Deletion mapping of a short polyoma virus middle T antigen segment important for transformation.

Viable polyoma virus mutants were constructed that had small deletions in the early region of the genome. The deletions together removed most of the segment missing from the genome of the nontransforming mutant dl23 (N. Smolar and B. E. Griffin, J. Virol. 38:958-967, 1981). The transformation properties, as measured by colony formation in soft agar, of mutants with overlapping or contiguous deletions showed that part or all of the middle T antigen segment, consisting of the short amino acid sequence Glu4-Tyr-Met-Pro-Met, was essential for the activity of the protein in transformation. However, the segment could be deleted without significant effect on the in vitro protein kinase activity associated with the middle T antigen.     

Purified polyoma virus medium T antigen has tyrosine-specific protein kinase activity but no significant phosphatidylinositol kinase activity.

Medium T antigen, the transforming protein of polyoma virus, is associated with pp60c-src and strongly activates its tyrosine-specific protein kinase activity. We investigated whether the medium T-pp60c-src complex is also associated with an activity that phosphorylates the membrane phospholipid phosphatidylinositol, as shown for pp60v-src and p68v-ros, the transforming proteins of Rous sarcoma virus and avian sarcoma virus UR2, respectively. Medium T was purified by affinity chromatography from extracts of polyoma virus-infected mouse fibroblasts. It was bound to antibodies against a peptide corresponding to the carboxy terminus of medium T and released from the immune complex with an excess of the same peptide. In a second step, the partially purified medium T was bound to antibodies against another peptide corresponding to an internal region of medium T and released with excess peptide. Further purification was carried out with a monoclonal antibody against pp60c-src. Samples from each purification step were examined for protein kinase and phosphatidylinositol kinase activity. The highly purified preparations of the medium T-pp60c-src complex showed very low levels of phosphatidylinositol kinase activity, and no difference between medium T from transforming viruses and nontransforming hr-t mutants was detected. In contrast, protein kinase activity was associated with medium T purified from transforming viruses but not from hr-t mutants.     

Purification and N-terminal amino acid sequence of the human T cell surface antigen T8

Monoclonal antibodies allow for the detection of structures on the cell surface of human cytotoxic T lymphocytes (CTL) that are involved in their effector function. Among these cell surface components, T8 is of particular interest because it is required during the recognition of target cells by a subset of CTL. An understanding of its role during CTL:target adhesion requires detailed biochemical structural analysis of the T8 molecule. This has been hindered by the small amounts of protein currently available. Here we describe the development of a purification scheme that will permit the accumulation of larger quantities of T8. We studied the binding of T8 to several lectins and determined that one of these, wheat germ agglutinin, bound T8 quantitatively. Experiments designed to test the properties of T8 in a phase separation system with the use of Triton X-114 were performed. These indicated that T8 partitions into the aqueous phase rather than the detergent phase during this procedure. With this in mind, we developed a protocol that resulted in a significant purification of T8 after affinity chromatography. Upon preparative SDS-PAGE followed by electroelution, the T8 antigen was purified to homogeneity and used for N-terminal acid acid sequencing. This analysis yielded the amino terminal 22 amino acids of T8. On purification, it was observed that the protein existed as two bands of Mr 33 and 34 kilodaltons after SDS-PAGE analysis. The relationship between these chains was investigated by limited radio-sequencing and tryptic peptide map analysis; our results indicated that the two polypeptides were identical. The two chains were treated with trifluoromethane sulfonic acid to determine whether carbohydrates accounted for the difference in m.w. This reagent, which cleaves both N-linked and O-linked sugars, cleaved approximately 2000 daltons of oligosaccharides from the T8 molecule. These oligosaccharides are most likely of the O-linked rather than the N-linked variety.     

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.     

Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation

We used site-specific mutagenesis to introduce a termination codon, TGA, into the reading frame for the polyoma virus medium T antigen. We induced this mutation in a region of the polyoma genome in which the overlapping coding regions for the large and medium TE antigens are translated in different reading frames. Therefore, the mutation terminated translation of the medium T antigen, but it caused only a single amino acid substitution in the large T antigen and did not affect the small T antigen. Cells infected by the mutant virus produced normal-size small and large T antigens. The infected cells produced a 28,000-dalton fragment of the 48,000-dalton medium T antigen, whose size and tryptic peptide map were consistent with its being a truncated N-terminal fragment terminating at the new termination codon of the mutant. Immunoprecipitates of mutant-infected cell extracts did not show medium-T-antigen-associated protein kinase activity. The mutant virus replicated normally in mouse 3T6 cells and induced cellular DNA synthesis in resting mouse 3T3 cells, but it failed to transform rat or hamster cells, as judged by focus formation and growth in agar. The mutant complemented a tsA mutant which affects the large T antigen for transformation, implying that the mutant defect for transformation was in the medium T antigen. These results imply that the small T antigen and the large T antigen together are insufficient to cause transformation and support the conclusion that the medium T antigen is essential for cell transformation by polyoma virus.     

N-terminal amino acid sequences and amino acid compositions of the Spirochaeta aurantia flagellar filament polypeptides.

The amino-terminal sequences and amino acid compositions of the three major and two minor polypeptides constituting the filaments of Spirochaeta aurantia periplasmic flagella were determined. The amino-terminal sequence of the major 37.5-kDa outer layer polypeptide is identical to the sequence downstream of the proposed signal peptide of the protein encoded by the S. aurantia flaA gene. However, the amino acid composition of the 37.5-kDa polypeptide is not in agreement with that inferred from the sequence of flaA. The 34- and 31.5-kDa major filament core polypeptides and the 33- and 32-kDa minor core polypeptides show a striking similarity to each other, and the amino-terminal sequences of these core polypeptides show extensive identity with homologous proteins from members of other genera of spirochetes. An additional 36-kDa minor polypeptide that occurs occasionally in preparations of S. aurantia periplasmic flagella appears to be mixed with the 37.5-kDa outer layer polypeptide or a degradation product of this polypeptide.     

Significance of the gastrin homology and surrounding sequences in polyomavirus middle T antigen for cell transformation.

Deletion of residues 305 to 327 of polyomavirus middle T antigen, including the (Glu)6-Tyr-315 sequence that is a preferred site of phosphorylation in vitro by pp60c-src, markedly altered viral transformation of rat cells. The efficiency of transformation by the deletion mutant depended on how it was introduced into cells, and the resulting transformants displayed limited growth rates in monolayer and in suspension. Substitution of the polyomavirus residues 305 to 327 with a homologous region (containing [Glu]5-Ala-Tyr) from porcine gastrin did not restore wild-type transforming activity. These mutant middle T antigens interacted with pp60c-src and were phosphorylated in vitro. Thus, although a sequence of consecutive glutamic acid residues followed by a tyrosine is a dominant structural element which strongly influences the physical properties of middle T antigen, its presence did not ensure the biological activity of the protein. Other elements in this region of middle T antigen also contributed substantially to the transforming capacity of polyomavirus.     

Antibody to the nonapeptide Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu is specific for polyoma middle T antigen and inhibits in vitro kinase activity

Middle T antigen of polyoma virus has an associated tyrosine kinase activity which phosphorylates tyrosine residue 315 on middle T in immunoprecipitates. A peptide representing the sequence of middle T from residue 311 to 319 has been synthesized. This peptide acts as a weak inhibitor of the kinase reaction. An antiserum has been raised against this peptide after conjugation to bovine serum albumin. The antibody is middle T-specific. Middle T antigen precipitated by this serum is largely inactive in the kinase reaction. Dissociation of the immune complex with peptide releases middle T in a kinase-active form.     

Human IFN-alpha protein engineering: the amino acid residues at positions 86 and 90 are important for antiproliferative activity

Human IFN-alpha is a family of structurally related proteins that exhibit a wide range of antiproliferative activities. To understand the structural basis for these different antiproliferative activities, eight recombinant human IFN-alpha hybrids (HY) of alpha21a/alpha2c (HY-4, HY-5) and mutants (site-directed mutagenesis (SDM)-1, 2 and cassette mutagenesis (CM)-1, 2, 3, and 4) have been expressed, purified, and characterized. The data showed that the amino acid region 81-95 is important for antiproliferative activity. Site-directed mutagenesis and cassette mutagenesis studies showed that if serine (S) 86 and asparagine (N) 90 were replaced by tyrosine (Y), the antiproliferative activity was increased. We have also observed that if Y86 was replaced by isoleucine (I), the antiproliferative activity was comparable. However, if Y86 was replaced by aspartic acid (D), lysine (K), or alanine (A), the antiproliferative activity was substantially decreased. Our results indicate that Y and/or I at position 86 and Y at position 90 are very important in antiproliferative activity of human IFN-alpha. Circular dichroism spectra showed that the amino acid replacements at position 86 did not change the secondary structure. Thus the biological activity changes among those mutants do not appear to be due to conformational changes. The results also suggest that hydrophobic residue(s) at position 86 may be important for the interaction of the molecule with its receptor. The competitive binding data correlated with the antiproliferative activity. The N-terminal region of the molecule and the hydrophobic residues (including Y and I) on the C-helix region at positions 86 and/or 90 are important for binding and antiproliferative activities of human IFN-alphas.     

A polyoma mutant that encodes small T antigen but not middle T antigen demonstrates uncoupling of cell surface and cytoskeletal changes associated with cell transformation.

The hr-t gene of polyoma virus encodes both the small and middle T (tumor) antigens and exerts pleiotropic effects on cells. By mutating the 3' splice site for middle T mRNA, we have constructed a virus mutant, Py808A, which fails to express middle T but encodes normal small and large T proteins. The mutant failed to induce morphological transformation or growth in soft agar, but did stimulate postconfluent growth of normal cells. Cells infected by Py808A became fully agglutinable by lectins while retaining normal actin cable architecture and normal levels of extracellular fibronectin. These properties of Py808A demonstrated the separability of structural changes at the cell surface from those in the cytoskeleton and extracellular matrix, parameters which have heretofore been linked in the action of the hr-t and other viral oncogenes.     

Purification and N-terminal amino acid sequences of Chlamydia trachomatis histone analogs.

DNA-binding proteins specific to Chlamydia trachomatis elementary bodies have been described and recently characterized as procaryotic histone analogs. I have developed an affinity purification procedure for the 18-kDa histone analog, Hc1, based on its affinity for polyanions. The availability of highly purified Hc1 has allowed for determination of its N-terminal amino acid sequence and should prove useful in studies of its biological function. The variable C. trachomatis histone analog not obtained by this procedure was electrophoresed onto Immobilon paper for sequencing. The N terminus of the variable histone was conserved among C. trachomatis serotypes L2, D, and B and was distinct from that of Hc1.     

In vitro association and phosphorylation of polyoma virus middle T antigen by cellular tyrosyl kinase activity

We have observed increased phosphorylation of tyrosine residues on the polyoma virus middle tumor antigen (MTAg) in in vitro kinase assays of the immune complexes immunoprecipitated from lysates of polyoma virus-infected mouse embryo cells to which increasing amounts of uninfected mouse embryo cell lysate had been added. The components from uninfected mouse cells responsible for increased MTAg phosphorylation were localized by subcellular fractionation to the plasma membrane and found to be sensitive to protease digestion, N-ethylmaleimide, and 5'-p-fluorosulfonylbenzoyladenosine inactivation. The majority of the membrane-associated activity responsible for the increased MTAg phosphorylation in these assays could be cleared from lysates of uninfected mouse cell lysates by centrifugation after reaction with Sepharose-bound monoclonal antibodies which recognize pp60c-src. These results suggest that MTAg can associate with cellular tyrosyl kinases in vitro and be phosphorylated by these enzymes in immune-complex kinase assays. The identity of at least one of these cellular tryosyl kinases which can associate with MTAg in vitro is likely to be pp60c-src.     

Determinants on simian virus 40 large T antigen are important for recognition and phosphorylation by casein kinase I.

Casein kinase I has been shown to phosphorylate Ser123 and possibly Thr124, in simian virus 40 (SV40) large T antigen; the same sites are also modified in cultured cells incubated with 32Pi [Friedrich A. Gr?sser, Karl H. Scheidtmann, Polygena T. Tuazon, Jolinda A. Traugh & Gernot Walter (1988) Virology 165, 13-22]. The peptide, A-D-S-Q-H-S-T-P-P, which corresponds to the amino acid sequence 118-125 of SV40 large T antigen, was synthesized together with peptides containing changes in specific amino acid residues on either side of Ser123. These peptides were used as model substrates to determine the amino acids in the SV40 large T antigen important for recognition by casein kinase I. The native peptide identified above, with aspartate at the -4 position, was a poor substrate for casein kinase I in vitro. Peptides with acidic residues added at the -2 and -3 positions, preceding Ser123, were phosphorylated by casein kinase I with apparent Km values around 2 mM and Vmax values up to 500 pmol.min-1.ml-1. When acidic residues were added at both sides of the phosphorylatable serine, the peptide had a first-order rate constant over 20-fold higher than peptides with acidic amino acid residues at the N-terminus only; the apparent Km value was 0.65 mM with a Vmax of 2900 pmol.min-1.ml-1. The effects of modifying Ser120 to phosphoserine were examined by addition of a recognition sequence for the cAMP-dependent protein kinase prior to Ser120. Prior phosphorylation of the peptide at Ser120 lowered the apparent Km to 0.061 mM and increased the Vmax to 360 pmol.min-1.ml-1, a 50-fold decrease in Km for casein kinase I and a 6-fold increase in Vmax as compared to the non-phosphorylated peptide. This indicates that Ser120, which has been shown to be phosphorylated in vivo, provides an appropriate recognition determinant for casein kinase I.     

Internalization and cycling of the T cell antigen receptor. Role of protein kinase C

The dynamics of the T cell antigen receptor on a murine antigen specific T cell hybridoma have been analyzed using a monoclonal anti-receptor antibody. When this antibody, A2B4-2, is bound to surface receptors, no internalization is seen at 4 degrees C. Upon warming to 37 degrees C, between 20 and 30% of the antibody molecules are internalized over 20-30 min as measured by sensitivity to external acid. This level of internalization is identical if monovalent Fab fragments are used. In contrast, cross-linking of the anti-receptor antibody with a second antibody leads to rapid internalization of 100% of prebound surface A2B4-2. Phorbol 12-myristate 13-acetate (PMA) leads to the rapid internalization of up to 65% of the surface A2B4-2 or A2B4-2 Fab fragments. This effect requires protein kinase C and can be completely inhibited by depleting this kinase from the cells by long term treatment with high doses of PMA. Pretreatment of the T cells with PMA leads to a 40-50% drop in surface T cell antigen receptor expression. Despite the loss of surface receptors, the uptake of A2B4-2 in PMA-treated cells at 37 degrees C is identical to that seen in control cells. The total uptake of A2B4-2 at 37 degrees C is 25-30% greater than the number of surface receptors in control cells and about 100-150% greater than the number of surface receptors in PMA-treated cells. At steady state the percentage of total A2B4-2 on the cell surface is 75% for control cells and 38% for PMA-treated cells. The good agreement of these numbers with the percent internalization of a cohort of surface receptors suggests that all receptors are constantly cycling. The effect of PMA is to alter the kinetic parameters of this cycling, thus changing the steady state distribution of receptors between the plasma membrane and internal, presumably endosomal compartments. Measurement of initial rates of internalization suggests that the PMA effect can be largely explained by an increase in the internalization rate constant.     

Inhibition of protein kinase activity and amino acid and alpha-methyl-D-glucoside transport by diamide.

Dizene dicarboxylic acid bis-(N,N-dimethylamide), commonly called diamide, is known to oxidize stoichiometrically intracellular pools of reduced glutathione and inhibit the accumulation of sugars and amino acids by rat kidney slices. Incubation of rat cortical slices in diamide also leads to a significant decrease in the level of endogenous protein kinase activity. The inhibition of sugar and amino acid transport and protein kinase activity by diamide is partially reversible by the addition of exogenous glutathione or other thiols. A comparison of protein kinase activity with amino acid and sugar transport at various concentrations of diamide indicates that there is a high degree of correlation between these two processes.     

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.     

Mutants of Fujinami sarcoma virus which are temperature sensitive for cellular transformation and protein kinase activity.

Two temperature-sensitive mutants of Fujinami sarcoma virus were isolated and characterized. Cells infected with the mutants were temperature sensitive in focus formation, colony formation, increased sugar uptake, and synthesis of plasminogen activator. The changes between transformed and nontransformed states of cultures were completely reversible by shifting the temperature. A Fujinami sarcoma virus-specific protein of 130,000 daltons, p130, was synthesized in mutant-infected cells regardless of the temperature, but the immunoprecipitates of p130 from extracts of infected cells were active in protein kinase only when cells had been incubated at the permissive temperature. These results appear to indicate that p130 is the transforming protein of Fujinami sarcoma virus, and that its protein kinase activity plays a crucial role in cell transformation by this virus.