses have small genomes and are relatively easy to manipulate in the laboratory, they offered tractable systems for molecular analysis. Thus, many of the early efforts to understand how eukaryotes replicate their DNA, regulate expression of their genes, and translate mRNA were focused on viral systems. The discovery that SV40 induces tumors in certain laboratory animals and transforms many types of cultured cells offered the first opportunity to explore the molecular basis for cancer. The goal of this article is to highlight some of the experiments that have led to our current view of SV40-induced transformation and to provide some context as to how they contributed to basic research in molecular biology and to our understanding of cancer. Wright, C.M., S.P. Seguin, S.W. Fewell, H. Zhang, C. Ishwad, A. Vats, C.A. Lingwood, P. Wipf, E. Fanning, J.M. Pipas, and J.L. Brodsky (2009) Inhibition of Simian Virus 40 replication by targeting the molecular chaperone function and ATPase activity of T antigen. Virus Res. 0: Polyomaviruses such as BK virus and JC virus have been linked to several diseases, but treatments that thwart their propagation are limited in part because of slow growth and cumbersome culturing conditions. In contrast, the replication of one member of this family, Simian Virus 40 (SV40), is robust and has been well-characterized. SV40 replication requires two domains within the viral-encoded large tumor antigen (TAg): The ATPase domain and the N-terminal J domain, which stimulates the ATPase activity of the Hsp70 chaperone. To assess whether inhibitors of polyomavirus replication could be identified, we examined a recently described library of small molecules, some of which inhibit chaperone function. One compound, MAL2-11B, inhibited both TAg's endogenous ATPase activity and the TAg-mediated activation of Hsp70. MAL2-11B also reduced SV40 propagation in plaque assays and compromised DNA replication in cell culture and in vitro. Furthermore, the compound significantly reduced the growth of BK virus in a human kidney cell line. These data indicate that pharmacological inhibition of TAg's chaperone and ATPase activities may provide a route to combat polyomavirus-mediated disease. Cantalupo, P.G., M.T. Saenz-Robles, A.V. Rathi, R.W. Beerman, W.H. Patterson, R.H. Whitehead, and J.M. Pipas (2009) Cell-type specific regulation of gene expression by simian virus 40 T antigens. Virology 0: SV40 transforms cells through the action of two oncoproteins, large T antigen and small t antigen. Small t antigen targets phosphatase PP2A, while large T antigen stimulates cell proliferation and survival by action on multiple proteins, including the tumor suppressors Rb and p53. Large T antigen also binds components of the transcription initiation complex and several transcription factors. We examined global gene expression in SV40-transformed mouse embryo fibroblasts, and in enterocytes obtained from transgenic mice. SV40 transformation alters the expression of approximately 800 cellular genes in both systems. Much of this regulation is observed in both MEFs and enterocytes and is consistent with T antigen action on the Rb-E2F pathway. However, the regulation of many genes is cell-type specific, suggesting that unique signaling pathways are activated in different cell types upon transformation, and that the consequences of SV40 transformation depends on the type of cell targeted. Wright, C.M., R.J. Chovatiya, N.E. Jameson, D.M. Turner, G. Zhu, S. Werner, D.M. Huryn, J.M. Pipas, B.W. Day, P. Wipf, and J.L. Brodsky (2008) Pyrimidinone-peptoid hybrid molecules with distinct effects on molecular chaperone function and cell proliferation. Bioorg. Med. Chem. 16:3291-3301 The Hsp70 molecular chaperones are ATPases that play critical roles in the pathogenesis of many human diseases, including breast cancer. Hsp70 ATP hydrolysis is relatively weak but is stimulated by J domain-containing proteins. We identified pyrimidinone-peptoid hybrid molecules that inhibit cell proliferation with greater potency than previously described Hsp70 modulators. In many cases, anti-proliferative activity correlated with inhibition of J domain stimulation of Hsp70. Zhao, X., R.J. Madden-Fuentes, B.X. Lou, J.M. Pipas, J. Gerhardt, C.J. Rigell, and E. Fanning (2008) Ataxia telangiectasia-mutated damage-signaling kinase- and proteasome-dependent destruction of Mre11-Rad50-Nbs1 subunits in Simian virus 40-infected primate cells. J. Virol. 82:5316-5328 Although the mechanism of simian virus 40 (SV40) DNA replication has been extensively investigated with cell extracts, viral DNA replication in productively infected cells utilizes additional viral and host functions whose interplay remains poorly understood. We show here that in SV40-infected primate cells, the activated ataxia telangiectasia-mutated (ATM) damage-signaling kinase, gamma-H2AX, and Mre11-Rad50-Nbs1 (MRN) assemble with T antigen and other viral DNA replication proteins in large nuclear foci. During infection, steady-state levels of MRN subunits decline, although the corresponding mRNA levels remain unchanged. A proteasome inhibitor stabilizes the MRN complex, suggesting that MRN may undergo proteasome-dependent degradation. Analysis of mutant T antigens with disrupted binding to the ubiquitin ligase CUL7 revealed that MRN subunits are stable in cells infected with mutant virus or transfected with mutant viral DNA, implicating CUL7 association with T antigen in MRN proteolysis. The mutant genomes produce fewer virus progeny than the wild type, suggesting that T antigen-CUL7-directed proteolysis facilitates virus propagation. Use of a specific ATM kinase inhibitor showed that ATM kinase signaling is a prerequisite for proteasome-dependent degradation of MRN subunits as well as for the localization of T antigen and damage-signaling proteins to viral replication foci and optimal viral DNA replication. Taken together, the results indicate that SV40 infection manipulates host DNA damage-signaling to reprogram the cell for viral replication, perhaps through mechanisms related to host recovery from DNA damage. Saenz-Robles, M.T., J.A. Markovics, J.L. Chong, R. Opavsky, R.H. Whitehead, G. Leone, and J.M. Pipas (2007) Intestinal hyperplasia induced by simian virus 40 large tumor antigen requires E2F2. J. Virol. 81:13191-13199 The simian virus 40 large T antigen contributes to neoplastic transformation, in part, by targeting the Rb family of tumor suppressors. There are three known Rb proteins, pRb, p130, and p107, all of which block the cell cycle by preventing the transcription of genes regulated by the E2F family of transcription factors. T antigen interacts directly with Rb proteins and disrupts Rb-E2F complexes both in vitro and in cultured cells. Consequently, T antigen is thought to inhibit transcriptional repression by the Rb family proteins by disrupting their interaction with E2F proteins, thus allowing E2F-dependent transcription and the expression of cellular genes needed for entry into S phase. This model predicts that active E2F-dependent transcription is required for T-antigen-induced transformation. To test this hypothesis, we have examined the status of Rb-E2F complexes in murine enterocytes. Previous studies have shown that T antigen drives enterocytes into S phase, resulting in intestinal hyperplasia, and that the induction of enterocyte proliferation requires T-antigen binding to Rb proteins. In this paper, we show that normal growth-arrested enterocytes contain p130-E2F4 complexes and that T-antigen expression destroys these complexes, most likely by stimulating p130 degradation. Furthermore, unlike their normal counterparts, enterocytes expressing T antigen contain abundant levels of E2F2 and E2F3a. Concomitantly, T-antigen-induced intestinal proliferation is reduced in mice lacking either E2F2 alone or both E2F2 and E2F3a, but not in mice lacking E2F1. These studies support a model in which T antigen eliminates Rb-E2F repressive complexes so that specific activator E2Fs can drive S-phase entry. Rathi, A.V., M.T. Sáenz-Robles, and J.M. Pipas (2007) Enterocyte proliferation and intestinal hyperplasia induced by simian virus 40 T antigen require a functional J domain. J. Virol. 81:9481-9489 Transgenic mice expressing the simian virus 40 large T antigen (TAg) in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. This induction requires TAg action on the retinoblastoma (Rb) family of tumor suppressors and is independent of the p53 pathway. In cell culture systems, the inactivation of Rb proteins requires both a J domain in TAg that interacts with hsc70 and an LXCXE motif that directs association with Rb proteins. Together these elements are sufficient to release E2Fs from their association with Rb family members. We have generated transgenic mice that express a J domain mutant (D44N) in villus enterocytes. In contrast to wild-type TAg, the D44N mutant is unable to induce enterocyte proliferation. Histological and morphological examination revealed that mice expressing the J domain mutant have normal intestines without loss of growth control. Unlike mice expressing wild-type TAg, mice expressing D44N do not reduce the protein levels of p130 and are also unable to dissociate p130-E2F DNA binding complexes. Furthermore, mice expressing D44N in a null p130 background are still unable to develop hyperplasia. These studies demonstrate that the ectopic proliferation of enterocytes by TAg requires a functional J domain and suggest that the J domain is necessary to inactivate all three pRb family members. Sullivan, C.S., A. Grundhoff, R. Treisman, J.M. Pipas, and D. Ganem (2007) Expression and function of microRNAs in viruses great and small. Cold Spring Harb. Sym. 71:351-365 Wright, C.M., S.W. Fewell, M.L. Sullivan, J.M. Pipas, S.C. Watkins, and J.L. Brodsky (2007) The hsp40 molecular chaperone, Ydj1p, along with the protein kinase C pathway, impact cell wall integrity in the yeast Saccharomyces cerevisiae. Genetics 175:1649-1664 Molecular chaperones, such as Hsp40, regulate cellular processes by aiding in the folding, localization, and activation of multi-protein machines. To identify new targets of chaperone action we performed a multi-copy suppressor screen for genes that improved the slow growth defect of yeast lacking the YDJ1 chromosomal locus and expressing a defective Hsp40 chimera. Among the genes identified were MID2, which regulates cell wall integrity, and PKC1, which encodes protein kinase C and is linked to cell wall biogenesis. We found that ydj1Delta yeast exhibit phenotypes consistent with cell wall defects and these phenotypes were improved by Mid2p or Pkc1p over-expression or by over-expression of activated down-stream components in the PKC pathway. Yeast containing a thermosensitive allele in the gene encoding Hsp90 also exhibited cell wall defects, and Mid2p or Pkc1p over-expression improved the growth of these cells at elevated temperatures. To determine the physiological basis for suppression of the ydj1Delta growth defect, wild type and ydj1Delta yeast were examined by electron microscopy and we found that Mid2p over-expression thickened the mutant's cell wall. Together, these data provide the first direct link between cytoplasmic chaperone function and cell wall integrity, and suggest that chaperones orchestrate the complex biogenesis of this structure. Fanning, E., and J.M. Pipas (2006) Polyomaviruses. Pp 141-154 in DNA replication and Human Disease, DePamphilis, M.L., Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, NY Ahuja, D., M.T. Saenz-Robles, and J.M. Pipas (2005) SV40 large T antigen targets multiple cellular pathways to elicit cellular transformation. Oncogene 24:7729-7745 DNA tumor viruses such as simian virus 40 (SV40) express dominant acting oncoproteins that exert their effects by associating with key cellular targets and altering the signaling pathways they govern. Thus, tumor viruses have proved to be invaluable aids in identifying proteins that participate in tumorigenesis, and in understanding the molecular basis for the transformed phenotype. The roles played by the SV40-encoded 708 amino-acid large T antigen (T antigen), and 174 amino acid small T antigen (t antigen), in transformation have been examined extensively. These studies have firmly established that large T antigen's inhibition of the p53 and Rb-family of tumor suppressors and small T antigen's action on the pp2A phosphatase, are important for SV40-induced transformation. It is not yet clear if the Rb, p53 and pp2A proteins are the only targets through which SV40 transforms cells, or whether additional targets await discovery. Finally, expression of SV40 oncoproteins in transgenic mice results in effects ranging from hyperplasia to invasive carcinoma accompanied by metastasis, depending on the tissue in which they are expressed. Thus, the consequences of SV40 action on these targets depend on the cell type being studied. The identification of additional cellular targets important for transformation, and understanding the molecular basis for the cell type-specific action of the viral T antigens are two important areas through which SV40 will continue to contribute to our understanding of cancer. Markovics, J.A., P.A. Carroll, M.T. Sáenz-Robles, H. Pope, C.M. Coopersmith, and J.M. Pipas (2005) Intestinal dysplasia induced by simian virus 40 T antigen is independent of p53. J. Virol. 79:7492-7502 Transgenic mice expressing simian virus 40 large T antigen in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. Hyperplasia is dependent on T antigen binding to the retinoblastoma (pRb) family of tumor suppressor proteins. Mice expressing a truncated T antigen that inactivates the pRb-family, but is defective for binding p53, exhibit hyperplasia but do not progress to dysplasia. We hypothesized that the inhibition of the pRb family leads to entry of enterocytes into the cell cycle, resulting in hyperplasia, while inactivation of p53 is required for progression to dysplasia. Therefore, we examined T antigen/p53 complexes from the intestines of transgenic mice. We found that T antigen did not induce p53 stabilization, and we could not detect T antigen/p53 complexes in villus enterocytes. In contrast, T antigen expression led to a large increase in the levels of the cyclin-dependent kinase inhibitor p21. Furthermore, mice in which pRb was inactivated by a truncated T antigen in a p53 null background exhibited intestinal hyperplasia but no progression to dysplasia. These data indicate that loss of p53 function does not play a role in T antigen-induced dysplasia in the intestine. Rather, some unknown function of T antigen is essential for progression beyond hyperplasia.
Sullivan, C.S., A.T. Grundhoff, S. Tevethia, J.M. Pipas, and D. Ganem (2005) SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells. Nature 435:682-686 MicroRNAs (miRNAs) are small (approximately 22-nucleotide) RNAs that in lower organisms serve important regulatory roles in development and gene expression, typically by forming imperfect duplexes with target messenger RNAs. miRNAs have also been described in mammalian cells and in infections with Epstein-Barr virus (EBV), but the function of most of them is unknown. Although one EBV miRNA probably altered the processing of a viral mRNA, the regulatory significance of this event is uncertain, because other transcripts exist that can supply the targeted function. Here we report the identification of miRNAs encoded by simian virus 40 (SV40) and define their functional significance for viral infection. SVmiRNAs accumulate at late times in infection, are perfectly complementary to early viral mRNAs, and target those mRNAs for cleavage. This reduces the expression of viral T antigens but does not reduce the yield of infectious virus relative to that generated by a mutant lacking SVmiRNAs. However, wild-type SV40-infected cells are less sensitive than the mutant to lysis by cytotoxic T cells, and trigger less cytokine production by such cells. Thus, viral evolution has taken advantage of the miRNA pathway to generate effectors that enhance the probability of successful infection. Cantalupo, P., A. Doering, C.S. Sullivan, A. Pal, K.W. Peden, A.M. Lewis, and J.M. Pipas (2005) Complete nucleotide sequence of polyomavirus SA12. J. Virol. 79:13094-13104 The Polyomaviridae have small icosahedral virions that contain a genome of approximately 5,000 bp of circular double-stranded DNA. Polyomaviruses infect hosts ranging from humans to birds, and some members of this family induce tumors in test animals or in their natural hosts. We report the complete nucleotide sequence of simian agent 12 (SA12), whose natural host is thought to be Papio ursinus, the chacma baboon. The 5,230-bp genome has a genetic organization typical of polyomaviruses. Sequences encoding large T antigen, small t antigen, agnoprotein, and the viral capsid proteins VP1, VP2, and VP3 are present in the expected locations. We show that, like its close relative simian virus 40 (SV40), SA12 expresses microRNAs that are encoded by the late DNA strand overlapping the 3' end of large T antigen coding sequences. Based on sequence comparisons, SA12 is most closely related to BK virus (BKV), a human polyomavirus. We have developed a real-time PCR test that distinguishes SA12 from BKV and the other closely related polyomaviruses JC virus and SV40. The close relationship between SA12 and BKV raises the possibility that these viruses circulate between human and baboon hosts.
Sullivan, C.S., A.E. Baker, and J.M. Pipas (2004) Simian virus 40 infection disrupts p130-E2F and p107-E2F complexes but does not perturb pRb-E2F complexes. Virology 320:218-228 In its native host species, the Rhesus Macaque, simian virus 40 (SV40) forms a persistent infection in the kidneys with no apparent harmful side effects. We show that SV40 infection of growth-arrested monkey kidney epithelial cells results in the specific disruption of certain Rb-E2F family complexes. Throughout the course of infection, p130-E2F and p107-E2F complexes are disrupted, but surprisingly pRb-E2F complexes remain intact. This suggests that the presence of some pRb-E2F complexes is not inhibitory to productive infection. Additionally, while a decrease of p130 steady state levels is observed during the later time points of infection, early during infection, p130 is readily detectable. This suggests SV40 infection overrides p130-mediated growth arrest through a mechanism(s) in addition to the well-documented T antigen-mediated degradation of p130. Finally, infection induces a dramatic relocalization of E2F4 from the nucleus to the cytoplasm. The implications of these observations to the life cycle of the virus are addressed. Chromy, L.R., J.M. Pipas, and R.L. Garcea (2003) Chaperone-mediated in vitro assembly of Polyomavirus capsids. Proc. Natl. Acad. Sci., USA 100:10477-10482 The polyomavirus coat protein viral protein 1 (VP1) has the intrinsic ability to self-assemble in vitro into polymorphic capsid-like structures on addition of calcium. In contrast, polyomavirus assembly in vivo is rigorously controlled, such that virions of uniform size are formed only in the cell nucleus. During viral infection, the 72 kDa cellular chaperone heat shock cognate protein (hsc70) binds VP1 posttranslation and colocalizes with VP1 to the nucleus, thereby suggesting a role for approximately 70-kDa heat shock protein (hsp70) family chaperones in regulating the quality and location of capsid assembly. We found that, after expression of recombinant VP1 in Escherichia coli, the prokaryotic hsp70 chaperone DnaK copurified with the VP1 C-terminal domain that links pentamers in an assembled capsid. When stably bound to VP1, DnaK inhibited in vitro assembly induced by calcium. However, in the presence of ATP, the hsp70 chaperone system comprised of DnaK, DnaJ, and GrpE assembled VP1 into uniform capsids without requiring calcium. Chaperone-mediated assembly was similarly catalyzed by the eukaryotic hsc70 protein, in combination with the J-domain function of the simian virus 40 large T-antigen protein. Thus, polyomavirus capsid assembly can be recapitulated with high-fidelity in vitro using either prokaryotic or eukaryotic hsp70 chaperone systems, thereby supporting a role for cellular chaperones in the in vivo regulation of virion assembly.
Genevaux, P., F. Lang, F. Schwager, J.V. Vartikar, K. Rundell, J.M. Pipas, C. Georgopoulos, and W.L. Kelley (2003) Simian virus 40 T antigens and J domains: analysis of Hsp40 cochaperone functions in Escherichia coli. J. Virol. 77:10706-10713 The N-terminal exon of DNA tumor virus T antigens represents a J domain that can direct interaction with the host-encoded Hsp70 chaperones. We have taken advantage of rapid Hsp40 cochaperone assays with Escherichia coli to assess simian virus 40 (SV40)-encoded J-domain loss of function. We found a strong correlation between loss of cochaperone function in E. coli and defective SV40 growth, suggesting that the major role of the J domain in DNA tumor viruses is to provide cochaperone function. We also report the expression of native SV40 virus T antigens in E. coli. Our results show that small t antigen, but not large T antigen (LT) or LT truncation TN125 or TN136, can functionally replace under limited growth conditions DnaJ (Hsp40) function in vivo. In addition, purified small t antigen can efficiently stimulate E. coli DnaK's (Hsp70) ATPase in vitro, thus behaving like a bona fide cochaperone. Furthermore, small t amino acids 83 to 174, which are adjacent to the viral J domain, can replace the E. coli DnaJ J-domain glycine-phenylalanine-rich domain, immediately adjacent to the J-domain sequences, even in the absence of significant amino acid similarity to their DnaJ counterpart. Taken together, our studies demonstrate that functionally related Hsp40 proteins from mammalian viral systems can be rapidly studied in bacteria and exploited to probe the universally conserved Hsp70 chaperone machine mechanism.
Sullivan, C.S., and J.M. Pipas (2002) T antigens of simian virus 40: molecular chaperones for viral replication and tumorigenesis. Microbiol. Mol. Biol. Rev. 66:179-202 . Simian virus 40 (SV40) is a small DNA tumor virus that has been extensively characterized due to its relatively simple genetic organization and the ease with which its genome is manipulated. The large and small tumor antigens (T antigens) are the major regulatory proteins encoded by SV40. Large T antigen is responsible for both viral and cellular transcriptional regulation, virion assembly, viral DNA replication, and alteration of the cell cycle. Deciphering how a single protein can perform such numerous and diverse functions has remained elusive. Recently it was established that the SV40 T antigens, including large T antigen, are molecular chaperones, each with a functioning DnaJ domain. The molecular chaperones were originally identified as bacterial genes essential for bacteriophage growth and have since been shown to be conserved in eukaryotes, participating in an array of both viral and cellular processes. This review discusses the mechanisms of DnaJ/Hsc70 interactions and how they are used by T antigen to control viral replication and tumorigenesis. The use of the DnaJ/Hsc70 system by SV40 and other viruses suggests an important role for these molecular chaperones in the regulation of the mammalian cell cycle and sheds light on the enigmatic SV40 T antigen-a most amazing molecule.
Fewell, S.W., J.M. Pipas, and J.L. Brodsky (2002) Mutagenesis of a functional chimeric gene in yeast identifies mutations in the simian virus 40 large T antigen J domain. Proc. Natl. Acad. Sci., USA 99:2002-2007 Simian virus 40 large T antigen contains an amino terminal J domain that catalyzes T antigen-mediated viral DNA replication and cellular transformation. To dissect the role of the J domain in these processes, we exploited the genetic tools available only in the yeast Saccharomyces cerevisiae to isolate 14 loss-of-function point mutations in the T antigen J domain. This screen also identified mutations that, when engineered into simian virus 40, resulted in T antigen mutants that were defective for the ability to support viral growth, to transform mammalian cells in culture, to dissociate the p130-E2F4 transcription factor complex, and to stimulate ATP hydrolysis by hsc70, a hallmark of J domain-containing molecular chaperones. These data correlate the chaperone activity of the T antigen J domain with its roles in viral infection and cellular transformation and support a model by which the viral J domain recruits the cytoplasmic hsc70 molecular chaperone in the host to rearrange multiprotein complexes implicated in replication and transformation. More generally, this study presents the use of a yeast screen to identify loss-of-function mutations in a mammalian virus and can serve as a widely applicable method to uncover domain functions of mammalian proteins for which there are yeast homologues with selectable mutant phenotypes.
Sullivan, C.S., and J.M. Pipas (2001) The virus-chaperone connection. Virology 287:1-8 Maya Angelou once said, "love is like a virus". Indeed, viruses, like love, sometimes require chaperones (of the molecular variety) to foster proper development. Molecular chaperones play an important role in the life cycles of a diverse array of viruses. This is perhaps not surprising given the numerous cellular activities that involve chaperones, and the dependence of viruses on host apparatuses for their propagation. This review is intended to introduce the reader to some viral activities where chaperones play a role, as well as to provide examples of the better studied virus±chaperone interactions. In-depth reviews of chaperones and their functions can be found elsewhere. Sáenz-Robles, M.T., C.S. Sullivan, and J.M. Pipas (2001) Transforming functions of Simian Virus 40. Oncogene 26:7899-7907 Tremblay, J.D., K.F. Sachsenmeier, and J.M. Pipas (2001) Propagation of wild-type and mutant SV40. Methods Mol. Biol. 165:1-7 Pipas, J.M., and A.J. Levine (2001) Role of T antigen interactions with p53 in tumorigenesis. Semin. Cancer Res. 11:23-30 SV40 induces neoplastic transformation by disabling several key cellular growth regulatory circuits. Among these are the Rb- and p53-families of tumor suppressors. The multifunctional, virus-encoded large T antigen blocks the function of both Rb and p53. Large T antigen uses multiple mechanisms to block p53 activity, and this action contributes to tumorigenesis, in part, by blocking p53-mediated growth suppression and apoptosis. Since the p53 pathway is inactivated in most human tumors, T antigen/p53 interactions offer a possible mechanism by which SV40 contributes to human cancer. Sachsenmeier, K.F., and J.M. Pipas (2001) Inhibition of Rb and p53 is insufficient for SV40 T-Antigen transformation. Virology 283:40-48 The SV40 large T-antigen (TAg) has proven useful in studying pathways involved with cell division and tissue homeostasis. TAg disrupts the normal action of tumor suppressors pRb and p53. It is unclear whether T-antigen inhibition of p53 and pRb is sufficient for oncogenic transformation or if additional T-antigen activities are required. To pursue this question, cell lines were generated that coexpress an amino-terminal fragment of T-antigen (TAgN136), which has been shown to be sufficient to block pRb function, together with a dominant-negative p53. Neither focus formation nor saturation density was enhanced by coexpression of the dominant-negative p53 molecule, p53DD, along with TAgN136. Furthermore, a full-length TAg mutant incapable of binding p53 was capable of relieving contact inhibition, a hallmark of transformation. These results suggest the presence of a novel transforming activity in addition to the binding and inactivation of p53, requiring TAg amino acids 137 to 708. Sullivan, C.S., S.P. Gilbert, and J.M. Pipas (2001) ATP-Dependent Simian Virus 40 T-Antigen-Hsc70 Complex Formation. J. Virol. 75:1601-1610 Simian virus 40 large T antigen is a multifunctional oncoprotein that is required for numerous viral functions and the induction of cellular transformation. T antigen contains a J domain that is required for many of its activities including viral DNA replication, transformation, and virion assembly. J-domain-containing proteins interact with Hsc70 (a cellular chaperone) to perform multiple biological activities, usually involving a change in the conformation of target substrates. It is thought that Hsc70 associates with T antigen to assist in performing its numerous activities. However, it is not clear if T antigen binds to Hsc70 directly or induces the binding of Hsc70 to other T-antigen binding proteins such as pRb or p53. In this report, we show that T antigen binds Hsc70 directly with a stoichiometry of 1:1 (dissociation constant = 310 nM Hsc70). Furthermore, the T-antigen-Hsc70 complex formation is dependent upon ATP hydrolysis at the active site of Hsc70 (ATP dissociation constant = 0.16 ?M), but T-antigen-Hsc70 complex formation does not require nucleotide hydrolysis at the T-antigen ATP binding site. N136, a J domain-containing fragment of T antigen, does not stably associate with Hsc70 but can form a transient complex as assayed by centrifugation analysis. Finally, T antigen does not associate stably with either of two yeast Hsc70 homologues or an amino-terminal fragment of Hsc70 containing the ATPase domain. These results provide direct evidence that the T-antigen-Hsc70 interaction is specific and that this association requires multiple domains of both T antigen and Hsc70. This is the first demonstration of a nucleotide requirement for the association of T antigen and Hsc70 and lays the foundation for future reconstitution studies of chaperone-dependent tumorigenesis induced by T antigen.
Rempel, R.E., M.T. Sáenz-Robles, R. Storms, S. Morham, S. Ishida, A. Engel, L. Jakoi, M.F. Melhem, J.M. Pipas, C. Smith, and J.R. Nevin (2000) Loss of E2F4 activity leads to abnormal development of multiple cellular lineages. Mol. Cell 6:293-306 We have generated mice deficient in E2F4 activity, the major form of E2F in many cell types. Analysis of newborn pups deficient in E2F4 revealed abnormalities in hematopoietic lineage development as well as defects in the development of the gut epithelium. Specifically, we observed a deficiency of various mature hematopoietic cell types together with an increased number of immature cells in several lineages. This was associated with an increased frequency of apoptotic cells. We also found a substantial reduction in the thickness of the gut epithelium that normally gives rise to crypts as well as a reduction in the density of villi. These observations suggest a critical role for E2F4 activity in controlling the maturation of cells in a number of tissues. Sullivan, C.S., P. Cantalupo, and J.M. Pipas (2000) The molecular chaperone activity of simian virus 40 large T antigen is required to disrupt Rb-E2F family complexes by an ATP-dependent mechanism. Mol. Cell. Biol. 20:6233-6243 The simian virus 40 large T antigen (T antigen) inactivates tumor suppressor proteins and therefore has been used in numerous studies to probe the mechanisms that control cellular growth and to generate immortalized cell lines. Binding of T antigen to the Rb family of growth-regulatory proteins is necessary but not sufficient to cause transformation. The molecular mechanism underlying T-antigen inactivation of Rb function is poorly understood. In this study we show that T antigen associates with pRb and p130-E2F complexes in a stable manner. T antigen dissociates from a p130-E2F-4-DP-1 complex, coincident with the release of p130 from E2F-4-DP-1. The dissociation of this complex requires Hsc70, ATP, and a functional T-antigen J domain. We also report that the "released" E2F-DP-1 complex is competent to bind DNA containing an E2F consensus binding site. We propose that T antigen disrupts Rb-E2F family complexes through the action of its J domain and Hsc70. These findings indicate how Hsc70 supports T-antigen action and help to explain the cis requirement for a J domain and Rb binding motif in T-antigen-induced transformation. Furthermore, this is the first demonstration linking Hsc70 ATP hydrolysis to the release of E2F bound by Rb family members.
Sullivan, C.S., J.D. Tremblay, S.W. Fewell, J.A. Lewis, J.L. Brodsky, and J.M. Pipas (2000) Species-specific elements in the large T-antigen J domain are required for cellular transformation and DNA replication by simian virus 40. Mol. Cell. Biol. 20:5749-5757 The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo.
Cantalupo, P., M.T. Sáenz-Robles, and J.M. Pipas (1999) Expression of SV40 large T antigen in baculovirus systems and purification by immunoaffinity chromatography. Methods Enzymol. 306:297-307 Slinskey, A., D. Barnes, and J.M. Pipas (1999) Simian virus 40 large T antigen J domain and Rb-binding motif are sufficient to block apoptosis induced by growth factor withdrawal in a neural stem cell line. J. Virol. 73:6791-6799 Serum-free mouse embryo (SFME) cells are a neural stem cell line that is dependent upon epidermal growth factor (EGF) for survival. Removal of EGF results in the G1 arrest and apoptosis of SFME cells. We have shown that the expression of simian virus 40 large T antigen in SFME cells blocks apoptosis and allows cell survival and division in the absence of EGF. Therefore the presence of T antigen abrogates the EGF requirement. The steady-state levels of p53, p21, and mdm-2 do not increase as SFME cells undergo apoptosis upon EGF withdrawal. Furthermore, the amino-terminal 136 amino acids (N136) of T antigen are sufficient to block death and to promote proliferation in the absence of EGF, while the carboxy-terminal fragment (C251-708), which contains the p53 binding site, is unable to block death. Taken together, these data suggest that SFME cells deprived of EGF undergo p53-independent apoptosis. Mutations that disrupt either the J domain or Rb family binding abolish the ability of T antigen to block SFME cell apoptosis and to promote cell growth. We conclude that T antigen must act on one or more members of the Rb family to inhibit SFME cell apoptosis.
Ibaraki, N., S.C. Chen, L.-R. Lin, H. Okamoto, J.M. Pipas, and V.N. Reddy (1998) Human lens epithelial cell line. Exp. Eye Res. 67:577-585 Robinson, C.G., and J.M. Pipas (1998) SV40 large tumor antigen (T antigen): database of mutants. Nucleic Acids Res. 26:295-296 The SV40 T antigen database (http://www.pitt.edu/~pipaslab/) lists viruses and plasmids expressing mutant forms of large T antigen. Each entry contains information regarding the mutant designation, mutant type, virus strain, nucleotide change, amino acid change and pertinent references. The database is now available as an internet searchable index.
Brodsky, J.L., and J.M. Pipas (1998) Polyomavirus T antigens: molecular chaperones for multiprotein complexes. J. Virol. 72:5329-5334
Pipas, J.M. (1998) Molecular chaperone function of the SV40 large T antigen. Dev. Biol. Stand. 94:313-319
The SV40 large T antigen (T antigen) is a potent viral oncogene capable of inducing tumours in test animals and transforming cells in culture. T antigen possesses multiple transforming functions that act in a cell-type dependent manner. One of these transforming functions requires a physical association between T antigen and the cellular tumour suppressor p53, while another requires T antigen binding to the retinoblastoma family of tumour suppressors. A third transforming function, < Higashino, F., J.M. Pipas, and T. Shenk (1998) Adenovirus E4orf6 oncoprotein modulates the function of the p53-related protein, p73. Proc. Natl. Acad. Sci., USA 95:15683-15687 Recently, several proteins have been identified that are related in their sequence to the p53 tumor-suppressor protein. One of these proteins, which is termed p73, exhibits sequence homology to the p53 transcriptional activation, DNA binding, and oligomerization domains. The adenovirus E1B 55-kDa protein, the adenovirus E4orf6 protein, and SV40 T antigen each can bind to p53 and inhibit p53 function. Here we demonstrate that the adenovirus E4orf6 protein, but not the E1B 55-kDa protein or T antigen, interacts with p73. The E4orf6 protein inhibits p73-mediated transcriptional activation and cell killing in a manner similar to its effect on p53. Thus, only a subset of viral oncoproteins that antagonize p53 function also interacts with the related p73 protein.
Peden, K.W.C., A. Srinivasan, J.V. Vartikar, and J.M. Pipas (1998) Effects of mutations within the SV40 large T antigen ATPase/p53 binding domain on viral replication and transformation. Virus Genes 16:153-165 The simian virus 40 (SV40) large T antigen is a 708 amino-acid protein possessing multiple biochemical activities that play distinct roles in productive infection or virus-induced cell transformation. The carboxy-terminal portion of T antigen includes a domain that carries the nucleotide binding and ATPase activities of the protein, as well as sequences required for T antigen to associate with the cellular tumor suppressor p53. Consequently this domain functions both in viral DNA replication and cellular transformation. We have generated a collection of SV40 mutants with amino-acid deletions, insertions or substitutions in specific domains of the protein. Here we report the properties of nine mutants with single or multiple substitutions between amino acids 402 and 430, a region thought to be important for both the p53 binding and ATPase functions. The mutants were examined for the ability to produce infectious progeny virions, replicate viral DNA in vivo, perform in trans complementation tests, and transform established cell lines. Two of the mutants exhibited a wild-type phenotype in all these tests. The remaining seven mutants were defective for plaque formation and viral DNA replication, but in each case these defects could be complemented by a wild-type T antigen supplied in trans. One of these replication-defective mutants efficiently transformed the REF52 and C3H10T1/2 cell lines as assessed by the dense-focus assay. The remaining six mutants were defective for transforming REF52 cells and transformed the C3H10T1/2 line with a reduced efficiency. The ability of mutant T antigen to transform REF52 cells correlated with their ability to induce increased levels of p53. Rushton, J.J., D. Jiang, A. Srinivasan, J.M. Pipas, and P.D. Robbins (1997) Simian virus 40 T antigen can regulate p53-mediated transcription independent of binding p53. J. Virol. 71:5620-5623 A simian virus 40 (SV40) T-antigen mutant containing only the N-terminal 136 amino acids, able to bind to Rb and p300 but not p53, partially inhibited p53-mediated transcription without affecting the ability of p53 to bind DNA. These results suggest that SV40 T antigen can regulate p53-mediated transcription either directly through protein-protein association or indirectly through interaction with factors which may function to confer p53-mediated transcription.
Castellino, A.M., P. Cantalupo, I.M. Marks, J.V. Vartikar, K.W. Peden, and J.M. Pipas (1997) trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP. J. Virol. 71:7549-7559 Simian virus 40 (SV40) DNA replication requires the coordinated action of multiple biochemical activities intrinsic to the virus-encoded large tumor antigen (T antigen). We report the preliminary biochemical characterization of the T antigens encoded by three SV40 mutants, 5030, 5031, and 5061, each of which have altered residues within or near the ATP binding pocket. All three mutants are defective for viral DNA replication in cultured cell lines. However, while 5030 and 5031 can be complemented in vivo by providing a wild-type T antigen in trans, 5061 exhibits a strong trans-dominant-negative phenotype. In order to determine the basis for their replication defects and to explore the mechanisms of trans dominance, we purified the T antigens encoded by each of these mutants and examined their activities in vitro. The 5061 T antigen had no measurable ATPase activity and failed to hexamerize in response to ATP, and its affinity for the SV40 origin of DNA replication (ori) DNA was not increased by ATP. In contrast, the 5030 and 5031 T antigens exhibited at least some ATPase activity and both readily formed hexamers in the presence of ATP. These mutants differed in that 5030 was very defective in an ori-dependent unwinding assay while 5031 retained significant activity. Both the 5030 and 5031 T antigens bound to ori-containing DNA, but the binding was less efficient than that of wild-type T antigen and was not affected by the presence of ATP. These results suggest that 5030 and 5031 are defective in some aspect of communication between the ATP binding and DNA binding domains and that the ability of ATP to induce T-antigen hexamerization is distinct from its action to increase the affinity for ori. Finally, all three mutants were defective for the ability to support SV40 DNA replication in vitro. Both the 5031 and 5061 T antigens inhibited wild-type-T-antigen-stimulated replication in vitro, while the 5030 T antigen did not. The fact that the 5031 T antigen was trans dominant in the in vitro assays but not in vivo indicates that the in vitro system does not accurately reflect events occurring in vivo.
Srinivasen, A., A.J. McClellan, J. Vartikar, I. Marks, P. Cantalupo, Y. Li, P. Whyte, K. Rundell, J.L. Brodsky, and J.M Pipas (1997) The amino-terminal transforming region of SV40 large and small T antigens function as a J-domain. Mol. Cell. Biol. 17:4761-4773 Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of large T antigen to transform mammalian cells. To examine whether a purified 136-amino-acid fragment from the T antigen amino terminus acts as a DnaJ-like chaperone, we investigated whether this fragment stimulates the ATPase activity of two hsc70s and discovered that ATP hydrolysis is stimulated four- to ninefold. In addition, ATPase-defective mutants of full-length T antigen, as well as wild-type small t antigen, stimulated the ATPase activity of hsc70. T antigen derivatives were also able to release an unfolded polypeptide substrate from an hsc70, an activity common to DnaJ chaperones. Because the J domain of T antigen plays essential roles in viral DNA replication, transcriptional control, virion assembly, and tumorigenesis, we conclude that this region may chaperone the rearrangement of multiprotein complexes.
Campbell, K.S., K. Mullane, I. Aksoy, J.M. Pipas, P.A. Silver, T.M. Roberts, B.S. Schaffhausen, and J.A. DeCaprio (1997) DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes efficient viral DNA replication. Genes Dev. 11:1098-1110 The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family. Genetic analyses demonstrated that amino-terminal sequences of TAg comprise a novel J-domain that mediates a specific interaction with the constitutively expressed hsc70 and show that the J-domain is also required for efficient viral DNA replication in vivo. Furthermore, we demonstrated that the J-domain of two human DnaJ homologs, HSJ1 or DNAJ2, could substitute functionally for the amino-terminus of TAg in promoting viral DNA replication. Together, our findings suggest that TAg uses its J-domain to support SV40 DNA replication in a manner that is strikingly similar to the use of Escherichia coli DnaJ by bacteriophage lambda in DNA replication. However, TAg has evolved a more efficient strategy of DNA replication through an intrinsic J-domain to associate directly with a partner chaperone protein. Our observations provide evidence of a role for chaperone proteins in the process of eukaryotic DNA replication. Kierstead, T.D., and J.M. Pipas (1996) Database of mutations that alter the large tumor antigen in simian virus 40. Nucleic Acids Res. 24:125-126 The SV40 T antigen database is a listing of plasmids and/or viruses that express mutant forms of the virus-encoded large T antigen protein. The parental virus strain, nucleic acid sequence of the mutations, the effect of the mutation on the T antigen amino acid sequence, and key references are included in the listing. The database is available from the authors as a Macintosh FileMaker Pro file, and as a hard copy printout.
Okamoto, S., M. Ohji, K. Kosaku, N. Sundarraj, J.R. Hassell, R.A. Thoft, and J.M. Pipas (1995) Characterization of immortalized rabbit corneal epithelial cells with SV40 large T antigen. Jpn. J. Ophthalmol. 39:323-333 The authors developed a line of immortalized rabbit corneal epithelial cells by transfecting a primary culture of New Zealand White (NZW) rabbit corneal epithelial cells with Simian virus 40 (SV40) T antigen gene using a calcium phosphate precipitation method. The transfected cells, which survived more than 40 passages and 400 population doublings, showed some ability to form colonies in soft agar, and had doubling times and plating efficiencies not significantly different from those of untransfected cells. The epithelial nature of the transfected cells was confirmed by immunofluorescence staining with the antibody to 64 kD keratin (AE5), a specific marker for corneal type epithelial cells. Immunofluorescence microscopic examination revealed that the transfected cells expressed major basement membrane components of epithelial cells, including collagen types IV and VII and laminin. Electron microscopic studies demonstrated the presence of microvilli and intercellular in cultured transfected cells. These results indicate that the transfected cells retain some of the normal phenotypic characteristics of corneal epithelial cells and may therefore be used to study the corneal epithelium in vitro. Collins, B.S., and J.M. Pipas (1995) T antigens encoded by replication-defective simian virus 40 mutants dl1135 and 5080. J. Biol. Chem. 270:15377-15384 We present a preliminary biochemical characterization of two simian virus 40 mutants that affect different T antigen replication functions. SV40 T antigen mutants dl1135 (delta 17-27 amino acids) and 5080 (P-L) have been studied extensively with regard to their ability to transform cells in culture and induce tumors in transgenic mice. Both mutants are defective for viral DNA replication in vivo. In order to assess in more detail the molecular basis for the in vivo replication defects of 5080 and dl1135, we expressed the mutant proteins using the baculovirus system and purified them by immunoaffinity chromatography. With each of the purified proteins, we examined some of the biochemical activities of T antigen required for replication, viz. ATPase, binding to the origin of replication (ori) and assembly on ori, DNA helicase and unwinding, and replication in in vitro assays. Consistent with previous studies, we found that the 5080 protein is defective for multiple biochemical activities including ATPase, helicase, ori-specific unwinding, and ATP-induced hexamerization. However, this mutant retains some sequence-specific DNA binding activity. In contrast, the dl1135 protein exhibited significant levels of activity in all assays, including the ability to drive SV40 DNA replication in vitro. Thus, dl1135 is one of several mutants with an altered amino-terminal domain which can replicate DNA in vitro, but not in vivo. Thus, while the 5080 mutation affects a T antigen enzymatic function directly required for viral DNA synthesis, dl1135 may alter an activity required to prepare the cell for viral replication.
Quartin, R.S., C.N. Cole, J.M. Pipas, and A.J. Levine (1994) The amino-terminal functions of the simian virus 40 large T antigen are required to overcome wild-type p53-mediated growth arrest of cells. J. Virol. 68:1334-1341 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.
Spence, J.T., and J.M. Pipas (1994) Simian virus 40 large T antigen host range domain functions in virion assembly. J. Virol. 68:4227-4240 The simian virus 40 (SV40) T antigen host range mutants dl1066 and dl1140 display a postreplicative block to plaque formation which suggests a novel role for T antigen late in the viral life cycle. The host range mutants dl1066 and dl1140 are able to grow in and plaque on BSC but not on CV1 monkey kidney cells, a normally permissive host. Previous work showed that in CV1 cells infected with dl1066 and dl1140, levels of viral DNA replication and of late capsid protein accumulation were only slightly reduced and the failure to accumulate agnoprotein was not likely to be the major factor responsible for the mutants' growth defect. Here we show that the host range mutants are defective in the assembly of viral particles. SV40 assembly proceeds as the progressive conversion of 75S viral chromatin complexes to 200S-240S assembled virions. When virus-infected cell extracts are separated on 5 to 40% sucrose gradients, wild-type extracts show the greatest accumulation of viral late protein in the 200S-240S fractions corresponding to the assembled virus peak and lesser amounts in the 75S-150S fractions corresponding to immature assembly intermediates. The host range mutants dl1066 and dl1140 grown in nonpermissive CV1 cells, however, failed to assemble any appreciable amounts of mature 200S-240S virions and accumulate 75S intermediates, whereas in permissive BSC cells, levels of assembly were more slightly reduced than those of the wild type. Analysis of the protein composition of gradient fractions suggests that SV40 assembly proceeds by a mechanism similar to that proposed for polyomavirus and suggests that the host range blockage may result from a failure of such mutants to add VP1 to 75S assembly intermediates. Spence, S.L., and J.M. Pipas (1994) SV40 large T antigen functions at two distinct steps in virion assembly. Virology 204:200-209 The SV40 large T antigen mutant 5002 has two amino acid substitutions (L19-F; P28-S) and is defective for productive viral infection as demonstrated by its small plaques that arise very late and by a 100-fold reduced yield of infectious progeny. 5002 replicates viral DNA at the same time postinfection as wild-type SV40, and the production of progeny DNA molecules is only marginally reduced. Furthermore, the viral capsid proteins accumulate to near normal levels following infection with 5002. In this manuscript we report evidence that 5002 infection is blocked at a specific stage of viral assembly. The SV40 viral assembly pathway involves conversion of 75S chromatin complexes to 240S virions. Unlike mutants within the T antigen host range (HR) domain, that are also defective for viral assembly and accumulate 75S particles (Spence and Pipas, 1994), 5002 particles are blocked as 150S previrions containing viral DNA and capsid proteins. We have previously shown that 5002 and HR mutants cooperate to produce viable progeny in trans complementation tests. Thus, by two criteria, SV40 large T antigen encodes two distinct activities that function at different steps in virion assembly.
Kim, S.H., K.A. Roth, C.M. Coopersmith, J.M. Pipas, and J.I. Gordon (1994) Expression of wild-type and mutant simian virus 40 large tumor antigens in villus-associated enterocytes of transgenic mice. Proc. Natl. Acad. Sci, USA 91:6914-6918 The four principal gut epithelial cell lineages undergo continuous and rapid renewal during a geographically well-organized migration along the crypt-to-villus axis. The molecules that regulate their proliferation and differentiation programs are largely unknown. The large tumor antigen (TAg) of wild-type (wt) simian virus 40 (SV40) and its mutant derivatives represent tools for describing the contributions of regulators of the cell cycle to the proliferative state of each lineage. Expression of SV40 TAgwt in postmitotic, villus-associated enterocytes of transgenic mice causes them to reenter the cell cycle without an apparent effect on their state of differentiation. When human KRAS with a Val-12 substitution ([Val12]KRAS) is coexpressed with SV40 TAgwt in villus enterocytes of bitransgenic animals, the two oncoproteins cooperate to produce dedifferentiation (dysplasia). SV40 mutant d11137 expresses a TAg that is unable to complex with p53 but retains N-terminal transforming functions, including the ability to complex pRB, p107, and p300. When SV40 TAgd11137 is expressed in villus enterocytes, they reenter into the cell cycle. However, coexpression of SV40 TAgd11137 and [Val12]KRAS does not produce dysplastic changes. Thus, the N-terminal 121 residues of TAg are sufficient to perturb the proliferative state of the enterocyte but not to produce detectable changes in the state of differentiation when coexpressed with [Val12]KRAS.
Finley, G.G., R.A. Koski, M.F. Melhem, J.M. Pipas, and A.I. Meisler (1993) Expression of the gastrin gene in the normal human colon and colorectal adenocarcinoma. Cancer Res. 53:2919-2926 Gastrin, produced in the G-cells of the gastric antrum and regulating acid secretion in the stomach, also acts as a trophic factor in the gastrointestinal tract. Because of its possible role in colon cell proliferation and differentiation, evidence for its presence in normal colorectal mucosa and adenocarcinoma was sought. Utilizing tumors and matched normal mucosa from 26 patients, mature gastrin and progastrin were studied by immunohistochemistry. In normal colonic mucosal crypts, occasional cells stained concordantly for gastrin, progastrin, and chromogranin A, suggesting that they are of neuroendocrine origin. Adenomatous polyps stained neither for gastrin nor chromogranin A. In 22 of 23 adenocarcinomas, more than 50% of tumor cells stained for gastrin and progastrin. The expected gastrin transcript was demonstrable by polymerase chain reaction and RNase protection in tumors and by polymerase chain reaction in normal mucosa. Its identity was confirmed by sequencing the polymerase chain reaction product. A larger transcript containing Intron II was present in both cancers and normal mucosa but was barely discernible in the gastric antrum. Aberrant expression of gastrin may contribute to deregulated proliferation of many colorectal carcinomas. Pipas, J.M., K. Pogue-Geile, G.G. Finley, C.A. Cartwright, and A.I. Meiesler (1993) Gene structure and expression in colorectal cancer. Adv. Exp. Med. Biol. 330:67-75 Pipas JM, Pogue-Geile K, Finley GG, Cartwright CA, Meiesler AI Mack, D.H., J. Vartikar, J.M. Pipas, and L.A. Laimins (1993) Specific repression of TATA-mediated but not initiator-mediated transcription by wild-type p53. Nature 363:281-283 The p53 protein is apparently central to the development of human cancers because both alleles are often found to be mutated in different tumour types. In addition, wild-type p53 can inhibit transformation by viral and cellular oncogenes in vitro, so p53 has been classified as a tumour suppressor. Investigations of the normal function of p53 have indicated that at least one of its functions could involve the activation of gene expression through the binding of specific DNA-regulatory sequences. Also, overexpression of p53 can mediate growth arrest and repress transcription from a variety of promoters. We demonstrate here both in vivo and in vitro that expression of wild-type p53 specifically represses the activity of promoters whose initiation is dependent on the presence of a TATA box. Promoters whose accurate transcription is directed by a pyrimidine-rich initiator element, however, are immune to the effects of p53. Furthermore, we observe that repression is mediated by an interaction of p53 with basal transcription factor(s). Thus, p53 appears to repress the activity of certain promoters through direct communication with TATA box-dependent basal transcription machinery. Symonds, H.S., S.A. McCarthy, J. Chen, J.M. Pipas, and T. Van Dyke (1993) Use of transgenic mice reveals cell-specific transformation by a simian virus 40 T-antigen amino-terminal mutant. Mol. Cell. Biol. 13:3255-3265 We have used the multifunctional transforming protein, simian virus 40 T antigen, as a probe to study the mechanisms of cell growth regulation in the intact organism. T antigen appears to perturb cell growth, at least in part, by stably interacting with specific cellular proteins that function to maintain normal cell growth properties. Experiments in cultured cells indicate that at least three distinct regions of simian virus 40 T antigen have roles in transformation. Two regions correlate with the binding of known cellular proteins, p53, pRB, and p107. A third activity, located near the amino terminus, has been defined genetically but not biochemically. By targeting expression of wild-type and mutant forms of T antigen to distinct cell types in transgenic mice, we have begun to systematically determine which activities play a role in tumorigenesis of each cell type. In this study, we sought to determine the role of the amino-terminal transformation function with such an analysis of the T-antigen mutant dl1135. This protein, which lacks amino acids 17 to 27, retains the p53-, pRB-, and p107-binding activities yet fails to transform cells in culture. To direct expression in transgenic mice, we used the lymphotropic papovavirus transcriptional signals that are specific for B and T lymphocytes and the choroid plexus epithelium of the brain. We show here that although defective in cell culture, dl1135 specifically induced the development of thymic lymphomas in the mouse. Expression of the protein was routinely observed in B- and T-lymphoid cells, although B-cell abnormalities were not observed. Choroid plexus tumors were observed only infrequently; however, dl1135 was not consistently expressed in this tissue. Within a given transgenic line, the penetrance of T-cell tumorigenesis was 100% but appeared to require secondary events, as judged from the clonal nature of the tumors. These experiments suggest that the amino-terminal region of T antigen has a role in the transformation of certain cell types (such as fibroblasts in culture and B lymphocytes) but is dispensable for the transformation of T lymphocytes.
Peden, K.W., and J.M. Pipas (1992) Simian virus 40 mutants with amino-acid substitutions near the amino terminus of large T antigen. Virus Genes 6:107-118 A series of amino-acid substitution mutants has been made with changes in the region of simian virus 40 large tumor antigen (T antigen) that is shared with the small tumor antigen (t antigen). Both single and multiple amino-acid replacements were obtained using the heteroduplex deletion loop method and sodium bisulfite as the mutagen. The mutants could be divided into five phenotypic classes on the basis of their biological properties: a) mutants whose changes did not affect their ability to propagate on permissive monkey cells, nor to transform nonpermissive rodent cells; b) mutants that were not viable, replicated their DNA to 5% or less of wild type, but were positive for transformation; c) mutants that were not viable, replicated their DNA to 5% or less of wild type, and were defective for transformation; and d) mutants that completely lost all three activities coordinately. In addition, one mutant with changes in this region, 5002, replicated its DNA to about 50% of wild type, had an impaired transformation activity, and produced virions at a level of about 4% that of wild type. Chen, J., G.J. Tobin, J.M. Pipas, and T. Van Dyke (1992) T-antigen mutant activities in vivo: roles of p53 and pRB binding in tumorigenesis of the choroid plexus. Oncogene 7:1167-1175 To study the mechanism by which SV40 large T antigen transforms cells under physiological conditions, we analysed several mutant forms of T antigen for their ability to induce cell proliferation and tumorigenesis in transgenic mice. These mutant proteins, which differ in their ability to form complexes with the tumor suppressors pRB and p53, were analysed under conditions in which wild-type T antigen induces choroid plexus papillomas as a result of uniform proliferation of the entire choroid plexus epithelium. The results presented here show that binding of T antigen to p53 is not required for induction of choroid plexus tumors. However, tumorigenesis does appear to require the binding of T antigen to pRB/p107. An additional activity, resident in the amino-terminal one-fifth of the protein, may also play a role. These experiments indicate the importance of whole-animal assays in determining the molecular basis of transformation, since each of these mutants possessed similar transformation phenotypes in culture but showed distinct phenotypes in the choroid plexus of the animal. Pipas, J.M. (1992) Common and unique features of T antigens encoded by the polyomavirus group. J. Virol. 66:3979-3985 Although 12 different members of the polyomavirus group have now been identified, only SV40 and PyV have been studied extensively. Whereas each member of the group shows a restricted host range, viruses infecting species from birds to humans have been reported. Although little is known concerning the biology of natural infections in the wild, it is apparent that these viruses exhibit various cell-type tropisms. Some viruses, such as LPV (B lymphocytes) or KV (pulmonary endothelium), are tightly restricted to specific cell types, while others, such as PyV, infect a variety of tissues in the animal. Despite these differences, all polyomaviruses share a common strategy of productive infection, expressing T antigens which act both on cellular targets, preparing cellular metabolism for supporting optimal viral replication, and then on targets within the viral genome, to regulate viral DNA replication, transcription, and assembly. Presumably, this common replication strategy restricts the degree to which the sequences of these viruses can diverge. Thus, sequence motifs conserved among these different viruses may indicate key structural elements essential for biochemical function. In this article I have compared the sequences of all polyomavirus-encoded large and small T antigens sequenced to date. This has led to the following conclusions and speculations. (i) Comparison of the domain organization of different large T antigens reveals that these proteins fall into two structural classes. Members of the SV40 class, which include SV40, JCV, BKV, and SA12, possess a carboxyl-terminal domain, which in SV40 has been shown to be dispensable for viral DNA replication but essential for virion assembly. The PyV class lacks the carboxyl-terminal domain and carries additional amino acids within the amino-terminal domain. When total amino acid identity is examined, members of the SV40 class show the highest degree of conservation (65 to 85%), while sequence identity among the remaining viruses varies from 18 to 55%. (ii) The DNA binding domains of most large T antigens are closely related, with amino acid identities ranging from 35 to 86%. Several residues within this domain are invariant among all T antigens. All of these viruses have multiple copies of the consensus T-antigen-binding pentanucleotide (GAGGC) in their ori region, suggesting that all T antigens recognize this sequence. The single exception is the large T antigen encoded by the avian virus BFDV. The putative DNA binding domain of this protein shows little or no sequence relation to that of other T antigens. Furthermore, the GAGGC motif is not found in the ori region of this virus.
Melhem, M.F., A.I. Meisler, G.G. Finley, W.H. Bryce, M.O. Jones, I.I. Tribby, J.M. Pipas, and R.A. Koski (1992) Distribution of cells expressing myc proteins in human colorectal epithelium, polyps, and malignant tumors. Cancer Res. 52:5853-5864 The myc gene family encodes nuclear phosphoproteins that are thought to play a role in the control of cellular proliferation and differentiation. We have undertaken an immunohistochemical study assessing the expression of myc gene family proteins in individual cells of normal colonic mucosa, colorectal polyps, and colorectal adenocarcinomas. We screened a panel of mouse monoclonal antibodies that we raised against recombinant human c-myc and N-myc proteins for recognition of myc proteins in paraffin tissue sections. Two of these antibodies, H120C69 and H8C150, were selected for indirect immunoperoxidase staining of tissue sections from 16 normal mucosas, 24 polyps, and 30 adenocarcinomas. In normal colon, about 25% of the cells in the lower one-third of the crypts of Lieberkuhn stain for myc-related protein. This distribution resembles that of proliferating cells in the crypt. Benign hyperplastic polyps resemble normal mucosa in their myc staining pattern, with about 25% of the cells positive. In adenomatous polyps, the putative precursors of adenocarcinomas, from 50 to 100% of the cells stain positively for myc protein. In these cases, stained cells extend to the luminal surface, consistent with the previously reported expansion of the proliferation zone in these lesions. All adenocarcinomas examined had increased levels of myc protein relative to normal mucosa. The tumor cells exhibited markedly heterogeneous myc staining patterns, both among different tumors and, in some cases, within a single tumor. Comparison with Ki-67 monoclonal antibody staining indicates that myc protein expression in many tumors is uncoupled from cellular proliferation. Surprisingly, we observed increased numbers of myc-expressing cells and increased levels of myc protein in histologically normal colon directly adjacent to tumor, suggesting that many colorectal carcinomas secrete growth factors that activate gene expression in neighboring normal mucosa. Yaciuk, P., M.C. Carter, J.M. Pipas, and E. Moran (1991) Simian virus 40 large-T antigen expresses a biological activity complementary to the p300-associated transforming function of the adenovirus E1A gene products. Mol. Cell Biol. 11:2116-2124 In this report we present evidence that simian virus 40 T antigen encodes a biological activity that is functionally equivalent to the transforming activity lost by deletion of the E1A p300-binding region. T-antigen constructs from which the pRb-binding region has been deleted are virtually unable to induce foci of transformed cells in a ras cooperation assay in primary baby rat kidney cells. Nevertheless, such a construct can cooperate with an E1A N-terminal deletion mutant, itself devoid of transforming activity, to induce foci in this assay. The heterologous trans-cooperating activity observed between E1A and T-antigen deletion products is as efficient as trans cooperation between mutants expressing individual E1A domains. The cooperating function can be impaired by a deletion near the N terminus of T antigen. Such a deletion impairs neither the p53-binding function nor the activity of the pRb-binding region.
Pogue-Geile, K., J.R. Geiser, M. Shu, C. Miller, I.G. Wool, A.I. Meisler, and J.M. Pipas (1991) Ribosomal protein genes are overexpressed in colorectal cancer: isolation of a cDNA clone encoding the human S3 ribosomal protein. Mol. Cell Biol. 11:3842-3849 We have isolated a cDNA clone encoding the human S3 ribosomal protein from a normal human colon cDNA library. The clone was identified as one of many that detected genes whose level of expression was increased in adenocarcinoma of the colon relative to normal colonic mucosa. Increased levels of the S3 transcript were present in the tumors of all eight patients examined. Moreover, the S3 mRNA was also more abundant in 7 of 10 adenomatous polyps, the presumed precursor of carcinoma. Additional studies demonstrated that increased levels of mRNAs encoding several other ribosomal proteins, including S6, S8, S12, L5, and P0, were present in colorectal tumors and polyps. These results suggest that there is increased synthesis of ribosomes in colorectal tumors and that this increase is an early event in colon neoplasia.
Ludlow, J.W., J. Shon, J.M. Pipas, D.M. Livingston, and J.A. DeCaprio (1990) The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T. Cell 60:387-396 Synchronized monkey cells pulse-labeled with [35S]-methionine and chased for various lengths of time were extracted, and immunoprecipitations were performed using monoclonal antibodies directed against the retinoblastoma protein (RB) and SV40 T antigen (T). By following a discrete population of these two proteins through the cell cycle, the following information was obtained. RB, which is wholly unphosphorylated in G1, became phosphorylated at the beginning of S and remained phosphorylated through S and G2. RB was, then, completely dephosphorylated between the end of G2 and the beginning of G1. Second, while all of the detectable unphosphorylated RB can be found complexed with T, these complexes present during G1 dissociated in S and reformed again in M or early G1. Finally, T molecules appeared to oligomerize prior to binding RB. Thus, complex formation between T and RB may be regulated in part by the cell cycle-dependent phosphorylation and dephosphorylation of RB and by the quaternary structure of T. Dyson, N., R. Bernards, S.H. Friend, L.R. Gooding, J.A. Hassell, E.O. Major, J.M. Pipas, T. Vandyke, and E. Harlow (1990) Large T antigens of many polyomaviruses are able to form complexes with the retinoblastoma protein. J. Virol. 64:1353-1356 Stable protein complexes between the large T antigens of mouse, monkey, baboon, or human polyomaviruses and the retinoblastoma protein were detected by an in vitro coimmunoprecipitation assay. All of the large T antigens tested were able to bind to both human and mouse retinoblastoma polypeptides, showing that these interactions have been conserved during evolution.
Peden, K.W., S.L. Spence, L.C. Tack, C.A. Cartwright, A. Srinivasan, and J.M. Pipas (1990) A DNA replication-positive mutant of simian virus 40 that is defective for transformation and the production of infectious virions. J. Virol. 64:2912-2921 Simian virus 40 (SV40) mutant 5002 carries base pair substitutions of C-5109----T and C-5082----T. These mutations lie in a region of the genome that encodes amino acids common to the large and small viral tumor antigens (T and t antigens, respectively) and result in amino acid substitutions of Leu-19----Phe and Pro-28----Ser. In contrast to wild-type SV40, which produces large plaques that are clearly visible 8 days postinfection, mutant 5002 is defective for productive infection, producing tiny plaques that arise at around 21 days postinfection. However, 5002 is capable of replicating viral DNA and producing normal amounts of capsid proteins, indicating that the mutations alter an activity of T antigen that is required subsequent to DNA synthesis, such as maturation, viral assembly, or release of virions. The mutant T antigen has normal ATPase activity, is phosphorylated in a manner that is indistinguishable from that of the wild-type T antigen, and retains the ability to oligomerize. 5002 complements mutants defective in T antigen host range-adenovirus helper function for productive infection. Thus, T antigen encodes two activities that affect at least two different steps in viral infection other than DNA replication, one inactivated by mutations in the host range-adenovirus helper domain and one inactivated by the mutations present in 5002. The 5002-encoded T antigen is also defective for transformation of REF52 cells when expressed from the normal SV40 early promoter, although this defect can be partially overcome by expressing the protein from stronger promoters.
Spence, S.L., L.C. Tack, J.H. Wright, S. Carswell, and J.M. Pipas (1990) Infection of CV1 cells expressing the polyoma virus middle T antigen or the SV40 agnogene product with simian virus 40 host-range mutants. In Vitro Cell Dev. Biol. 26:604-611 SV40 viruses bearing mutations at the carboxy-terminus of large T antigen exhibit a host-range phenotype: such viruses are able to grow in BSC monkey kidney cells at 37 degrees C, but give at least 10,000-fold lower yields than wild type virus in BSC cells at 32 degrees C or in CV1 monkey kidney cells at either temperature. The block to infection in the nonpermissive cell type occurs after the onset of viral DNA replication. Infectious progeny virions are produced at very low efficiency. Although capsid proteins are synthesized at decreased levels, this does not account for the magnitude of the defect. Presumably some step of virion assembly or maturation is affected in these mutants. We have previously reported that the viral agnogene product, a protein thought to be involved in viral assembly or release, fails to accumulate in CV1 cells infected with host-range mutants. In polyoma virus the middle T antigen plays a role in virion maturation by influencing the phosphorylation of capsid proteins. In this communication we show that host-range mutants fail to undergo productive infection of CV1 cells expressing middle T antigen. These mutants do form plaques on an agnoprotein-expressing cell line. However, the agnoprotein does not seem to act by correcting the mutational block but rather increases the efficiency of plaque formation. Peden, K.W., A. Srinivasan, J.M. Farber, and J.M. Pipas (1989) Mutants with changes within or near a hydrophobic region of simian virus 40 large tumor antigen are defective for binding cellular protein p53. Virology 168:13-21 SV40 mutants bearing either amino acid substitution or in-frame deletion/insertion mutations in a region of the gene for large T antigen encoding a stretch of hydrophobic residues were analyzed for their behavior in permissive and nonpermissive cells. One of the mutants, with an Ile(573)-Phe substitution had a phenotype indistinguishable from that of wild-type SV40. The remaining three mutants were not viable and were defective for DNA replication. In addition, they displayed a cell-type specificity with respect to transformation; namely, they transformed the mouse C3H10T1/2 cell line, although with a reduced efficiency relative to wild-type, but were unable to transform the rat REF52 cell line. None of the T antigens from the defective mutants formed a complex with the cellular protein p53, indicating that the T-antigen-p53 complex is not required for the transformation of C3H10T1/2 cells. Cartwright, C.A., M.P. Kamps, A.I. Meisler, J.M. Pipas, and W. Eckhart (1989) pp60c-src activation in human colon carcinoma. J. Clin. Invest. 83:2025-2033 We measured the in vitro protein-tyrosine kinase activity of pp60c-src from human colon carcinoma cell lines and tumors. The activity of pp60c-src from six of nine carcinoma cell lines was higher (on average, fivefold as measured by enolase phosphorylation, or eightfold as measured by autophosphorylation) than that of pp60c-src from normal colonic mucosal cells, or human or rodent fibroblasts. Similarly, the activity of pp60c-src from 13 of 21 primary colon carcinomas was five- or sevenfold higher than that of pp60c-src from normal colonic mucosa adjacent to the tumor. The increased pp60c-src activity did not result solely from an increase in the level of pp60c-src protein, suggesting the specific activity of the pp60c-src kinase is elevated in the tumor cells. pp60c-src from colon carcinoma cells and normal colonic mucosal cells was phosphorylated at similar sites. We used immunoblotting with antibodies to phosphotyrosine to identify substrates of protein-tyrosine kinases in colonic cells. Three phosphotyrosine-containing proteins were detected at significantly higher levels in most colon carcinoma cell lines than in normal colonic mucosal cells or human or rat fibroblasts. All colon carcinoma cell lines with elevated pp60c-src in vitro kinase activity, showed increased phosphorylation of proteins on tyrosine in vivo, suggesting the presence of an activated protein-tyrosine kinase(s).
Tack, L.C., C.A. Cartwright, J.H. Wright, W. Eckhart, K.W. Peden, A. Srinivasan, and J.M. Pipas (1989) Properties of a simian virus 40 mutant T antigen substituted in the hydrophobic region: defective ATPase and oligomerization activities and altered phosphorylation accompany an inability to complex with cellular p53. J. Virol. 63:3362-3367 We have analyzed the biochemical properties of a nonviable simian virus 40 (SV40) mutant encoding a large T antigen (T) bearing an amino acid substitution (Pro-584-Leu) in its hydrophobic region. Mutant 5080 has an altered cell type specificity for transformation (transforming mouse C3H10T1/2 but not rat REF52 cells), is defective for viral DNA replication, and encodes a T that is unable to form a complex with the cellular p53 protein (K. Peden, A. Srinivasan, J. Farber, and J. Pipas, Virology 168:13-21, 1989). In this article, we show that 5080-transformed C3H10T1/2 cell lines express an altered T that is synthesized at a significantly higher rate but with a shorter half-life than normal T from wild-type SV40-transformed cells. 5080 T did not oligomerize beyond 5 to 10S in size compared with normal T, which oligomerized predominantly to 14 to 20S species. In addition, the 5080 T complex had significantly decreased ATPase activity and had a 10-fold-lower level of in vivo phosphorylation compared with that of normal T. Two-dimensional phosphopeptide analysis indicated several changes in the specific 32P labeling pattern, with altered phosphorylation occurring at both termini of the mutant protein compared with the wild-type T. Loss of p53 binding is therefore concomitant with changes in ATPase activity, oligomerization, stability, and in vivo phosphorylation of T and can be correlated with defective replication and restricted transformation functions. That so many biochemical changes are associated with a single substitution in the hydrophobic region of T is consistent with its importance in regulating higher-order structural and functional relationships in SV40 T.
Finley, G.G., N.T. Schulz, S.A. Hill, J.R. Geiser, J.M. Pipas, and A.I. Meisler (1989) Expression of the myc gene family in different stages of human colorectal cancer. Oncogene 4:963-971 Colorectal carcinoma serves as a model system for the study of changes in gene expression and structure relating to tumorigenesis. In this study, the levels of c-myc, N-myc and L-myc mRNAs were assessed in normal human colonic mucosa in 33 cases representing different stages of adenocarcinoma and in 36 adenomatous polyps, the presumed premalignant stage. Consistent with the findings of Erisman et al. (1985), we found that the c-myc gene was overexpressed (3-24-fold) in approximately two-thirds of the tumors examined. Amplification of the gene (3-4-fold) was observed in 2 of 12 tumors examined and did not correlate with the level of expression. Greater amounts of c-myc-specific mRNA than occur in normal tissue was also found in about two-thirds of the polyps examined demonstrating that premalignant lesions also overexpress the gene. N-myc and L-myc specific transcripts can be detected at low abundance in normal colonic mucosa. These genes were found to be frequently overexpressed in tumors and polyps, but in most cases the level of overexpression was modest. A single case of adenocarcinoma showed an approximately 30-fold increase in the level of N-myc mRNA without gene amplification. Adenomatous polyps more frequently overexpressed the L-myc gene than tumors. Srinivasan, A., K.W. Peden, and J.M. Pipas (1989) The large tumor antigen of simian virus 40 encodes at least two distinct transforming functions. J. Virol. 63:5459-5463 The large tumor antigen (T antigen) of simian virus 40 is necessary and sufficient for the neoplastic transformation of a number of established cell lines. Mutational analysis has revealed that a biochemical activity residing within the amino-terminal 121 amino acids of T antigen is sufficient to induce the transformation of some cell lines, such as C3H10T1/2. The same domain of the molecule also encodes the transactivation function of T antigen and the ability to complex with the retinoblastoma susceptibility gene product. However, the transformation of other lines, such as REF52, requires an additional activity that is affected by mutations in other portions of the molecule.
Greenwood, D., A. Srinivasan, S. McGoogan, A.I. Meisler, and J.M. Pipas (1988) Defined media as a system for detecting oncogene activity and isolation of novel oncogenes. Pp 37-60 in Inflammatory Bowel Disease, Macdermott, R.P., Ed. Excerpta Medica, New York Tevethia, M.J., J.M. Pipas, T. Kierstead, and C. Cole (1988) Requirements for immortalization of primary mouse embryo fibroblasts probed with mutants bearing deletions in the 3' end of SV40 gene A. Virology 162:76-89 The influence of specific contiguous stretches of amino acids predominantly in the carboxy terminal third of the SV40 large T antigen on the immortalization of cells in culture was investigated. Mutants that bear either small in-phase or frameshift deletions in the large T antigen coding sequence were transfected into primary mouse embryo fibroblasts of C57Bl/6 origin (B6/MEF). The frequency of immortalization was determined as the number of colonies that developed from cells escaping senescence. The results indicated that the terminal 81 amino acids of large T antigen are not needed for efficient immortalization or tumorigenicity. In contrast removal of as few as three amino acids encoded in the vicinity of the Dde-1 site at 0.234 map units (m.u.) severely restricted immortalization, suggesting that this region of the coding sequence either structurally or functionally is essential to at least one parameter of the transformed cell phenotype. The T antigen produced by dlA2433 which bears a deletion of nine nucleotides at 0.234 m.u. fails to associate stably with the cellular protein p53. The results showed that the addition of long stretches of amino acids (96 or 97 residues) from the open reading frame at the 3' end of the early region inactivated immortalizing functions, although the addition of as many as 18 amino acids from other reading frames was not detrimental. The evidence presented also confirmed that wild-type levels of ATPase activity are not necessary for immortalization or tumorigenicity of B6/MEF. Finally, we show that one of the mutants that immortalized primary cells did not produce dense foci on a cell monolayer. This last result indicated that independent functions are required for these two parameters of the transformed cell phenotype. Khalili, K., J. Brady, J.M. Pipas, S.L. Spence, M. Sadofsky, and G. Khoury (1988) Carboxyl-terminal mutants of the large tumor antigen of simian virus 40: a role for the early protein late in the lytic cycle. Proc. Natl. Acad. Sci., USA 85:354-358 Simian virus 40 (SV40) mutants dl1066 and dl1140 contain deletions within the region encoding the carboxyl terminus of the large tumor (T) antigen. Although these mutations have little effect on the efficiency of viral DNA replication, they decrease the yield of infectious virus particles by 3-4 orders of magnitude [Pipas, J. (1985) J. Virol. 54, 569-575]. Here we show that the level of late RNA is lower by a factor of 5-15 in CV-1P monkey cells infected with these mutants compared to cells infected with wild-type SV40. Consistent with this decrease in RNA, synthesis of late viral structural proteins VP1 and VP3 decreases by a factor of 5-15. In contrast, the synthesis of SV40 agnoprotein decreases by a factor greater than 100. Intercistronic complementation of these mutants with pm1493 and dl121, two SV40 mutants that are defective in agnoprotein but encode wild-type T antigen, results in an increased synthesis of agnoprotein in the infected cells. These results suggest that the carboxyl-terminal portion of T antigen participates in the posttranscriptional regulation of agnoprotein.
Hunter, L.A., and J.M. Pipas (1988) Isolation of a simian virus 40-simian agent 12 recombinant papovavirus that synthesizes a hybrid large tumour antigen. J. Gen. Virol. 69:683-687 The simian virus 40 (SV40) mutant dl1055 carries a 55 base pair deletion in the viral early region coding sequences that causes premature termination of large tumour (T) antigen translation resulting in a protein fragment consisting of the amino-terminal 399 residues. The mutation renders the virus defective. We report the characterization of hyb4001, which was isolated as a single plaque following cotransfection of permissive cells with dl1055 DNA and the DNA of a related papovavirus, simian agent 12 (SA12). Hyb4001 arose by two homologous recombination events involving crossovers in regions of 7 and 12 base pairs of perfect homology between the two viruses. Hyb4001 synthesizes a hybrid T antigen with the amino-terminal 382 residues encoded by SV40, residues 383 to 449 encoded by SA12, and the carboxy-terminal 259 residues encoded by SV40. Pipas, J.M. (1988) SV40 large T antigen mutant data base. In Vitro Cell Dev. Biol. 24:1147 Michalovitz, D., L. Fischer-Fantuzzi, C. Vesco, J.M. Pipas, and M. Oren (1987) Activated Ha-ras can cooperate with defective simian virus 40 in the transformation of nonestablished rat embryo fibroblasts. J. Virol. 61:2648-2654 We studied the ability of activated Ha-ras to cooperate with simian virus 40 (SV40) in the transformation of nonestablished rat embryo fibroblasts. Cotransfection with Ha-ras greatly accelerated the rate of focus induction by wild-type SV40. Moreover, a series of transformation-defective SV40 mutants could be partially complemented by Ha-ras. This was true not only for mutants retaining an intact N-terminal immortalization-competent domain, but also for a nonkaryophilic SV40 mutant. In the latter case, all detectable T antigen was cytoplasmic, indicating that efficient transformation can be achieved through the interaction of two nonnuclear proteins. By employing cell lines derived with various SV40 mutants, it was determined that the ability to complex with p53 depends on the integrity of a relatively large region in the C-terminal half of large T. Finally, we report that nonkaryophilic SV40 large T forms a complex with the major heat shock protein HSP70, and we discuss its possible implications.
Cunningham, T.P., and J.M. Pipas (1985) Simian agent 12 is a BK virus-like papovavirus which replicates in monkey cells. J. Virol. 54:483-492 We have begun to characterize the genomic structure and replication of the baboon papovavirus simian agent 12 (SA12). We have defined a wild-type clone of SA12 (SA12 wt100) by plaque purification from a heterogeneous stock. The functional map of SA12 wt100 can be aligned with those of the other primate papovaviruses by assigning one of the two EcoRI sites as 0/1.0 map units. The origin of bidirectional viral DNA replication maps near 0.67 map units, consistent with the limits of sequences homologous to origin sequences in the other papovaviruses. DNA sequence analysis shows that the organization of the SA12 genome is similar to that of the other primate papovaviruses studied. The arrangement and sequence of functional elements in the origin of replication region, as well as the sequences of the N-terminal regions of early protein products, indicate that SA12 is most closely related to the human virus BK, next most closely related to JC virus, and less closely related to simian virus 40. Unlike BK virus, SA12 is capable of productive infection of African green monkey kidney cells.
Pipas, J.M. (1985) Mutations near the carboxyl terminus of the simian virus 40 large tumor antigen alter viral host range. J. Virol. 54:569-575 We report the characterization of three mutants of simian virus 40 with mutations that delete sequences near the 3' end of the gene encoding large tumor antigen (T antigen). Two of these mutants, dl1066 and dl1140, exhibit an altered viral host range. Wild-type simian virus 40 is capable of undergoing a complete productive infection on several types of established African green monkey kidney lines, including BSC40 and CV1P. dl1066 and dl1140 grow on BSC40 cells at 37 degrees C. However, both mutants fail to form plaques on BSC40 cells at 32 degrees C or on CV1P cells at any temperature. These mutants are capable of replicating viral DNA in the nonpermissive cell type, indicating a defect in an activity of T antigen not related to its replication function. Furthermore this defect can be complemented in trans by the wild type or by a variety of DNA replication-negative T antigen mutants, so long as they produce a normal carboxyl-terminal region of the molecule. Our data are consistent with the hypothesis that the C-terminal region of T antigen constitutes a functional domain. We propose that this domain encodes an activity that is required for simian virus 40 productive infection on the CV1P cell line, but not on BSC40.
Peden, K.W., and J.M. Pipas (1985) Site-directed mutagenesis of the simian virus 40 large T-antigen gene: replication-defective amino acid substitution mutants that retain the ability to induce morphological transformation. J. Virol. 55:1-9 We used a heteroduplex deletion loop mutagenesis procedure for directing sodium bisulfite-induced mutations to specific sites on viral or plasmid DNA to generate a series of SV40 large T-antigen point mutants. The mutations were directed to a region of the T-antigen gene, 0.5 map units, that is thought to be important for interaction of the protein with the viral origin of DNA replication. Of the 16 mutants reported here, 10 had lost the ability to replicate their DNA, and 3 others showed a reduced level of replication compared to wild type. All of the mutants tested were capable of transforming rat cells in culture by the dense focus assay. We conclude that the sequences of the early region around 0.5 map units are critical for the replication of viral DNA but not for the transformation function of T antigen.
Chiang, L.C., J. Silnutzer, J.M. Pipas, and D.W. Barnes (1985) Selection of transformed cells in serum-free media. In Vitro Cell Dev. Biol. 21:707-712 NIH3T3 cells grow in a serum-free basal nutrient medium supplemented with fibronectin, transferrin, insulin, epidermal growth factor (EGF) and high density lipoprotein (HDL). The individual omission from the serum-free medium of insulin, EGF, or HDL results in greatly reduced cell growth. These growth-restrictive conditions can be used to select for cells transformed with SV40, the polyomavirus middle T antigen gene, the activated human ras gene, and the mouse c-myc gene. Pipas, J.M., L.-C. Chinag, and D.W. Barnes (1984) Influence of genetic, cellular and hormonal factors on Simian Virus 40-induced transformation. Cold Spring Harbor Conf. Cell Prolif. Cancer 11:355-362 Chiang, L.-C., J. Silnutzer, J.M. Pipas, and D.W. Barnes (1984) Serum-free cell culture for growth of NIH3T3 and 10T1/2 mouse embryo fibroblast cell lines: SV40 virus propagation and selection of SV40-transformed cells. Cell Culture Meth. Mol. Cell Biol. 3:265-276 Nathans, D., K. Peden, and J.M. Pipas (1983) Mutational analysis of a viral regulatory protein, the T antigen of simian virus 40. Pontificae Academiae Scientiarvm Scripta Varia 51:117-122 Pipas, J.M., K.W. Peden, and D. Nathans (1983) Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene. Mol. Cell Biol. 3:203-213 A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein.
Soprano, K.J., N. Galanti, G.J. Jonak, S. McKercher, J.M. Pipas, K.W. Peden, and R. Baserga (1983) Mutational analysis of simian virus 40 T antigen: stimulation of cellular DNA synthesis and activation of rRNA genes by mutants with deletions in the T-antigen gene. Mol. Cell Biol. 3:214-219 The biological activity of several deletion mutants of simian virus 40, cloned in pBR322, was determined. Three functions of the simian virus 40 A gene were studied: (i) the ability to express T antigen; (ii) the ability to induce cell DNA replication; and (iii) the ability to reactivate silent rRNA genes in hybrid cells. Recombinant plasmid DNA was introduced into cells by manual microinjection or by transfection. The results (together with previous reports) indicate that the critical sequences for these three functions are located separately on the simian virus 40 A gene, as follows: (i) the sequences necessary for the detection of the common antigenic determinant of T antigen extend from nucleotide 4147 to nucleotide 4001 (map units 0.45 to 0.42); (ii) the sequences critical for the stimulation of cell DNA synthesis extend from nucleotide 4327 to nucleotide 4001 (map units 0.49 to 0.42); and (iii) those critical for the reactivation of rRNA genes extend approximately from nucleotide 3827 to nucleotide 3526 (map units 0.39 to 0.33).
Clark, R., K. Peden, J.M. Pipas, D. Nathans, and R. Tjian (1983) Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants. Mol. Cell Biol. 3:220-228 We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.
Wilson, J.H., P.B. Berget, and J.M. Pipas (1982) Somatic cells efficiently join unrelated DNA segments end-to-end. Mol. Cell Biol. 2:1258-1269 Molecular substrates for probing nonhomologous recombination in somatic cells were constructed by inserting pBR322 sequences at selected sites on the simian virus 40 (SV40) genome. The chimeric products are too large to be packaged into an SV40 capsid. Therefore, production of viable progeny requires that most of the pBR322 sequences be deleted without altering any SV40 sequences that are essential for lytic infection. As judged by plaque assay, these recombination events occur at readily detectable frequencies after transfection into CV1 monkey kidney cells. Depending on the site of pBR322 insertion, the infectivities of the full-length circular or linear chimeras ranged from 0.02 to 2% of the infectivity of linear wild-type SV40 DNA. Nucleotide sequence analysis of several recombinant progeny revealed three distinct classes of recombination junction and indicated that the causative recombination events were minimally dependent on sequence homology. Potential mechanisms involving recombination at internal sites or at ends were distinguished by measuring the infectivity of chimeric molecules from which various lengths of pBR322 had been removed. These data support end-to-end joining as the primary mechanism by which DNA segments recombine nonhomologously in somatic cells. This end joining appears to be very efficient, since SV40 genomes with complementary single-stranded tails or with short non-complementary pBR322 tails were comparably infectious. Overall, this study indicates that mammalian somatic cells are quite efficient at the willy-nilly end-to-end joining of unrelated DNA segments.
Pipas, J.M., S.P. Adler, K.W. Peden, and D. Nathans (1980) Deletion mutants of SV40 that affect the structure of viral tumor antigens. Cold Spring Harb. Sym. 44:285-291 Peden, K.W., J.M. Pipas, S. Pearson-White, and D. Nathans (1980) Isolation of mutants of an animal virus in bacteria. Science 209:1392-1396 Mutants of animal viruses can be isolated in bacteria by recombinant DNA methods. Since no viral functions are required for propagation of recombinants in bacteria, viral mutants with lethal changes in cis- or trans-acting elements can be isolated, as well as partially or conditionally defective mutants. In the cases of viruses with small DNA genomes, such as the tumorigenic simian virus 40 (SV40), the entire viral DNA can be inserted into the bacterial plasmid pBR322 and cloned in Escherichia coli. Recombinant plasmids with a single copy of SV40 DNA cause morphological transformation of mouse cells in culture with the same efficiency as SV40 DNA isolated from virus-infected monkey cells, but the recombinant DNA is noninfectious and replicates poorly in permissive cells. However, SV40 DNA excised from the plasmid replicates as well as authentic viral DNA and is fully infectious. SV40 mutants with small deletions or base substitutions have been isolated by in vitro site-specific or random local mutagenesis of recombinant DNA followed by cloning in E. coli. Many of the mutants thus isolated are defective in specific viral functions. Shortle, D., J.M. Pipas, S. Lazarowitz, D. DiMaio, and D. Nathans (1979) Constructed mutants of Simian Virus 40. Pp 73-92 in Genetic Engineering, Setlow, J., and A. Hollaender, Ed. Plenum Publishing Co., New York Pipas, J.M., and R.H. Reeves (1979) Host transcription in bacteriophage P22-infected Salmonella typhimurium. J. Virol. 32:822-831 The kinetics of host RNA synthesis, as measured by pulse-label kinetics and RNA-DNA hybridization, is inhibited 10-fold shortly after infection with bacteriophage P22. This early inhibition lasts through the first 6 min of infection and affects not only RNA synthesis but several other energy-requiring cellular functions. In lysogenic infections, the rate of bacterial transcription rapidly recovers to the value of uninfected controls. In lytic infections, the rate of host transcription increases only to about 20 to 25% of the original level, indicating a second mechanism for the inhibition of RNA synthesis in the lytic response. The early inhibition is multiplicity dependent, bhloramphenicol insensitive, and independent of phage gene 24. The lytic inhibition is dependent upon the expression of gene 24 but independent of gene 23.
Pipas, J.M., and R.H. Reeves (1977) Patterns of transcription in bacteriophage P22-infected Salmonella typhimurium. J. Virol. 21:825-828 Two peaks of RNA synthesis (early and late) are directed by bacteriophage P22 in lytic infections of Salmonella typhimurium. Late RNA synthesis is not seen in P22 23- infections; neither early nor late RNA synthesis occurs in P22 24- infections. Genes 23 and 24 of P22 appear to be analogous to genes Q and N of lambda, respectively.
McMahon, J.E., and J.M. Pipas (1975) Predicting RNA secondary interactions from primary structure. Int. J. Quantum Chem. 2:129-131 Pipas, J.M., and J.E. McMahon (1975) Method for predicting RNA secondary structure. Proc. Natl. Acad. Sci., USA 72:2017-2021 We report a method for predicting the most stable secondary structure of RNA from its primary sequence of nucleotides. The technique consists of a series of three computer programs interfaced to take the nucleotide sequence of any RNA and (a) list all possible helical regions, using modified Watson-Crick base-pairing rules; (b) create all possible secondary structures by forming permutations of compatible helical regions; and (c)evaluate each structure for total free energy of formation from a completely extended chain. A free energy distribution and the base-by-base bonding interactions of each possible structure are catalogued by the system and are readily available for examination. The method has been applied to 62 tRNA sequences. The total free-energy of the predicted most stable structures ranged from -19 to -41 kcal/mole (-22 to -49 kJ/mole). The number of structures created was also highly sequence-dependent and ranged from 200 to 13,000. In nearly all cases the cloverleaf is predicted to be the structure with the lowest free energy of formation.
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