Phosphorylation of polyomavirus large T antigen: effects of viral mutations and cell growth state.

Phosphorylation is responsible for the shift in electrophoretic mobility of polyomavirus large T antigen observed in pulse-chase or continuous-labeling experiments. Phosphorylated forms migrated more slowly than newly synthesized [35S]methionine large T antigen, and alkaline phosphatase treatment reversed the mobility shift. Analysis of phosphopeptides with Staphylococcus aureus V8 protease showed that large T antigen forms of intermediate mobility were enriched in peptides 1 to 4, 8, and 9, while the slower migrating species had all nine phosphopeptides, including peptides 5 and 7. The phosphorylations represented by phosphopeptides 5 and 7 were of particular interest. These phosphopeptides were entirely lacking in large T antigen from tsa mutants such as ts616 labeled at the nonpermissive temperature. Also, the phosphorylation of peptides 5 and 7 depends on the growth state of the cell. Early in infection of quiescent cells intermediate mobility forms of large T antigen with little or no phosphorylation, particularly of peptides 5 and 7, were seen, whereas peptides 5 and 7 were well represented at the same time in patterns from growing cells. Later in infection of growth-arrested cells, these phosphorylations were observed, suggesting that infection stimulates the relevant kinase. Because large T antigen of hrt mutants, which lack middle and small T antigens, showed phosphorylation of peptides 5 and 7, large T antigen was apparently responsible for the stimulation. Because some differences in the distribution of phosphopeptides were noted between hrt mutants and the wild type, middle T antigen, small T antigen, or both may play a modulating role in large T antigen phosphorylation.     

Altered phosphorylation pattern of simian virus 40 T antigen expressed in insect cells by using a baculovirus vector.

The phosphorylation pattern of simian virus 40 (SV40) large tumor (T) antigen purified from insect cells infected with a recombinant baculovirus was compared with that reported previously for T antigen from SV40-infected monkey cells. The specific activity of metabolic phosphate labeling of baculovirus T antigen was reduced, and the phosphopeptide map of the baculovirus protein, while qualitatively similar to that of lytic T, revealed several quantitative differences. The most striking difference was the prominence in the baculovirus map of peptides containing phosphothreonine 124. These peptides are known to arise from other phosphopeptides upon dephosphorylation of neighboring serines, suggesting that baculovirus T may be underphosphorylated at these serines and perhaps other sites. Functional assays used to further investigate the phosphorylation state of the baculovirus protein included SV40 DNA binding after enzymatic dephosphorylation with alkaline phosphatase and after phosphorylation by a murine homolog of cdc2 protein kinase. The results imply that baculovirus T antigen is underphosphorylated, in particular at those serine residues whose phosphorylation is responsible for down regulation of DNA-binding activity at site II in the core origin of DNA replication. In contrast, no evidence for a functionally significant underphosphorylation at threonine 124 could be found.     

Simian Virus 40 Large T Antigen Is Phosphorylated at Multiple Sites Clustered in Two Separate Regions

The phosphorylation sites of simian virus 40 large T antigen were determined within the primary structure of the molecule. Exhaustive digestion of ³²P-labeled large T antigen with trypsin generated six major phosphopeptides which could be separated in a newly developed isobutyric acid-containing chromatography system. By partial tryptic digestion, large T antigen was cleaved into an amino-terminal fragment of 17,000 daltons and overlapping fragments from the carboxy-terminal region ranging in size between 71,000 and 13,000 daltons. The location of the phosphopeptides was then determined by fingerprint analyses of individual fragments. Their physical properties were analyzed by sizing on polyacrylamide gels and by sequential digestion and peptide mapping; their amino acid composition was determined by differential labeling with various amino acids. The amino-terminal 17,000-dalton fragment gave rise to only one phosphopeptide (phosphopeptide 3) that contained half of the phosphate label incorporated into large T antigen. It contained phosphoserine and phosphothreonine sites, all of which were clustered within a small segment between Cys₁₀₅ and Lys₁₂₇. This segment contained five serines and two threonines. Among these, Ser₁₀₆, Ser₁₂₃, and Thr₁₂₄ were identified as phosphorylated residues; in addition, either one or both of Ser₁₁₁ and Ser₁₁₂ were phosphorylated. The neighboring residues, Ser₁₂₃ and Thr₁₂₄, were found in three different phosphorylation states in that either Ser₁₂₃ or Thr₁₂₄ or both were phosphorylated. Phosphopeptides 1, 2, 4, 5, and 6 were all derived from a single fragment extending 26,000 daltons upstream from the carboxy terminus of large T antigen. Phosphopeptide 6 was identical with the previously determined phosphothreonine peptide phosphorylated at Thr₇₀₁. Phosphopeptides 1, 2, 4, and 5 contained only serine-bound phosphate. Phosphopeptides 1, 2, and 4 represented overlapping peptides, all of which were phosphorylated at Ser₆₃₉ located next to a cluster of six acidic residues. In phosphopeptide 5, a large peptide ranging from Asn₆₅₃ to Arg₆₉₁, at least two of seven serines were phosphorylated. Thus, large T antigen contains at least eight phosphorylation sites. Their clustering within two separate regions might correlate with structural and functional domains of this protein.     

Phosphorylation of rabbit skeletal muscle glycogen synthase by casein kinase 1: Evidence of phosphorylation sites specific for casein kinase 1

Casein kinase 1 phosphorylated rabbit skeletal muscle glycogen synthase at both seryl and threonyl residues. With glycogen synthase phosphorylated up to 7.5 mol phosphate/mol subunit, about 26% of the phosphate was present in the N-terminal cyanogen bromide fragment (CB1) and 74% in the C-terminal fragment (CB2). Both fragments contained phosphothreonine (11 to 14%) in addition to phosphoserine. When ³²P-labeled glycogen synthase was totally digested with trypsin and chromatographed on reversephase high-performance liquid chromatography, seven phosphopeptides were observed. Peptide I eluted in the vicinity of the peptide containing site 1a, peptide II coincided with sites 4 + 5, peptides III and IV eluted in the region corresponding to sites 3a + 3b + 3c, peptide V appeared slightly after the peptide containing site 1b and peptide VII behaved as the peptide containing site 2, whereas peptide VI did not coincide with any of the known phosphopeptides. Limited trypsinization prior to analysis by HPLC led to the disappearance of peaks V and VI without altering peaks I to IV and VII. Only peaks I and VII remained when limited chymotrypsinization was performed prior to HPLC analysis. Chromatography on HPLC of the fragments derived from complete trypsinization of CB2 showed the presence of peaks II to VI. Phosphoamino acid analysis of the different peptides demonstrated the presence of quantitative amounts of phosphothreonine in peptides V, VI, and VII. These results indicate that multiple phosphorylation sites for casein kinase 1 must exist in both the N-terminal and C-terminal regions of glycogen synthase, some of which would only be labeled by casein kinase 1.     

Phosphorylation pattern of large T antigens in mouse cells infected by simian virus 40 wild type or deletion mutants

The phosphorylation sites of simian virus 40 (SV40) large tumor (T) antigens have been extensively studied in productive infection of monkey cells. In this study, we analyzed the phosphorylation sites of large T antigen from SV40-infected nonpermissive mouse cells by partial proteolysis fingerprints and analysis of the phosphoamino acids present in the resulting fragments. The wild-type virus and deletion mutants (dl1263, dl1265, dl2194, and dl2198) were used for infection. On the basis of our results and published data (M. Schwyzer, R. Weil, and H. Zuber, J. Biol. Chem. 225:5627-5634, 1980), a cleavage map of large T antigen was established. It was reported that at least four sites of phosphorylation were present. The amino-terminal part of the molecule contained both phosphoserine and phosphothreonine. One phosphothreonine residue was located in the prolinerich C-terminal end of the molecule at position 701 or 708. On the basis of the concensus as to the amino acid sequence surrounding the recognition sites for protein kinases, it was possible to more precisely locate this phosphothreonine at residue 701. Moreover, the C-terminal part of the molecule contained phosphoserine at a more internal position. In addition, this study firmly established the presence of a phosphothreonine in the N-terminal part of large T antigen. In conclusion, it was shown that the location of phosphorylation sites of large T antigen produced by nonpermissive mouse cells infected by SV40 is strikingly similar to that reported by other groups for large T antigen produced by SV40-infected permissive cells.     

The T cell response to the glycoprotein D of the herpes simplex virus: the significance of antigen conformation

Synthetic peptides corresponding to the first 23 amino acids of the glycoprotein D molecule of herpes simplex virus have been used to immunize mice and examine the role of antigen conformation in T cell responses. The structure of the peptides was determined by circular dichroism studies and was shown to be consistent with theoretical structure predictions. T cell clones were found that could respond to peptides that were nonhelical, and this response, which was directed to the C-terminal determinant (residues 8-23), could be modified by a constrained N-terminal sequence (residues 1-7) of the peptide. Namely, substitution of Ala for Pro at position 7 induced an alpha-helix and inhibited the response. Furthermore, a response to the N-terminal part of the molecule seemed to be directed to the alpha-helix and correlated positively with calculated degree of helicity. This response was also modified, in this case, by the C-terminal part of the molecule. These results suggest that local secondary structure of an antigen may regulate T cell responses and that structural changes in the peptide antigen downstream from the determinant modify recognition of that determinant. Furthermore, antigen processing by the macrophage and antigen presentation of T cells appears to conserve antigen structural integrity.     

Myc oncoproteins are phosphorylated by casein kinase II.

Casein kinase II (CK-II) is a ubiquitous protein kinase, localized to both nucleus and cytoplasm, with strong specificity for serine residues positioned within clusters of acidic amino acids. We have found that a number of nuclear oncoproteins share a CK-II phosphorylation sequence motif, including Myc, Myb, Fos, E1a and SV40 T antigen. In this paper we show that cellular myc-encoded proteins, derived from avian and human cells, can serve as substrates for phosphorylation by purified CK-II in vitro and that this phosphorylation is reversible. One- and two-dimensional mapping experiments demonstrate that the major phosphopeptides from in vivo phosphorylated Myc correspond to the phosphopeptides produced from Myc phosphorylated in vitro by CK-II. In addition, synthetic peptides with sequences corresponding to putative CK-II phosphorylation sites in Myc are subject to multiple, highly efficient phosphorylations by CK-II, and can act as competitive inhibitors of CK-II phosphorylation of Myc in vitro. We have used such peptides to map the phosphorylated regions in Myc and have located major CK-II phosphorylations within the central highly acidic domain and within a region proximal to the C terminus. Our results, along with previous studies on myc deletion mutants, show that Myc is phosphorylated by CK-II, or a kinase with similar specificity, in regions of functional importance. Since CK-II can be rapidly activated after mitogen treatment we postulate that CK-II mediated phosphorylation of Myc plays a role in signal transduction to the nucleus.     

Mapping of phosphorylation sites in polyomavirus large T antigen.

The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, 32Pi-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino-terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed.     

Altered phosphorylation of free and bound forms of monkey p53 and simian virus 40 large T antigen during lytic infection.

We have identified the phosphorylation sites in monkey p53 as well as specific changes in the phosphorylation state of free and complexed forms of simian virus 40 (SV40) large T antigen (T) and monkey p53 isolate from SV40 lytically infected CV1 cells. Phosphopeptide analyses of free T and p53 (To and p53o) and complexed T and p53 (T+ and p53+) fractions indicated several quantitative increases in the specific phosphorylation of complexed forms of both proteins. The N terminus of monkey p53+ is phosphorylated at Ser-9, Ser-15, Ser-20, either Ser-33 or Ser-37, and at least one of Ser-90 to Ser-99. The C-terminal sites are Ser-315 and Ser-392. On comparing p53+ with p53o, we found that labeling of the two N-terminal phosphotryptic peptides encompassing residues 1 to 20 and 33 to 101 was increased fivefold and that Ser-315 was sevenfold more labeled than was Ser-392. When T+ was compared with To, the N-terminal peptide containing phosphorylation sites Ser-106 through Thr-124 was twofold more labeled, the peptide containing Ser-657 through Ser-679 was sixfold more labeled and contained up to four phosphorylated serine residues, and Ser-639 and Thr-701 appeared unchanged. Overall, T+ molecules appeared to contain 3.5 mol more of labeled phosphate than did To, with the N-terminal peptide appearing fully phosphorylated. The phosphopeptide patterns obtained for lytic T+ and To fractions were nearly identical to those found for wild-type SV40 T (stably complexed with mouse p53) and mutant 5080 T (defective for p53 binding) expressed in transformed C3H10T1/2 cells (L. Tack, C. Cartwright, J. Wright, A. Srinivasan, W. Eckhart, K. Peden, and J. Pipas, J. Virol. 63:3362-3367, 1989). These results indicate that increases in specific phosphorylation sites in both T+ and p53+ correlate with the association of T with p53. The enhanced phosphorylation state may be a consequence of complex formation between T and p53 or reflect an increased affinity of p53 for highly phosphorylated forms of T.     

Monoclonal antibody analysis of simian virus 40 small t-antigen expression in infected and transformed cells.

The monoclonal antibody PAb280 binds to small t antigen but not to large T antigen. Its binding site within the unique region of small t antigen was localized by studying its reaction with simian virus 40 mutants, other papovaviruses, and bacterial expression vectors coding for fragments of small t antigen. The antibody was used to define the cellular location of small t antigen by immunocytochemistry and by immunoprecipitation of subcellular extracts of infected cells. PAb280 reacts strongly with a cytoplasmic form of small t antigen that appears to be associated with the cytoskeleton and is not detected by antibodies directed to the common N terminus of small t and large T antigens. Immunoperoxidase staining of cells infected by the simian virus 40 defective strain SV402 with PAb280 and other anti-T antibodies demonstrated that this virus produced an N-terminal fragment of large T antigen as well as small t antigen. In cells infected by the virus, this fragment was located in the cell nucleus but was very unstable. These results suggest that the activity of the SV402 virus in transformation assays may not be entirely due to the action of small t antigen alone.     

Sites of in vivo phosphorylation of histone H5

Previous studies have suggested that the phosphorylation and dephosphorylation of histone H5 play an important role in controlling the condensation of avian erythrocyte chromatin. The present work locates in the polypeptide chain the major sites at which H5 is phosphorylated in vivo. The majority of the radioactivity in 32P-labeled H5 is clustered in two regions of the molecule. Nearly 50% of the 32P is found in the amino-terminal N-bromosuccinimide (NBS) peptide (residues 1-28); the remainder is confined to three phosphopeptides arising from the C-terminal half of the molecule (residues 100-200). All phosphopeptides are found in a tryptic digest of monophosphorylated H5, indicating the phosphorylation of a given site is a random event. Automatic Edman degradation of the amino-terminal fragment shows that the radioactivity is equally divided between serines at positions 3 and 7. The C-terminal phosphorylated tryptic peptides share some features with the C-terminal phosphorylation sites in H1. If, as has been postulated, the sites of phosphorylation are in or near DNA combining regions, then H5 may have two DNA combining sites. The location of the phosphorylation sites is discussed in relation to a possible mechanism for controlling chromatin condensation.     

Phosphorylation sites in polyomavirus large T antigen that regulate its function in viral, but not cellular, DNA synthesis.

Polyomavirus large T antigen (large T) is a highly phosphorylated protein that can be separated by proteolysis into two domains that have independent function. A cluster of phosphorylation sites was found in the protease-sensitive region connecting the N-terminal and C-terminal domains. Edman degradation of 32P-labeled protein identified serines 267, 271, and 274 and threonine 278 as sites of phosphorylation. Analysis of site-directed mutants confirmed directly that residues 271, 274, and 278 were phosphorylated. Threonine 278, shown here to be phosphorylated by cyclin/cyclin-dependent kinase activity, is required for viral DNA replication in either the full-length large T or C-terminal domain context. The serine phosphorylations are unimportant in the C-terminal domain context even though their mutations activates viral DNA replication in full-length large T. This finding suggests that these sites may function in relating the two domains to each other. Although the phosphorylation sites were involved in viral DNA replication, none was important for the ability of large T to drive cellular DNA replication as measured by bromodeoxyuridine incorporation, and they did not affect large T interactions with the Rb tumor suppressor family.     

Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding.

The effect of phosphorylation on the ability of simian virus 40 large T antigen to stimulate DNA synthesis in vitro was tested. Treatment of affinity-purified large T antigen with calf intestinal alkaline phosphatase resulted in the removal of 70 to 80% of the phosphate residues. Only serine-bound phosphate residues were affected. Phosphatase-treated large T antigen stimulated in vitro DNA synthesis fourfold over the untreated control. The stimulation was strongest at early times of DNA replication. At later times, DNA replication proceeded at equal rates with dephosphorylated and untreated large T antigen. The ATPase activity of large T antigen was not affected by phosphatase treatment. The origin-binding activity of large T antigen was tested over a wide range of large T antigen to DNA ratios, including DNA excess, and in the presence and absence of carrier DNA. Under no condition was an effect of dephosphorylation of large T antigen on its DNA-binding activity observed. These findings might indicate that phosphorylation at serine residues modulates the interaction of large T antigen with cellular factors. During DNA synthesis large T antigen was substantially rephosphorylated by kinases in the HeLa cell extract. As shown by two-dimensional peptide mapping, this phosphorylation occurred at all known in vivo sites. No phosphatase and protease activities were detectable in the HeLa cell extract.     

DNA-binding activity of simian virus 40 large T antigen correlates with a distinct phosphorylation state

The state of phosphorylation and the relationship of various subclasses of simian virus 40 large T antigen (large T) differing in DNA-binding activity, degree of oligomerization, age, and subcellular distribution were investigated. Young large T (continuously labeled for 4 h late in infection) comprised about 20% of the total cellular large T. It was phosphorylated to a low degree and existed primarily in a monomeric form, sedimenting at 5S. More than 50% of this fraction bound to simian virus 40 DNA, preferentially to origin-containing sequences. Old large T (continuously labeled for 17 h, followed by a 4-h chase) represented the majority of the population. It was highly phosphorylated and predominantly in an oligomeric form, sedimenting at 15S to 23S. Only 10 to 20% of this fraction bound to simian virus 40 DNA. Another subclass of large T which was extracted from nuclei with 0.5 M salt resembled newly synthesized molecules in all properties tested; it was phosphorylated to a low degree, sedimented at 5S, and bound to viral DNA with high efficiency (greater than 70%). Two-dimensional phosphopeptide analysis of the individual subclasses revealed two distinct phosphorylation patterns, one characteristic for young, monomeric, and DNA-binding large T, the other for old, oligomeric, and non-DNA-binding large T. All sites previously identified in unfractionated large T (K.H. Scheidtmann et al., J. Virol. 44:116-133, 1982) were also phosphorylated in the various subclasses, but to different degrees. Peptide maps of the DNA-binding fraction, the 5S form, and the nuclear high-salt fraction showed two prominent phosphopeptides not previously characterized. Both peptides were derived from the amino-terminal region of large T, presumably involved in origin binding, and probably represent partially phosphorylated intermediates of known phosphopeptides. Our data show that the DNA-binding activity, age, and oligomerization of large T correlate with distinct states of phosphorylation. We propose that differential phosphorylation might play a role in the interaction of large T with DNA.     

Effects of in vitro dephosphorylation on DNA-binding and DNA helicase activities of simian virus 40 large tumor antigen.

Simian virus 40 large T antigen is a phosphoprotein with two clusters of phosphorylation sites. Each cluster includes four serine residues and one threonine residue. In vitro treatment with intestinal alkaline phosphatase removes the phosphate groups from the serine but not from the threonine residues. Potato acid phosphatase additionally dephosphorylates the phosphothreonine (Thr-124) in the N-terminal cluster but does not attack the phosphothreonine in the C-terminal cluster (Thr-701). Two biochemical functions of untreated and partially dephosphorylated T antigen were assayed, namely, its specific DNA-binding property and its DNA helicase activity. After treatment with alkaline phosphatase, T antigen had a severalfold higher affinity for the specific binding sites in the viral genomic control region, in particular, for binding site II in the origin of replication. However, T antigen, when dephosphorylated by acid phosphatase, had DNA-binding properties similar to those of the untreated control. Neither alkaline nor acid dephosphorylation affected the DNA helicase activity of T antigen.     

Simian virus 40 tumor antigen: isolation of the origin-specific DNA-binding domain

To localize the origin-specific DNA-binding domain on the simian virus 40 tumor (T) antigen molecule, we used limited proteolysis with trypsin to generate fractional peptides for analysis. A 17,000-Mr peptide was found to be capable of binding not only to calf thymus DNA, but also specifically to the simian virus 40 origin of DNA replication. This approximately 130-amino-acid peptide was derived from the extreme N-terminus of the T antigen and represented less than one-fifth of the entire molecule. The coding sequence for this tryptic peptide was located approximately between 0.51 and 0.67 map units (excluding the intron, which maps between 0.54 and 0.59). Since the first 82 amino acids are shared between large T and small t antigens, and since the latter does not bind DNA, it can be concluded that the sequence between isoleucine 83 and approximately arginine 130 is necessary for origin-specific binding by the T antigen. We also observed that in vivo phosphorylation of the T antigen within this region completely abolished the ability of the 17,000-Mr peptide to bind DNA. This observation is consistent with the idea that DNA binding by the T antigen is regulated by posttranslational modifications.     

Phosphorylation patterns of tumour antigens in cells lytically infected or transformed by simian virus 40.

The phosphorylation sites of simian virus 40 (SV40) large tumor (T) antigens have been analyzed by partial proteolysis peptide mapping and phosphoamino acid analysis of the resulting products. At least four sites were found to be phosphorylated. An amino-terminal part of the molecule contained both phosphoserine and phosphothreonine. One phosphothreonine residue was located in the proline-rich carboxy-terminal end of the molecule, either at position 701 or at position 708. The mutant dl 1265, which is defective in adenovirus helper function, lacked this phosphorylation site. In addition, the carboxy-terminal part of the molecule contained phosphoserine at a more central position. T-antigen-associated proteins of SV40-transformed cell (nonviral T; 51,000 to 55,000 daltons) also contained multiple phosphorylation sites involving at least two serine residues in mouse antigens and an additional threonine residue in rat, human, and monkey antigens. The latter residue and at least one phosphoserine residue were located near one terminus of the human NVT molecule. We did not find any evidence for phosphorylation of tyrosine residues in any of the multiple species of either large T or nonviral T molecules. Several forms of large T antigens were extracted from both SV40-transformed and SV40-infected permissive and nonpermissive cells, and their phosphorylation patterns were compared. No evidence was found for a different phosphorylation pattern of T antigen in transformed cells.     

Localization and characterization of phosphorylation sites of the Fujinami avian sarcoma virus and PRCII virus transforming proteins

Fujinami sarcoma virus (FSV) and PRCII are avian sarcoma viruses which share cellularly derived v-fps transforming sequences. The FSV P140gag-fps gene product is phosphorylated on three distinct tyrosine residues in transformed cells or in an in vitro kinase reaction. Three variants of FSV, and the related virus PRCII which lacks about half of the v-fps sequence found in FSV, encode gene products which are all phosphorylated at tyrosine residues contained within identical tryptic peptides. This indicates a stringent conservation of amino acid sequence at the tyrosine phosphorylation sites which presumably reflects the importance of these sites for the biologic activity of the transforming proteins. Under suitable conditions the proteolytic enzymes p15 and V8 protease each introduce one cut into FSV P140, p15 in the N-terminal gag-encoded region and V8 protease in the middle of the fps-encoded region. Using these enzymes we have mapped the major site of tyrosine phosphorylation to the C-terminal end of the fps region of FSV P140gag-fps. A second tyrosine phosphorylation site is found in the fps region of FSV P140 isolated from transformed cells, and a minor tyrosine phosphorylation site is found in the N-terminal gag-encoded region. Our results suggest that the C-terminal fps-encoded region is required for expression of the tyrosine-specific protein kinase activity.     

Rapid and sensitive quantitative immunoassay for the large simian virus 40 T antigen

A quantitative, enzyme-linked immunoadsorbent assay has been developed for the simian virus 40 large T antigen. When hamster anti-simian virus 40 tumor serum was used, this method permitted specific identification of large T antigen and its analog, the D2 hybrid protein, a molecule with the same C-terminal approximately 600 amino acids as large T antigen. The sensitivity limit of this test was 0.63 ng of protein. The slopes of the regression lines of the enzyme-linked immunosorbent assay titrations performed with highly purified D2 or simian virus 40 large T antigen and with crude extracts of simian virus 40-infected monkey and transformed human cells were identical. Thus, the curve generated with a purified protein, such as D2, can serve as a quantitative standard for the measurement of large T antigen in a wide variety of extracts. Furthermore, solutions containing high salt concentrations and buffers containing up to 0.1% Nonidet P-40 did not interfere with the assay, making it applicable to the measurement of large T antigen in a variety of chromatographic fractions. The enzyme-linked immunosorbent assay was three times more sensitive, was significantly faster to perform, and was quantitatively valid over a much broader large-T-antigen concentration range than the complement fixation test. As such, it should be useful in future studies of the structure and function of this protein.