Truncated forms of the polyomavirus middle T antigen can substitute for the small T antigen in lytic infection.

Cloned polyomavirus genomes encoding the small T antigen or truncated forms of the middle T antigen facilitated the growth of genomes encoding only the large T antigen in mouse 3T6 cells. We conclude that an N-terminal domain of the middle T antigen, in the appropriate cellular location, can substitute for the small T antigen during lytic infection.     

Cellular proteins that associate with the middle and small T antigens of polyomavirus.

We have used two-dimensional gel electrophoresis to analyze in more detail the cellular proteins which associate with the middle and small tumor antigens (MT and ST, respectively) of polyomavirus. Proteins with molecular masses of 27, 29, 36, 51, 61, 63, and 85 kilodaltons (kDa) that specifically coimmunoprecipitated with MT were identified on these gels. The 36-, 51-, 61-, 63-, and 85-kDa proteins are probably the same as the proteins of similar sizes previously reported by a number of groups, whereas the 27- and 29-kDa proteins represent proteins that are heretofore undescribed. The 27- and 29-kDa proteins were abundant cellular proteins, whereas the others were minor cellular constituents. The association of each of these proteins with MT was sensitive to one or more mutations in MT that rendered it transformation defective. The association of the 85-kDa protein was the most sensitive indicator of the transformation competence of MT mutants. In addition, the 85-kDa protein was the only associated protein whose association with MT changed consistently in parallel with MT-associated phosphatidylinositol kinase activity. Furthermore, the fraction of the 85-kDa protein which was found associated with the MT complex contained 15 to 20% of its phosphate content on tyrosine. The 36- and 63-kDa proteins complexed with both polyomavirus MT and ST and comigrated on two-dimensional gels with two simian virus 40 ST-associated proteins originally described by Rundell and coworkers (K. Rundell, E. O. Major, and M. Lampert, J. Virol. 37:1090-1093, 1981). None of the other MT-associated proteins associated significantly with ST.     

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.     

Altered susceptibility to tumor necrosis factor alpha-induced apoptosis of mouse cells expressing polyomavirus middle and small T antigens.

Infection with some virus types induces susceptibility to the cytotoxic effect of tumor necrosis factor alpha (TNF-alpha). To investigate whether expression of polyomavirus proteins has this effect on cells, the TNF-alpha sensitivity of C127 and L929 mouse cells transfected with viral DNA was analyzed. Expression of all three polyomavirus early proteins, the tumor (T) antigens, had no apparent effect. In contrast, middle T antigen by itself induced hypersensitivity to TNF-alpha. This effect was reversed by retransfection of the cells with DNA encoding small T antigen. Expression of this polypeptide also decreased the sensitivity of bovine papillomavirus type 1-transformed cells to TNF-alpha, showing that the protective function of the polyomavirus small T antigen was not strictly linked to a middle-T-antigen-induced event. Mouse and human TNF-alpha had the same effect on normal and transformed mouse cells, suggesting that this effect was mediated by TNF receptor 1. Consistent with this conclusion, all cell clones used in the experiments expressed TNF receptor 1 at similar levels, while we failed to detect TNF receptor 2. The amount of receptor on the cells was not influenced by binding of the ligand. Addition of TNF-alpha at cytotoxic concentrations to cells expressing middle T antigen by itself resulted in significant fragmentation of chromosomal DNA after only a few hours, indicating induction of apoptosis. Addition of the cytokine to these cells also leads to release of arachidonic acid, showing that phospholipase A2 was activated. However, production of arachidonic acid did not appear to significantly precede loss of cell viability.     

Natural biology of polyomavirus middle T antigen.

"It has been commented by someone that 'polyoma' is an adjective composed of a prefix and suffix, with no root between--a meatless linguistic sandwich" (C. J. Dawe). The very name "polyomavirus" is a vague mantel: a name given before our understanding of these viral agents was clear but implying a clear tumor life-style, as noted by the late C. J. Dawe. However, polyomavirus are not by nature tumor-inducing agents. Since it is the purpose of this review to consider the natural function of middle T antigen (MT), encoded by one of the seemingly crucial transforming genes of polyomavirus, we will reconsider and redefine the virus and its MT gene in the context of its natural biology and function. This review was motivated by our recent in vivo analysis of MT function. Using intranasal inoculation of adult SCID mice, we have shown that polyomavirus can replicate with an MT lacking all functions associated with transformation to similar levels to wild-type virus. These observations, along with an almost indistinguishable replication of all MT mutants with respect to wild-type viruses in adult competent mice, illustrate that MT can have a play subtle role in acute replication and persistence. The most notable effect of MT mutants was in infections of newborns, indicating that polyomavirus may be highly adapted to replication in newborn lungs. It is from this context that our current understanding of this well-studied virus and gene is presented.     

Cooperation of middle and small T antigens of polyomavirus in transformation of established fibroblast and epithelial-like cell lines.

We have reported recently that small T antigen of polyomavirus stimulates the growth of NIH 3T3 cells beyond their saturation density and induces weak anchorage-independent growth (T. Noda, M. Satake, T. Robins, and Y. Ito, J. Virol. 60:105-113, 1986). We examined whether small T antigen would cooperate with middle T antigen in the in vitro transformation of NIH 3T3 (fibroblasts) and NRK-52E (epitheliallike) cells. The small-T-antigen gene, when cotransfected with the middle-T-antigen gene, had no additional effect on the efficiency or size of dense foci formation induced by the middle-T-antigen gene on a monolayer of NIH 3T3 cells. However, the small-T-antigen gene dramatically increased the rate of growth of NIH 3T3 cells transformed by middle T antigen in semisolid medium. Introduction of the small-T-antigen gene into middle-T-antigen-transformed cells did not disturb the integrated middle-T gene, alter expression of the middle-T gene, or enhance middle-T-antigen-associated tyrosine protein kinase activity. For NRK-52E cells, the expression of middle T antigen alone resulted in small, slow-growing foci on a monolayer. These cells did not show anchorage-independent growth, despite the fact that middle-T-antigen-associated tyrosine protein kinase activity was clearly detected in these cells. NRK-52E cells expressing both middle and small T antigens formed faster growing foci on a monolayer than middle-T-antigen-expressing cells did and grew in semisolid medium, even when the amounts of middle T antigen and its associated kinase activities were lower than those of middle-T-antigen-expressing cells. We conclude that small T antigen cooperates with middle T antigen in the in vitro transformation of established cell lines of fibroblast and epitheliallike cells, that it does not share the middle-T-antigen function even though they are structurally related, and that it has a significantly more important role in the transformation of NRK-52E cells than that of NIH 3T3 cells.     

Identification and characterization of the hamster polyomavirus middle T antigen.

Hamster polyomavirus (HaPV) is associated with lymphoid and hair follicle tumors in Syrian hamsters. The early region of HaPV has the potential to encode three polypeptides (which are related to the mouse polyomavirus early proteins) and can transform fibroblasts in vitro. We identified the HaPV middle T antigen (HamT) as a 45-kDa protein. Like its murine counterpart, HamT was associated with serine/threonine phosphatase, phosphatidylinositol-3 kinase, and protein tyrosine kinase activities. However, whereas mouse middle T antigen associates predominantly with pp60c-src and pp62c-yes, HamT was associated with a different tyrosine kinase, p59fyn. The ability of HaPV to cause lymphoid tumors may therefore reside in its ability to associate with p59fyn, a potentially important tyrosine kinase in lymphocytes.     

Identification of regions in polyomavirus middle T and small t antigens important for association with protein phosphatase 2A.

Two subunits of protein phosphatase 2A (PP2A) have been shown previously to bind to the small t and middle T antigens (ST and MT, respectively) of polyomavirus. To determine sequences important for binding of PP2A to ST and MT, we first constructed a series of ST mutants in regions known to be important for biological activity of ST and MT. Several mutations in two small regions just amino terminal to the Cys-X-Cys-X-X-Cys motifs of ST and MT abolished PP2A binding to ST in vitro. Parallel mutations were constructed in MT to investigate the role of PP2A binding in the function of polyomavirus MT. Wild-type and mutant MT proteins were stably expressed in NIH 3T3 cells and analyzed (i) for their ability to induce transformation and (ii) for associated cellular proteins and corresponding enzymatic activities previously described as associating with wild-type MT. A number of the mutant MTs were found to be defective in binding of PP2A as assayed by coimmunoprecipitation. In contrast, a deletion of the highly conserved stretch of amino acids 42 to 47 (His-Pro-Asp-Lys-Gly-Gly) in the ST-MT-large T antigen common region did not affect PP2A binding to MT. MT mutants defective for PP2A binding were also defective in transformation, providing further evidence that association with PP2A is important for the ability of MT to transform cells. All mutants which were impaired for PP2A binding were similarly or more dramatically impaired for associated protein and lipid kinase activities, supporting the possibility that PP2A binding is necessary for the formation and/or stability of an MT-pp60c-src complex.     

Phosphatidylinositol metabolism in cells transformed by polyomavirus middle T antigen.

Associated with the middle T antigen of polyomavirus is a novel phosphatidylinositol (PtdIns) kinase activity which phosphorylates PtdIns at the D-3 position of the inositol ring. We have undertaken an analysis of myo-[3H]inositol-containing compounds in a panel of NIH 3T3 cell lines stably transfected with transforming and nontransforming middle T antigen mutants. All cell lines from which PtdIns 3-kinase activity coprecipitated with middle T antigen exhibited modestly elevated levels of PtdIns(3)P and compounds with predicted PtdIns(3,4)P2 and PtdIns(3,4,5)P3 structures. Complex formation between middle T antigen and PtdIns 3-kinase correlated not with an increase in total inositol phosphate levels but rather with elevated levels of InsP2 and InsP4. A specific increase in the level of an InsP2 species which comigrated in high-pressure liquid chromatography analysis with Ins(3,4)P2 was observed. These results suggest that association of the polyomavirus middle T antigen with PtdIns 3-kinase activates a distinct inositol metabolic pathway.     

Properties of T antigens induced by wild-type SV40 and tsA mutants in lytic infection

T antigen in extracts of cells infected with tsA mutants is 2 to 6 times more labile at 32°C or 41°C than the antigen in extracts of cells infected with wild-type SV40, as assayed by complement fixation. The stabilities of wild-type and mutant antigens are not altered by mixing the extracts, and thus the stability is an intrinsic property of each antigen and is not determined by another component of the extract. This observation indicates that T antigen is probably the virus-coded product of the A gene. In cells infected at the permissive temperature of 32°C with a high multiplicity of either wild-type or tsA mutant virus, the amounts of T antigen are approximately equivalent and increase logarithmically during the entire period of infection, up to 96 hr. Cells infected at 32°C for 96 hr with mixtures of wild-type and tsA virus produce T antigen with the stability of wild-type, even when the infection is carried out with up to a 5 fold excess of the mutant. The more stable wild-type antigen may repress, directly or indirectly, the synthesis of the more labile mutant antigen.     

Transformation by middle T antigens

The polyomaviruses, members of the papovavirus group of DNA tumor viruses, encode in the 'early' region of the genome, three proteins involved in transformation, the tumor (or T) antigens, called large (LT), middle (mT) and small (sT) T. LT is required for establishment of primary fibroblasts, and sT promotes the efficiency of transformation both in vivo and in vitro, but it is mT that carries the transforming ability. The mTs of the two known polyomaviruses, from mouse and hamster, possess no intrinsic catalytic activity, but rather interact with and change the activity of several cellular proteins, including Src family protein tyrosine kinases, protein phosphatase 2A, phosphatidylinositol 3-kinase and Shc. Some of these proteins are also involved in signal transduction events elicited by growth factors. Like activated growth factor receptors, mT brings its associated proteins to a membranous environment. Transformation by mT might result not only from allosteric effects of mT on its interacting proteins but also as a result of their subcellular relocalization.     

Large T antigens of simian virus 40 and polyomavirus efficiently establish primary fibroblasts.

Recombinant retroviruses that transduce the simian virus 40 (SV40) large T antigen or the polyomavirus large T antigen as well as encoding resistance to antibiotic G418 were used to investigate whether these genes alone were sufficient for immortalization of primary cells. The results provided definitive evidence that either viral gene can efficiently establish primary fibroblasts. The capability of the SV40 large T antigen to establish primary fibroblasts was undiminished by a mutation that alters its binding to sequences within the origin of replication. Surprisingly, most of the primary cells established by the expression of the SV40 large T antigen did not have a transformed phenotype. This suggests that transformation by SV40 is not simply due to a high level of expression of the SV40 large T antigen and stabilization of cellular p53.     

p56lck protein-tyrosine kinase is cytoskeletal and does not bind to polyomavirus middle T antigen.

p56lck and p60c-src are closely related protein-tyrosine kinases that are activated by similar oncogenic mutations. We have used fibroblast cell lines that express p56lck from introduced DNA molecules to compare the subcellular localizations of p60c-src and p56lck and their abilities to bind polyomavirus middle T antigen (mT). p56lck is associated with the detergent-insoluble matrix, as defined by extraction with solutions containing nonionic detergents, whereas p60c-src is soluble under these conditions. p56lck is also associated with detergent-insoluble structures in a lymphoid cell line, LSTRA. p60c-src binds to mT, but p56lck does not bind detectably. In terms of both solubility and mT interactions, the nononcogenic p56lck more closely resembles oncogenically activated p60c-src mutants than it resembles p60c-src. Because published results show that an intact carboxy terminus is required for p60c-src to bind mT and be soluble, we tested whether the different localization and mT binding properties of p56lck and p60c-src were dictated by their different carboxy termini. A protein consisting largely of p60c-src sequences but carrying a p56lck carboxy terminus was soluble and bound to mT. We suggest that both the solubility and mT-binding properties of p60c-src not only require sequences common to the carboxy termini of p60c-src and p56lck, but also require sequences unique to the body of p60c-src.     

Functional asymmetry of the regions juxtaposed to the membrane-binding sequence of polyomavirus middle T antigen.

The functional importance of the two clusters of positively charged amino acids which flank the hydrophobic membrane-anchoring sequence of polyomavirus middle T (mT) protein has been investigated by using site-directed mutagenesis. A clear asymmetry was apparent. No effect on transformation was seen following multiple alterations or complete removal of the cluster at the carboxyl end of the protein. In contrast, a single substitution replacing the first arginine amino terminal to the hydrophobic stretch with glutamic acid, but not with lysine, histidine, or methionine, produced a partially transformation-defective mutant with a novel phenotype. This mutant failed to confer anchorage-independent growth on F111 established rat embryo fibroblasts but induced foci with altered morphology compared with wild-type mT. Biochemical studies on this mutant revealed that F111 clones expressing levels of mutant mT equivalent to those of wild-type controls showed a 65% reduction in pp60c-src activation and an 87% reduction in mT-associated phosphatidylinositol 3-kinase activity. However, F111 clones expressing seven times more mutant mT than did wild-type controls showed equal or greater levels of kinase activities yet remained incompletely transformed. Possible mechanisms involving this transformation-sensitive region of mT are discussed.     

The amino terminus of polyomavirus middle T antigen is required for transformation.

In polyomavirus-transformed cells, pp60c-src is activated by association with polyomavirus middle T antigen. These complexes have a higher tyrosine kinase activity compared with that of unassociated pp60c-src. Genetic analyses have revealed that the carboxy-terminal 15 amino acids of pp60c-src and the amino-terminal half of middle T antigen are required for this association and consequent activation of the tyrosine kinase. To define in greater detail the borders of the domain in middle T antigen required for activation of pp60c-src, we constructed a set of unidirectional amino-terminal deletion mutants of middle T antigen. Analysis of these mutants revealed that the first six amino acids of middle T antigen are required for it to activate the kinase activity of pp60c-src and to transform Rat-1 fibroblasts. Analysis of a series of insertion and substitution mutants confirmed these observations and further revealed that mutations affecting the first four amino acids of middle T antigen reduced or abolished its capacity to activate the kinase activity of pp60c-src and to transform Rat-1 cells in culture. Our results suggest that the first four amino acids of middle T antigen constitute part of a domain required for activation of the pp60c-src tyrosyl kinase activity and for consequent cellular transformation.     

SV40 small T antigen enhances progression to >G2 during lytic infection.

The infection of monkey kidney (CV-1) cells with simian virus 40 (SV40) stimulates the cells into successive rounds of DNA synthesis without an intervening mitosis, leading to the acquisition of a >G2 DNA content. To elucidate the role of small t antigen in cell cycle progression and in viral replication during infection, studies were performed using an SV40 mutant (dl888) that lacks the ability to produce small t. Initially dl888-infected cells move through the first S phase at roughly the same rate as wild-type infected cells. Upon reaching G2, however, the dl888-infected cells progressed to >G2 at a reduced rate relative to wild-type. The slower rate of entry into >G2 of dl888-infected cells is associated with a decrease in total pRb and an increase in the ratio of hypophosphorylated to hyperphosphorylated pRb. The expression of cyclin D1 and p27(kip1) were elevated in dl888-infected cells compared to wild-type-infected CV-1 cells. Taken together, these results indicate that small t antigen plays a role in stimulating entry into >G2 in SV40-infected CV-1 cells, possibly by affecting the regulation of key cell cycle proteins.