The hr-t gene of polyoma virus

Malignant transformation of cells by polyoma virus results from the continual expression of a viral gene (hr-t) the normal function of which is to facilitate productive viral infection. The series of investigations described here on the polyoma hr-t gene originated with attempts to understand polyoma virus-cell interactions along lines suggested by temperate bacteriophage. Nucleic acid hybridization experiments indicated clearly that viral DNA persists in transformed cells and continues to be expressed. Radiobiological and other experiments, however, suggested a function for the expressed gene(s) which was not expected of a prophage: the promotion, rather than repression, of lytic virus growth. The hr-t gene acts pleiotropically to alter the physiological state of the host in a manner which facilitates virus production and induces a transformed cellular phenotype. The cellular alterations are manifested transiently during productive infection or abortive transformation, but permanently when the viral genome is integrated in stably transformed cells. hr-t mutants are defective in their growth in mice and in most cultured mouse cell lines. They are also unable to induce tumors or any of the morphological, structural, or growth-related changes which accompany cells transformation by the wild-type virus.The 22 kDa and 56 kDa proteins encoded in the early region of the viral DNA constitute dual products of the hr-t gene. hr-t mutants are localized in a narrow segment of the early region that specifies an amino acid sequence shared by these two overlapping proteins. Current efforts to link structural (i.e., mutational) changes with functional changes in these proteins center around the 56 kDa middle T antigen and its associated protein kinase activity. Assayed in vitro, this activity leads to phosphorylation of the 56 kDa protein itself, predominantly at a specific tyrosine residue in the C-terminal portion of the molecule. The middle T protein is anchored in cellular membranes by a hydrophobic tail close to the C-terminus. Membrane association is essential for transformation, as well as for the kinase activity. The common region of the 22 kDa/56 kDa proteins where hr-t mutants map has local regions of homology with highly conserved sequences in the pituitary glycoprotein hormones. The integrity of this region is also essential for transformation and for kinase activity. In vivo, the 56 kDa protein is a substrate for cellular kinase(s) and undergoes multiple phosphorylations (serine and/or threonine) that may affect the tyrosine-specific activity. These kinase reactions, originating in cellular membrane but potentially affecting pathways into the cytoplasm and nucleus, currently provide the most plausible biochemical mechanism underlying the pleiotropic effects of the hr-t gene.     

Cellular alterations dependent upon the polyoma virus Hr-t function: separation of mitogenic from transforming capacities.

Hr-t mutants of polyoma virus are restricted in their growth properties (host range) and defective in cell transformation and tumor induction. The present study indicates that these mutants have lost the ability to induce morphological transformation, but have retained a mitogenic function. Thus an early and dramatic difference between wild-type virus and hr-t mutant-infected cultures of rat fibroblasts is the morphological change in individual cells observed by light, fluorescence and scanning electron microscopy. Viruses containing an intact hr-t function (wild-type virus and ts-a mutants) induce a transformed phenotype consisting of stellate cell shape, loss of defined cytoplasmic actin architecture, cellular "underlapping," and increased nuclear and nucleolar sizes. These prominent alterations constitute an abortive transformation, peaking 24-48 hr post-infection, and subsequently resolving in most or all of the cells. In contrast, cells infected with hr-t mutants do not develop the above structural changes, but rather retain their preinfection appearance. Both wild-type virus and hr-t mutants induce cellular DNA synthesis in confluent monolayers of rat cells beginning 12-14 hr post-infection. Flow microfluorometric (FMF) analysis confirms the viral mediated transit of cells from the G1 to the S and G2 phases of the cell cycle, as well as an increase in the proportion of cells with an 8N (octaploid) DNA content. Approximately 50% of the clones isolated from wild-type-infected cultures are polyploid. Stable transformants are found among these polyploid clones, but the majority of the latter resemble the parental cells in their morphology and growth properties. Polyploid clones are derived from hr-t mutant-infected cultures at a much lower frequency, similar to that of mock-infected cultures. Data obtained by sequential labeling of infected cultures with 3 H-thymidine and 5-bromo-deoxyuridine, together with cell number quantitation, indicate that hr-t mutants promote only a single round of cell division, while the wild-type virus and ts-a mutants promote multiple rounds. Loss of the hr-t function in polyoma virus therefore reveals a residual viral mitogenic activity, but prevents the virus from effecting morphological transformation of cells with concomitant loss of defined actin cables, polyploidization and multiple cycles of cell division in confluent cultures.     

Phosphorylation of polyoma T antigens.

The T antigens of polyoma virus have been examined for phosphorylation in vivo and associated protein kinase activities in vitro. The 100K "large" T antigen is the major phosphoprotein among the T antigen species in vivo as determined by labeling virus-infected cells with 32P-orthophosphate. Hr-t mutants show normal phosphorylation of their 100K T antigens. The wild-type 56K plasma membrane-associated "middle" T antigen is also phosphorylated in the cell, but to a lesser extent than the 100K; this low level phosphorylation is also observed in the presumably altered 56K protein induced by hr-t mutant NG59 and in the 50K truncated "middle" T of hr-t mutant SD15. Addition of dibutyryl cyclic AMP to the medium does not affect labeling of either large or middle T antigens in wild-type- or mutant-infected cells. Thus no differences are observed in T antigen phosphorylation in vivo between wild-type virus and hr-t mutants. Hr-t mutants are defective in a protein kinase activity assayed in vitro by adding gamma-32P-ATP to T antigen immunoprecipitates. In the case of wild-type virus, the 56K protein is the major phosphate acceptor in the in vitro kinase reaction, with a somewhat lower level of phosphorylation observed in the 100K band. Hr-t mutants NG59 and SD15 show no labeling of the altered 56K or 50K, respectively, but do show detectable levels of 32P in the 100K bands. A wild-type virus carrying a small deletion affecting the 100K and 56k bands shows a normal level of kinase activity associated with the truncated T antigens. Ts-a mutants appear to be normal with respect to the middle T antigen-associated kinase. Photoaffinity labeling of infected cell extracts with 8-azido cyclic AMP shows that the two major classes of regulatory subunits of cyclic AMP-dependent protein kinases are present in the immunoprecipitates. Phosphorylation of histone H1 occurs when this substrate is added to immunoprecipitates of either mock-infected or virus-infected cells, again demonstrating the presence of cellular kinases. Further experiments will be required to determine whether the middle T antigen of polyoma virus is itself a protein kinase or simply a substrate for one or more cellular kinases.     

An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates.

Polyoma T antigen immunoprecipitates contain a protein kinase-like activity which preferentially phosphorylates material of 50-60,000 daltons molecular weight. Phosphorylation is not diminished in extracts of polyoma tsA mutant-infected cells shifted to the nonpermissive temperature late in infection, conditions which inactivate the large T antigen. Phosphorylation is reduced or absent in cells infected with polyoma host range nontransforming (hr-t) mutants, which have defective small and medium T antigens. The major acceptor of phosphate is not the heavy chain of immunoglobulin, but appears to be the polyoma medium T antigen. The large T antigen is also phosphorylated, but usually to a lower specific activity. In terms of acid and alkali sensitivity and electrophoretic and chromatographic mobility in one and two dimensions, the phosphorylated residue behaves identically to phosphotyrosine and differently than phosphorylated serine, threonine, lysine and histidine.     

Untransformed rat cells containing free and integrated DNA of a polyoma nontransforming (Hr-t) mutant.

The polyoma virus hr-t deletion mutant A185, when compared to wild-type (Py) virus, is at least 10⁵ fold inhibited in its transforming ability. Total cellular DNA from 50 cell lines derived from individual colonies formed after infection of Rat-1 cells with A185 virus was analyzed for the presence of viral sequences by “blot” hybridization (Southern, 1975). Viral sequences were detected in two of these cellular DNAs. One positive cell line (18–37) was studied in detail. The viral sequences present in 18–37 cells as well as the viral sequences present in virus rescued from 18–37 after fusion with permissive mouse cells were identified as A185 and not Py sequences. The A185 viral sequences in 18–37 cells were found to exist both covalently linked to host DNA sequences (integrated) and as free forms. The integrated A185 viral sequences were present in a partial head-to-tail tandem array, as has been observed for Py sequences in transformed rat cells (Birg et al., 1979). Both integrated and free forms of A185 viral sequences were retained in subclones of the parental 18–37 cell line although a simplification of the integrated viral sequence was observed. In the 18–37 cells the 100K large T antigen was synthesized but the 55K middle and 22K small T antigen species were not detected. The 18–37 cells had a normal morphology, were density-sensitive, anchorage-dependent and did not form tumors when injected into syngeneic animals. This normal phenotype of the 18–37 cells was not a result of the inability of the cells to express the transformed phenotype, since the 18–37 cells could be transformed at a high frequency upon infection with Py virus. These results show that integration of viral sequences per se or the presence of the 100K large T antigen is not sufficient for the transformed phenotype to be expressed, and strongly suggest that Py-induced transformation is mediated by the 55K middle and/or 22K small T antigens.     

Tumor antigens induced by nontransforming mutants of polyoma virus.

We have studied the tumor (T) antigens induced by wild-type polyoma virus and several nontransforming mutants using immunoprecipitation with antisera from animals bearing polyomya-induced tumors followed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. In a variety of mouse cells, wild-type virus induces a major T antigen species with apparent molecular weight of 100,000 daltons, and four minor T antigen species with apparent molecular weights of 63,000, 56,000, 36,000 and 22,000 daltons. Hr-t mutants, which have an absolute defect in transformation, induce a normal 100,000 dalton T antigen but are altered in the minor T antigen species. Hr-t deletion mutants induce none of the minor T antigen species seen in wild-type virus. In their place, these mutants induce T antigen species with molecular weights in the range of 6,000--9,000 daltons. The size of the very small T antigen products does not correlate in any simple way with the size or location of the deletions in the viral DNA. Point hr-t mutants induce two of the four minor T antigen species; they make apparently normal amounts of the 56,000 dalton product and reduced amounts of the 22,000 dalton product, but none of the 63,000 or 36,000 dalton species. Ts-a mutants, which have a temperature-sensitive defect in the ability to induce stable transformation, and which complement hr-t mutants, induce T antigens with the same mobility as wild-type; however, the 100,000 dalton T antigen of ts-a mutants is thermolabile compared to wild-type. A double mutant virus carrying both a ts-a mutation and a deletion hr-t mutation induces a thermolabile 100,000 dalton product and none of the minor T antigen species. Cell fractionation studies with productively infected cells have been carried out to localize the T antigen species.     

Comparison of phosphorylation of two polyoma virus middle T antigens in vivo and in vitro.

Two species of polyoma virus middle T antigen were detected in both lytically infected and transformed cells by in vitro kinase assay of immunoprecipitates. A minor species with an apparent molecular weight of 58,000 (58K) represented less than 10% of the total middle T protein. This species was roughly 10 times more active as a phosphate acceptor than was the predominant 56K form. Partial proteolytic mapping experiments showed that the same site was phosphorylated in both species. Mapping of the middle T antigens from a series of deletion mutants suggested that the major site of phosphorylation is tyrosine residue 315. Phosphorylation occurred on both middle T species in vivo, involving sites predominantly other than the tyrosine labeled in vitro. The 56K and 58K middle T forms differed from each other in their in vivo phosphorylation patterns. Some phosphate was incorporated into the 58K species in a region of the molecule to which at least part of the apparent molecular weight different could be mapped. hr-t mutant NG-59, which codes for a slightly altered middle T, produced only a single species (56K) which was inactive in the in vitro kinase reaction. Moreover, no 58K species appeared in vivo with this mutant. hr-t mutants are therefore defective in both aspects of phosphorylation. Phenotypically normal revertant cells of a polyoma transformed line failed to express any middle T antigens or associated kinase activity.     

Effect of Interferon on Some Aspects of Transformation by Polyoma Virus

WHEN BHK 21 hamster cells are infected with polyoma virus¹, there is no vegetative growth of virus, but stably transformed cells appear. These transformed cells are more easily transplanted than BHK 21 cells; they initiate their growth cycle in otherwise restrictive cultural conditions such as the absence of serum, high density and suspension; they grow with random orientation and have exposed on their surfaces receptor sites for certain glycoprotein agglutinins^2–5. The proportion of stably transformed cells is low, even after high doses of virus. But a much higher proportion (sometimes all cells) shows abortive transformation—changes characteristic of transformation, but which last only a few days. In suspension cultures, for example, most of the infected cells grow into small colonies of four to thirty-two cells⁶. In surface cultures deprived of serum DNA synthesis is initiated and the cells may then divide at least once⁷: they also temporarily lose the normal parallel orientation and develop the typical random appearance of transformed cells. Moreover, the polyoma nuclear T-antigen and also a surface antigen detected by immunofluorescence, appear temporarily in most polyoma infected BHK 21 cells⁸, while 3T3 cells exposed to SV40 virus show transient exposure of cell surface sites reacting with conconavalin A (ref. 9).     

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.     

Immunization against the polyoma virus-induced tumor-specific transplantation antigen by early region mutants of the virus

To investigate the relation between the polyoma tumor-specific transplantation antigen and the virus-coded proteins, mice were immunized by inoculation of a variety of viable polyoma virus mutants and then challenged with polyoma virus-induced tumors. Two classes of early region mutants were used. One class produces a normal small T-antigen and truncated middle and large T-antigens. The second class (hr-t mutants) forms a normal large T-antigen together with N-terminal fragments of small and middle T-antigens. All mutants, transforming as well as nontransforming, induced protection against polyoma virus tumors. However, there were quantitive differences between the mutants. The finding that an hr-t mutant could induce tumor rejection suggests that full-length middle and small T-antigens are not necessary for the induction of this response. Since intact middle T-antigen is the only virus-coded protein known to associate with the plasma membrane, the possibility must be considered that the polyoma virus tumor-specific transplantation antigen consists of cellular components.     

Distinct transformation phenotypes induced by polyoma virus and simian virus 40 in rat fibroblasts and their control by an early viral gene function.

Several transformed cell lines established from Fisher rat cells (FR 3T3) infected with wild-type polyoma virus or simian virus 40 or early temperature-sensitive mutants (polyoma tsa and simian virus 40 tsA30) were studied for their transformation phenotypes. The distinct patterns which were obtained for polyoma and simian virus 40 transformants led to the conclusion that these two viruses express different transforming abilities in rat cells. The results obtained with temperature-sensitive mutant-derived transformants indicate that all of the transformation characteristics studied so far may be under the control of a viral function in polyoma tsa-transformed cells.     

Transformation phenotype of polyoma virus-transformed rat fibroblasts: plasminogen activator production is modulated by the growth state of the cells and regulated by the expression of an early viral gene function

The expression of two transformation parameters, namely, ability to grow in agar and plasminogen activator production, was studied in several rat fibroblasts transformed by either wild-type or thermo-sensitive (tsa and ts25) polyoma viruses. The production of plasminogen activator was found to be dependent upon the growth state of the infected cells during a period of several days after infection. The analysis of the transformed phenotype of 25 tsa transformants and of 19 ts25 transformants independently isolated under various growth conditions led to the conclusion that there is no correlation between the regulation processes involved in plasminogen activator production and ability to grow without anchorage. The results obtained also suggested that the production of plasminogen activator is under the control of a functional large T antigen.     

Cooperation of p53 and polyoma virus middle T antigen in the transformation of primary rat embryo fibroblasts.

Cell transformation in vivo seems to be a multistep process. In in vitro studies certain combinations of two oncogenes, a cytoplasmic gene product together with a nuclear gene product, are sufficient to transform primary rodent cells. Polyoma virus large T antigen can immortalize and, in cooperation with polyoma virus middle T antigen, transform primary cells. On the other hand mutant mouse p53 can also immortalize and, in cooperation with an activated Ha-ras oncogene, transform primary cells. In the present study we analyzed whether mutant p53 can replace polyoma virus large T antigen in a cell transformation assay with polyoma virus middle T antigen. Transfection of mutant p53 alone resulted in a cell line which had retained the actin cable network, grew poorly in medium with low concentration of serum, and failed to grow in semisolid agar. Cotransfection of mutant p53 together with polyoma virus middle T led to cells which grew in medium containing low serum concentration, grew well in semisolid agar, and displayed an altered morphology with the tendency to overgrow the normal monolayer. By these criteria these cells were considered fully transformed. The rate of p53 synthesis was similar in both cell lines. However, only p53 from the transformed cell line turned out to be stable. Cells transformed by mutant p53 and polyoma virus middle T expressed nearly the same amount of the c-src-encoded pp60c-src protein as cells transformed by the same p53 and cotransfected activated Ha-ras oncogene. However, only the polyoma virus middle T/p53-transformed cells exhibited an elevated level of pp60c-src-specific tyrosine kinase activity. Thus, despite different mechanisms leading to cell transformation, mutant p53 can replace polyoma virus large T antigen and polyoma virus middle T can replace the activated Ha-ras oncogene in cell transformation.     

Structural prerequisites of simian virus 40 large T antigen for the maintenance of cell transformation.

We have investigated the functional roles of two structural subsets of simian virus 40 (SV40) large T antigen, namely homo-oligomers and complexes with the host cellular p53 protein, for the transformed phenotype. We examined T antigen produced in cells transformed by temperature-sensitive SV40 large T mutants: heat-sensitive or unrestricted SV40 tsA58-transformed rat cells and unrestricted tsA1499 transformants. In both unrestricted cell lines, T antigen was temperature-sensitive only for the formation of fast sedimenting homo-oligomers. Corresponding to our recent observations obtained with tsA1499-infected monkey cells, in tsA1499 transformants large T was competent to form stable T-p53 complexes independently of the temperature. However, T antigen coded for by tsA58, which is heat-sensitive for binding to p53, occurred in stable complexes with this protein in unrestricted tsA58 transformants under all conditions. Furthermore, in both unrestricted transformants T-p53 complexes arise in the absence of homo-oligomers of T antigen. In conclusion, T antigen homo-oligomers are not involved in cell transformation, whereas T-p53 complexes may be involved in the maintenance of this phenotype.     

Expression of a New Complement-fixing Antigen reactive with Murine Sarcoma Virus Rat Antiserum in Rat Cells transformed by Polyoma Virus

WE reported accelerated transformation by DNA viruses (SV40 and polyoma) in rat embryo (RE) cells chronically infected with a C-type RNA virus^1,2. Recently we found in RE cells transformed by polyoma virus a new complement-fixing (CF) antigen detectable by rat antisera having broad reactivity with the various intraspecies and interspecies antigens of the RNA tumour viruses^3–8; this antigen, however, was distinct from the murine intraspecies and interspecies group-specific (gs) antigens both immunologically and by virtue of other properties. It is also distinct from the polyoma virion (capsid) and tumour (“T”) antigens.     

Effects of large and small T antigens on DNA synthesis and cell division in simian virus 40-transformed BALB/c 3T3 cells.

The roles of the large T and small t antigens of simian virus 40 in cellular DNA synthesis and cell division were analyzed in BALB/c 3T3 mouse cells transformed by wild-type, temperature-sensitive A (tsA), or tsA-deletion (tsA/dl) double mutants. Assessment of DNA replication and cell cycle distribution by radioautography of [3H]thymidine-labeled nuclei and by flow microfluorimetry indicate that tsA transformants do not synthesize DNA or divide at the restrictive temperature to the same extent as they do at the permissive temperature or as wild-type transformants do at the restrictive temperature. This confirms earlier studies suggesting that large T induces DNA synthesis and mitosis in transformed cells. Inhibition of replication in tsA transformants at the restrictive temperature, however, is not complete. Some residual cell division does occur but is in large part offset by cell detachment and death. This failure to revert completely to the parental 3T3 phenotype, as indicated by residual cell cycling at the restrictive temperature, was also observed in cells transformed by tsA/dl double mutants which, in addition to producing a ts large T, make no small t protein. Small t, therefore, does not appear to be responsible for the residual cell cycling and plays no demonstrable role in the induction of DNA synthesis or cell division in stably transformed BALB/c 3T3 cells. Comparison of cell cycling in tsA and tsA/dl transformants, normal 3T3 cells, and a transformation revertant suggests that the failure of tsA transformants to revert completely may be due to leakiness of the tsA mutation as well as to a permanent cellular alteration induced during viral transformation. Finally, analysis of cells transformed by tsA/dl double mutants indicates that small t is not required for full expression of growth properties characteristic of transformed cells.     

Middle T antigen as primary inducer of full expression of the phenotype of transformation by polyoma virus.

A large number of polyoma virus-transformed cells of rat, mouse, and hamster origin were examined for presence of T-antigen species. The results showed that all lines of cells contained middle and small T antigens, but not all contained a full-sized large T antigen, in some cell lines large T antigen was absent, whereas in others it was present as truncated forms lacking various lengths of the carboxy-terminal part of the protein. Cells transformed by the new viable deletion mutants of polyoma virus, dl-8 and dl-23, formed larger and smaller colonies or foci, respectively, when they were suspended in semisolid medium or plated as monolayers together with untransformed cells on a plastic surface. The deletions in the DNA of these mutants resulted in the shortening of the large and middle T antigens simultaneously without affecting the size of the small T antigen. Variation of large T-related proteins in dl-8 and dl-23-transformed cells seemed to be the same as that observed in wild-type-transformed cells. Regardless of the amount and size of large T-related protein in mutant-transformed cells, the phenotype of the cells was entirely dependent on the mutant used. The results suggest that (i) persistence of large T antigen is not universally required for the maintenance of the transformation phenotype, (ii) small T antigen alone may not be sufficient for inducing the full expression of the transformation phenotype, and (iii) middle T antigen is implicated as being primarily responsible for the full expression of the phenotype of transformation. The results also provide the evidence that the carboxy-terminal region of middle T antigen and a part of large T antigen are encoded in the genome in the same DNA segment around map units 88 to 94 in different reading frames.     

Viable deletion mutant in the medium and large T-antigen-coding sequences of the polyoma virus genome.

A polyoma virus mutant that maps in the early region between the known hr-t and ts-a mutants has been isolated. Its 66-base-pair deletion results in structural changes in both medium and large T-antigens but causes no substantial alterations in viral replication or cell transformation.     

DNA sequence alterations in Hr-t deletion mutants of polyoma virus.

We have investigated the DNA sequence alterations in several hr-t mutants of polyoma virus. These mutants are defective in one of the two known viral functions essential for transformation and are altered with respect to several minor T antigen species. The lesions in some of these mutants have been mapped previously by marker rescue experiments to Hpa II fragment 4 (Hpa II-4, 78.4--91.7 map units) in the proximal part of the early region of the viral DNA. Thirteen of sixteen hr-t mutants examined carry deletions 2 to 5 map units (100--250 bp) long in Hpa 11-4. Three mutants carry either point mutations or very small deletions/insertions. Eight of the deletion mutants were mapped closely with restriction enzymes. Seven of them have deletions located entirely within the Hae III subfragment A of Hpa II-4 (the Hae A subfragment, 78.4--85.2 map units), and one extends just beyond this subfragment, ending at 85.5 map units. The complete sequence of the wild-type Hae A subfragment was determined and compared with those of four deletion mutants, NG-18, A-8, 6B5 and B-2. The deletion in each of these mutants is out-of-phase: NG-18, 187 bp; A-8, 127 bp; 6B-5, 179 bp; B-2, 241 bp. All are expected to remove protein sequences in the C terminal part of the small t antigen.     

Protein kinase activity associated with polyoma virus middle T antigen in vitro.

A protein kinase activity can be detected in immunoprecipitates of extracts from polyoma virus (Py)-infected cells using antiserum raised against Py-transformed cells (anti-T serum). The activity is not detected in uninfected cells or when using control serum. Using rat anti-T serum both Py middle T and the heavy chain of rat IgG are phosphorylated, whereas using hamster anti-T serum only Py middle T is phosphorylated. Experiments using a number of different mutants of Py indicate that the kinase activity detected is under viral control and is associated with Py middle T. Consistent with this the kinase, like middle T, can be detected in purified preparations of plasma membranes. The kinase can also be detected in a large number of Py-transformed cells, but not in untransformed cells or in cells transformed by other viruses. Some of the Pytransformed cells which contain kinase activity lack full sized Py large T but all contain middle T. Kinase activity is not detected in a cell line (18.37) which contains integrated viral DNA of a nontransforming hr-t deletion mutant and which contains Py large T but not middle T or small t. These results show that Py middle T or a protein which specifically binds to it has protein kinase activity in vitro. Although these results raise the possibility that protein kinases play an essential role in Py-induced transformation, however, thus far we have no data which show unequivocally that the results are physiologically significant.