A retrovirus carrying the promyelocyte-retinoic acid receptor PML-RARalpha fusion gene transforms haematopoietic progenitors in vitro and induces acute leukaemias.

The promyelocyte (PML)-retinoic acid receptor alpha (RARalpha) fusion gene results from a t(15;17) chromosome translocation in acute promyelocytic leukaemia. We have analysed the oncogenic potential of the human fusion PML-RARalpha product in chicken using retrovirus vectors. We show that PML-RARalpha transforms very early haematopoietic progenitor cells in vitro and induces acute leukaemias. Neither PML nor RARalpha domains alone achieve such a transformation. The PML-RARalpha viruses recovered from the transformed cells carry two point mutations in the PML domain, one of which alters both the pattern of intracellular localization of the fusion protein and its functional interference with AP-1, thus defining an essential domain in PML for oncogenic transformation.     

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.     

A retrovirus carrying the K-fgf oncogene induces diffuse meningeal tumors and soft-tissue fibrosarcomas.

The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family and transforms cells through an autocrine mechanism which requires extracellular activation of its receptor(s). To identify the cell and tissue targets of K-fgf oncogenic potential in vivo, we constructed a recombinant retrovirus carrying the human K-fgf cDNA and injected it, together with helper Moloney murine leukemia virus, into immunocompetent as well as nude mice. The original construct was highly transforming in tissue culture but produced no detectable pathologies in vivo with the exception of a single fibrosarcoma which arose after a long latency. The virus produced by this tumor appears to have undergone a complex series of recombination events involving the helper Moloney murine leukemia virus. It encodes an Env/K-FGF fusion protein whose expression is under the control of a hybrid long terminal repeat. This virus (designated MFS, for meningeal fibrosarcoma) induces tumors in mice with high frequency and short latency. These neoplasms consist of aggressive fibrosarcomas of soft tissue as well as diffuse meningeal tumors originating from the dura mater that surround the whole central nervous system and cause severe hydrocephalus. The Env/K-FGF fusion protein expressed by the MFS virus has retained all of the biological properties of native K-FGF, including secretion, mitogenic activity, heparin binding, and neutralization by anti-K-FGF antibodies. These and other results indicate that the tumors induced by the MFS virus result from the oncogenic potential of K-FGF.     

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.     

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.     

Endothelial cell tumors develop in transgenic mice carrying polyoma virus middle T oncogene

Inoculation of newborn mice with the murine polyoma (Py) virus leads to tumor formation in a wide range of tissues. In order to investigate viral oncogenesis, we generated transgenic mice carrying either the Py large T antigen (LT) gene or the Py middle T antigen (MT) gene linked to Py early region regulatory sequences. While Py LT mice exhibit no phenotype, Py MT mice develop multifocal tumors of the vascular endothelium. These hemangiomas are lethal to the animals and can be passaged in vivo. Transgene RNAs and protein are present in both hemangiomas and the testes of these mice, and the Py middle T protein in both tissues is complexed to a cellular tyrosine kinase. The expression of complexed middle T protein in both tumorigenic endothelial cells and unperturbed testes implies that endothelial cells may be particularly susceptible to the action of the middle T oncogene. These observations indicate that Py middle T disrupts the normal strict controls on vascular growth, and suggest that Py MT transgenic mice will provide a model for studying the control of angiogenesis.     

Characterization of the interaction of polyomavirus middle T antigen with type 2A protein phosphatase.

Two cellular proteins of 36 and 63 kDa which bind the small T and middle T antigens of polyomavirus recently have been identified as the catalytic and regulatory subunits of the phosphoserine/threonine-specific type 2A protein phosphatase (PP2A). We report here the presence of phosphoseryl phosphatase activity associated with polyomavirus small T and middle T antigens in immunoprecipitates prepared from virus-infected and transformed cells. Phosphatase activity was also found associated with middle T-antigen mutants, some of which had been defined previously to associate with 36- and 63-kDa cellular proteins. Middle T-antigen-associated phosphatase activity was sensitive to okadaic acid and microcystin-LR, inhibitors of PP2A, and insensitive to inhibitor 1 or 2, orthovanadate, or EDTA. Using antiserum specific for the catalytic subunit of PP2A, we found that unlike the majority of PP2A, middle T-antigen-bound PP2A was membrane associated. However, no gross change in the amount, activity, or localization of PP2A could be attributed to middle T-antigen expression in transformed cells. Anti-PP2A antibodies coprecipitated a 63-kDa protein from normal cells and in addition coprecipitated middle T antigen, 60- and 61-kDa proteins (identified as src family members), and an 81-kDa protein from middle T-antigen-transformed cells. Furthermore, we detected protein kinase activity in PP2A immunoprecipitates and protein phosphatase activity in src immune complexes from extracts of middle T-antigen-transformed, but not normal, cells. These results reinforce the notion that at least a portion of middle T antigen bridges a protein kinase with a protein phosphatase.     

mlt Mutation in the polyomavirus genome impairing a function of the middle T protein.

The DNA from polyomavirus mlt mutant P155 transforms cells in culture as efficiently as wild-type DNA but has a much lower tumorigenic potential when injected into newborn rodents. The mutant has a 12-base-pair deletion between nucleotides 1347 and 1360, i.e., in a region which encodes parts of the middle and large T antigens (G elinas et al., J. Virol. 43:1072-1081, 1982). To determine which of the two viral gene functions was affected by the mutation, we transferred the latter into a modified polyomavirus genome encoding exclusively the middle T protein. Our results show that the P155 mutation alters a function of the polyomavirus middle T protein required for the induction of the tumorigenic process in vivo. Beside the 12-base-pair deletion at 96.3 map units, there is no other alteration in the coding sequence of P155 middle T with respect to that of P16, the wild-type parental strain. We conclude, therefore, that the deletion is the lesion affecting the tumorigenic potential of mutant P155 .     

Evidence that the middle T antigen of polyomavirus interacts with the membrane skeleton.

The transforming protein of polyomavirus, middle T antigen, is associated with cellular membranes. We have examined the subcellular location of the middle T antigen in two different cell types by fractionation and detergent phase partitioning. Middle T antigen expressed in human cells by a recombinant adenovirus was detected primarily in the membrane skeleton. Sucrose gradient fractionation revealed that the middle T antigen was associated with complexes with molecular weights of 500,000 to 1,000,000. Several markers for cytoskeleton cofractionate with these complexes, including actin, tubulin, and vimentin. Electron micrographs of membrane skeleton prepared from cells expressing middle T antigen demonstrated that this material contained primarily fibrous structures and was clearly devoid of bilayer membranes. These structures were distinct from the filamentous structures observed in fractions enriched for cytoskeleton. Consistent with a role for membrane skeleton localization in transformation, middle T antigen was detected exclusively in fractions enriched for membrane skeleton in middle T antigen-transformed Rat-2 cells. Our results may resolve the apparent difference between middle T antigen localization as determined by immunomicroscopy and that determined by subcellular fractionation.     

Genetic analysis of myeloproliferative leukemia virus, a novel acute leukemogenic replication-defective retrovirus.

The myeloproliferative leukemia virus (MPLV) is a new acute leukemogenic, nonsarcomatogenic retroviral complex that is generated during the in vivo passage of a molecularly cloned Friend ecotropic helper virus. Examination of viral RNA expression in MPLV-producing cells revealed the presence of two distinct molecular species that hybridized with a long terminal repeat or an ecotropic env-specific probe but not with a xenotropic mink cell focus-forming virus env-specific probe derived from a spleen focus-forming virus: an 8.2-kilobase species corresponding to a full-length Friend murine leukemia virus (F-MuLV) and a deleted species with a genomic size of 7.4 kilobases. This deleted virus was biologically cloned by limiting dilutions and single cell cloning in Mus dunni fibroblasts. Three nonproducer clones with normal morphologies and containing one single integrated copy of the deleted virus were superinfected with F-MuLV, Moloney murine leukemia virus, Gross murine leukemia virus, mink cell focus-forming virus (HIX), or the amphotropic 1504 murine leukemia virus. All pseudotypes caused macroscopic and microscopic abnormalities in mice that were similar to those seen in the parental stock. A comparison of the physical maps of F-MuLV and MPLV, which was deduced from the restriction enzyme digests of unintegrated proviral DNAs, indicated that the MPLV-defective genome (i) is probably derived from F-MuLV, (ii) has conserved the F-MuLV gag and pol regions, and (iii) is deleted and rearranged in the env region in a manner that is clearly distinct from that of Friend or Rauscher spleen focus-forming viruses.     

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.     

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.     

Small and middle T antigens contribute to lytic and abortive polyomavirus infection.

Using three different polyomavirus hr-t mutants and two polyomavirus mlT mutants, we studied induction of S-phase by mutants and wild-type virus in quiescent mouse kidney cells, mouse 3T6 cells, and FR 3T3 cells. At different times after infection, we measured the proportion of T-antigen-positive cells, the incorporation of [3H]thymidine, the proportion of DNA-synthesizing cells, and the increase in total DNA, RNA, and protein content of the cultures. In permissive mouse cells, we also determined the amount of viral DNA and the proportion of viral capsid-producing cells. In polyomavirus hr-t mutant-infected cultures, onset of host DNA replication was delayed by several hours, and a smaller proportion of T-antigen-positive cells entered S-phase than in wild-type-infected cultures. Of the two polyomavirus mlT mutants studied, dl-23 behaved similarly to wild-type virus in many, but not all, parameters tested. The poorly replicating but well-transforming mutant dl-8 was able to induce S-phase, and (in permissive cells) progeny virus production, in only about one-third of the T-antigen-positive cells. From our experiments, we conclude that mutations affecting small and middle T-antigen cause a reduction in the proportion of cells responding to virus infection and a prolongation of the early phase, i.e., the period before cells enter S-phase. In hr-t mutant-infected mouse 3T6 cells, production of viral DNA was less than 10% of that in wild-type-infected cultures; low hr-t progeny production in 3T6 cells was therefore largely due to poor viral DNA replication.     

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.     

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.     

Amino-terminal regions of polyomavirus middle T antigen are required for interactions with protein phosphatase 2A.

Polyomavirus middle T antigen (MT) is the major transforming protein of the virus. It functions through interactions with a number of cellular proteins involved in cell proliferation. MT forms complexes with protein phosphatase 2A (PP2A), pp60c-src, phosphatidylinositol 3-kinase, and Shc. We introduced both deletion and point mutations into three regions of MT and examined their ability to associate with PP2A and pp60c-src. The first 25 amino acid residues of MT are required for association with PP2A and pp60c-src. Amino acids 105 to 111, comprising the sequence Cys-Arg-Met-Pro-Leu-Thr-Cys, is also required for complex formation between MT and PP2A. However, the sequence Asp-Lys-Gly-Gly (amino acids 44 to 47), also found in the B subunit of PP2A, is dispensable for complex formation between MT and PP2A. We find a strict correlation between the ability of MT to associate with PP2A and the ability of MT to associate with pp60c-src. One mutant, L5E, associates with a phosphatase other than PP2A, pp60c-src, and phosphatidylinositol 3-kinase in a manner similar to that of wild-type MT yet is reduced in its transforming ability on NIH 3T3 cells.     

Regulation of cellular phenotype and expression of polyomavirus middle T antigen in rat fibroblasts.

Polyoma middle T antigen (mT) was expressed in rat F-111 cells under control of the dexamethasone-regulatable mouse mammary tumor virus promoter. Graded phenotypic responses to levels of mT induction by the hormone were seen, with morphological transformation, focus formation, and anchorage-independent growth requiring increasing levels of mT expression. The ability of different clones to form tumors reflected their maximum level of induction of mT-associated kinase and their ability to grow in soft agar. Expression of transformation parameters and tumorigenicity correlates with the level of mT phosphorylated by pp60c-src in immune complexes and not with the total amount of mT determined by metabolic labeling. We suggest that cellular factors regulate mT activity by forming a kinase-active fraction of mT molecules that controls the transformed state.     

Abundant expression of polyomavirus middle T antigen and dihydrofolate reductase in an adenovirus recombinant.

A modular gene with a cDNA encoding the polyomavirus middle T antigen positioned behind the adenovirus type 2 major late promoter and tripartite leader was substituted for the E1a region in an adenovirus vector. Permissive human cells infected with this recombinant produce middle T protein at levels as high as those of the most abundant late adenoviral proteins, e.g., hexon or fiber. This level represents at least a 40-fold increase over that observed in a polyomavirus lytic infection of murine cells. Partial proteolytic mapping showed that this protein has the same primary structure as middle T protein produced in polyomavirus-infected murine cells. The adenovirus recombinant-generated middle T protein exhibited in vitro kinase activity, although at an approximately 10-fold-lower specific activity than that of middle T protein from polyomavirus-infected murine cells. Comparison of the expression levels of this middle T antigen-containing adenovirus vector with a similar construction encoding dihydrofolate reductase suggested that the translation efficiency of the inserted gene was dependent upon the proximity of its initiation codon to the tripartite leader. We tested this possibility by comparing three dihydrofolate reductase recombinants among which the spacing between the initiation codon and tripartite leader varied from 188 to 36 nucleotides. The efficiency of expression of dihydrofolate reductase protein dramatically increased as this spacing was reduced.