alternatively spliced third SV40 early mRNA - NCBI

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Aug 16, 1993 - encoded by a different reading frame. 17kT mRNA ..... encoded in a different reading frame, followed by a stop codon. ...... Biol., 11, 2116-2124.
The EMBO Journal vol.12 no.12 pp.4739-4746, 1993

Independent expression of the transforming aminoterminal domain of SV40 large T antigen from an alternatively spliced third SV40 early mRNA Jens Zerrahn, Uwe Knippschild, Thomas Winkler and Wolfgang Deppert1 Heinrich-Pette-Institut ftir Experimentelle Virologie und Immunologie an der Universitiit Hamburg, Martinistrasse 52, D-20251 Hamburg, Germany 'Corresponding author Communicated by M.Osborn

We found that simian virus 40 (SV40), in addition to the SV40 early proteins large T antigen (large T) and small t antigen (small t), codes for a third early protein with a molecular weight of 17 kDa. This protein (17kT) is expressed from an alternatively spliced third SV40 early mRNA, using a splice donor site at position 4425 and a splice acceptor site at position 3679 of the SV40 genome. The 17kT protein consists of 135 amino acids. Of these, 131 correspond to the amino-terminus of large T, while the four carboxy-terminal amino acids are unique and encoded by a different reading frame. 17kT mRNA, and the corresponding protein, were found in all SV40 transformed cells analyzed, as weli as in SV40 infected cells. Transfection of a cDNA expression vector encoding the 17kT protein into rat Flll fibroblasts induced phenotypic transformation of these cells. The expression of the transforming amino-terminal domain of large T as an independent 17kT protein might provide a means for individually regulating the various functions associated with this domain. Key words: alternative splicing/differential phosphorylation/ SV40 large T antigen/transforming domains

these processes are tightly controlled. Previous evidence from our laboratory indicated that as yet unknown cellular factors are involved in such control mechanisms, e.g. by mediating the functional interaction of large T with different structural systems of the cell (Staufenbiel and Deppert, 1983; Hinzpeter and Deppert, 1987) and by controlling large T phosphorylation (Deppert et al., 1991; Knippschild et al., 1991). Characteristic features of the phosphorylation-mediated control of large T functions are that the phosphorylation status of large T regarding its function in lytic infection is controlled by the concerted action of various kinases and phosphatases (reviewed in Prives, 1990) and that a coordinated phosphorylation of amino-terminal as well as of carboxy-terminal phosphorylation sites seems to be required to establish and maintain a biologically active conformation of large T (Schneider and Fanning, 1988; Hoss et al., 1990; Scheidtmann et al., 1991). During our studies on large T phosphorylation we found that all SV40 transformed cells analyzed, in addition to large T, expressed another SV40 specific phosphoprotein of 17 kDa. We demonstrate that this 17 kDa protein is authentically encoded by SV40, using an alternatively spliced third SV40 early mRNA. The 17 kDa protein corresponds to the aminoterminal transforming domain of large T, and seems to be preferentially expressed in SV40 transformed cells, although it is also found in SV40 lytically infected cells. The independent expression of the amino-terminal transforming domain of large T as a 17 kDa protein might provide a means for regulating the various activities ascribed to this domain, independent from the regulatory influence of the large T carboxy-terminus on amino-terminal functions.

Results Introduction Lytic viral infections and cellular transformation by simian virus 40 (SV40) are the result of complex interactions of the SV40 large T antigen (large T) with different cellular targets. Large T can influence viral and cellular gene expression by binding to (reviewed in Salzman, 1987) or by transactivating (Zhu et al., 1991 and references cited therein) viral and cellular DNA sequences. In addition, large T directly modulates the activities of cellular regulatory proteins by binding to them, e.g. by binding to DNA polymerase a (Gannon and Lane, 1987) or to the transcription factor AP2 (Mitchell et al., 1987). Furthermore, tight complex formation of large T with the putative tumor suppressor proteins pRb and p53 seems to be required for the transforming functions of large T (Levine, 1990; Hamel et al., 1992). Large T thus is a multifunctional protein, consisting of several functional domains which have been delineated by extensive mutational analyses (reviewed in Pipas, 1992). Considering the complexity of large T functions both in viral replication and cellular transformation, one has to assume that the various activities of large T in C Oxford University Press

Identification of a third SV40 early protein in SV40 transformed cells While analyzing a variety of 32P,-labeled SV40 transformed cells by immunoprecipitation of cellular extracts with the monoclonal antibody PAb419 specific for an amino-terminal epitope on large T (Harlow et al., 1981), we found that these cells, in addition to the SV40 large T antigen (large T), expressed another SV40 specific phosphoprotein of 17 kDa (Figure lA, lanes 1-4). Further analysis of [35S]methionine-labeled cells demonstrated that this 17 kDa protein is expressed in addition to the SV40 small t antigen (small t) and migrates close in front of it (Figure 1B, lane 1). Comparative analyses of tryptic peptides of 32Pi-labeled large T and 17 kDa proteins revealed that the 17 kDa protein contained all amino-temTinal phosphorylation sites of large T (Figure 2A and B). Thus this protein largely corresponds to the amino-terminus of large T, and therefore was termed -

M7kT. The SV40 17kT protein is encoded by an alternatively spliced SV40 early RNA

Generation of an amino-terminal fragment of large T in SV40 transformed cells could be due to proteolytic degradation 4739

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Fig. 1. Expression of SV40 17kT in various SV40 transformed cell lines and in SV40 17kT transformed Fl rat fibroblasts. (A) Immunoprecipitation of l7kT from FR(wt648) cells (ane 1), SVMEFcl.3 cells (lane 2), SV52 cells (lane 3) and mKSA cells (lane 4), metabolically labeled with 32p;. (B) Imnmunoprecipitation of l7kT from mKSA cells (lane 1) and control normal Fl rat fibroblast cells (lane 2) metabolically labeled with [35S]methionine. (C) Expression of SV40 17kT protein in Fl rat fibroblast cells. Fl cells were cotransfected with pUC18SV4OT4, coding for the 17kT protein, and phyg, mediating resistance to hygromycin B. After selection, colonies were picked, expanded and analyzed. Immunoprecipitation from control Fl cells (lane 1), of large T from F5 cells (lane 2), of an amino-terminal fragment of large T, comprising amino acids 1-147, from FR3 cells (lane 3), and of 17kT protein from a representative F 111 cell derived clone, FT4.5 cells (lane 4), metabolically labeled with [35S]methionine.

Fig. 3. Northern blot analysis of SV40 early mRNAs in various SV40 transformed and SV40 lytically infected cells. Total RNA was isolated from SV52 cells (lane 1), mKSA cells (lane 2), FR(wt648) cells (lane 3), COS cells (lane 4) and SV40 lytically infected TC7 cells at 48 h.p.i. (lane 5). Northern blotting and hybridization were as described in Materials and methods. Positions of small t and large T specific mRNAs are marked. High molecular weight SV40 specific RNAs in mKSA cells are derived from rearranged SV40 DNA (unpublished).

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Fig. 2. Comparison of the phosphorylation of the amino-termiinus of large T and of l7kT. FR(wt648) cells were labeled with 32p; for 4 h. Phosphopeptide analysis of large T (A) and 17kT (B) from FR(wt648) cells was as described in Materials and methods. The anode is at the left, and the origin is marked with a solid square at the bottom of each panel. Pi indicates the position of free radioactive phosphate. Numbering of the phosphopeptides was according to the nomenclature of Knippschild et al. (1991). 4740

of large T, or to the expression of rearranged copies of integrated SV40 DNA. Examples of the latter possibility have been described, but in all cases led to expression of amino-terminal large T fragments with differing molecular weights (McCormick et al., 1980; Spangler et al., 1980; Chaudry et al., 1982). In contrast, the 17kT protein under investigation had the same molecular weight in all cells analyzed (see Figure IA). Furthermore, as proteolytic degradation of large T under our experimental conditions (see Materials and methods) was extremely unlikely, and as no other intermediate proteolytic degradation products of large T could be detected, we considered the possibility that the 17kT protein might be authentically encoded by SV40. It has been reported that SV40 early RNA, in addition to the prominent large T and small t splices, contains another potential splice donor site at position 4425 on the SV40 genome, which was detected during analysis of the expression of truncated SV40 early genes (Sompayrac and Danna, 1985). We therefore considered the possibility that use of this alternative splice-site might have generated an mRNA encoding the 17kT protein. However, we were unable to detect a third SV40 early mRNA with certainty by Northern blot analyses of total RNA from SV40 transformed and lytically infected cells (Figure 3, lanes 1-5). However, this does not exclude the presence of such an RNA in low copy numbers, as small t was expressed in all SV40 transformed cells analyzed in Figure 3 (data not shown), but small t specific mRNA also could not be detected in these cells by Northern blot analysis. In contrast, small t specific mRNA was readily detected in SV40 lytically infected cells, and in these cells was at least as prominent as the large T specific mRNA (Figure 3, lane 5). Therefore, we tried to demonstrate this postulated RNA by the more sensitive method of PCR amplification of cDNA from SV40

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Fig. 4. PCR strategy for detection of the alternatively spliced mRNA encoding the SV40 l7kT protein and schematic overview of the SV40 early region and the alternatively spliced SV40 early mRNAs coding for large T, small t and l7kT. Positions of PCR primers used are indicated (upstream primers: open symbols; downstream primers: closed symbols). LE and EE indicate positions of late-early and early-early transcriptional initiation sites, respectively. Numbering refers to the SV40 genomic nucleotide numbers.

transformed cells. Knowing that the l7kT protein contained all amino-terminal phosphorylation sites of large T, we postulated that the l7kT protein must be colinear with large T at least up to Thrl24. Assuming a splice donor site at position 4425 for the l7kT mRNA, our strategy was to employ PCR primers which would generate amplification products for the large T specific exon 2, and to find an amplification product, which due to the postulated alternative RNA splice should differ in size from the authentic large T specific amplification product. Such an amplification product then could be further analyzed by sequencing. The primers T3E and T3B used for this strategy, and their location on SV40 early region DNA, are shown in Figure 4. cDNA from total RNA of FR(wt648) cells (1 ,ug) was prepared by reverse transcription. PCR amplification of this cDNA indeed generated two large T specific amplification products, one with the postulated size of the authentic large T amplification product (1875 bp), and another one of 1129 bp (data not shown). This amplification product was cloned into vector pUC18 and subjected to sequence analysis. The result of this sequence analysis is shown in Figure 5A and B and reveals that FR(wt648) cells harbored an SV40 specific RNA which had been generated by using the alternative splice donor site at position 4425 and a splice acceptor site at position 3679. The sequences around these postulated splice donor and splice acceptor sites fit well with the known sequence requirements for splice sites (Mount, 1982; see Figure SA). The protein encoded by such an alternatively spliced transcript is colinear with SV40 large T up to Lysl3 1, followed by four unique amino acids (Ala-Leu-Leu-Thr) encoded in a different reading frame, followed by a stop codon. It has a theoretical molecular weight of 15.5 kDa, -

which corresponds well to the molecular weight of the 17kT protein as determined by SDS-PAGE. To verify that generation of the l7kT protein by such an alternatively spliced SV40 early mRNA is a general phenomenon rather than the result of peculiar splicing properties of FR(wt 648) cells, we analyzed a variety of SV40 transformed cells, as well as SV40 lytically infected TC7 monkey cells (see Figure 6) for the presence of this transcript. To detect all early SV40 mRNAs in these cells, we used primers EN5 and HM2, allowing the amplification of cDNAs of all three postulated SV40 early mRNAs. These primers are depicted in Figure 4. Approximately 1 /kg of total RNA from each cell line was transcribed into cDNA by reverse transcription and analyzed for SV40 early region specific cDNAs by PCR using primers EN5 and HM2. Amplification products were analyzed by SDS - PAGE and visualized by silver staining (Hinzpeter et al., 1986). Figure 6 demonstrates that all cells analyzed contained the three postulated SV40 specific RNAs. However, grossly different ratios of amplification products from these RNAs were observed between transformed and lytically infected cells. Assuming similar levels of amplification of SV40 specific cDNAs, we conclude that in transformed cells the large T mRNA was the predominant SV40 specific RNA, whereas in lytically infected cells the small t mRNA was the predominant SV40 specific RNA species, as had already become evident from the Northern blot analysis of these cells shown in Figure 3. The amounts of the 17kT mRNA varied between the different SV40 transformed cells. However, the 17kT mRNA was generally more abundant in transformed cells than in lytically infected cells. Sequence analysis of each of these cDNA amplification products specific for the l7kT 4741

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Fig. 6. Detection of the alternatively spliced mRNA coding for the 17kT protein in productively SV40 infected cells and SV40 transformed cells. Total RNA was isolated from SV40 infected TC7 cells 48 h.p.i. (lane 1), FR(wt648) cells (lane 2), mKSA cells (lane 3), SV52 cells (lane 4) and COS cells (lane 5). After synthesis of first strand cDNA, PCR was performed using SV40 primers EN5 and HM2. Amplified DNA was separated by SDS-PAGE and visualized by silver staining.

Fig. 5. Sequence analysis of the SV40 l7kT protein. (A) Comparison of splice consensus sequences (DC = donor consensus sequence, AC = acceptor consensus sequence) with the SV40 sequence surrounding the detected splice sites in the second exon sequence of large T. The resulting amino acid sequence of the carboxy-terminus of the l7kT protein is shown. (B) Sequence of the splice junctions ()n.i

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Fig. 7. Comparison of overall phosphorylation of large T, l7kT and Ac mutant large T. In a parallel experiment equal numbers of FTPB.3 (lane 1) and FR(wt648) (lane 2) cells were labeled with either [35S]methionine (panel A) or 32p; (panel B) and extracted, and T specific proteins were immunoprecipitated with PAb419. Immunoprecipitates were analyzed by SDS-PAGE on a 12% gel.

coding sequences to generate vector pUC 18SV4OT4. Normal F 111 rat fibroblasts (Freeman et al., 1975), were cotransfected with pUC 18SV40T4 DNA and a hygromycin B resistance gene [vector phyg (Sugden et al., 1985)], as described recently (Zerrahn and Deppert, 1993) and colonies were selected for hygromycin B resistance. Cells of colonies positive for the expression of the l7kT protein were subcloned and analyzed for phenotypic alterations relative to fully SV40 transformed F 111 [FR(wt648)] cells (Pintel et al., 1981), and F 111 minimal transformants derived after infection with the large T retroviral expression vector pZIPTEX (Brown et al., 1986) (F5 cells), or the retroviral vector pRDD3 encoding a 147 amino acid amino-terminal fragment of large T (FR3 cells). Figure IC demonstrates that all transformants expressed the corresponding T specific

17kT: a third SV40 early protein Table I. Phenotypic characterization of F 111 cells transformed by authentic SV40 LT, 17kT and Ac mutant LT

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A detailed description of growth analysis in soft agar and analysis of actin cables is given in Materials and methods. aThe designations Flll + LT, Flll + TI-147, Flll + 17kT, Flll + TA539-682 or Flll + phyg represent various independent subclones of Flll cells expressing large T, an amino-terminal large T fragment (amino acids 1-147), the SV40 17kT protein, the large T with amino acids 539-682 deleted (Ac large T) or solely hygromycin B resistance as a control. bThe indicated ranges refer to the variability of the cloning efficiencies between individual subclones which correlated with the expression level of the respective transforming proteins. c +, well-developed actin cable system; -, weakly developed actin cable system.

proteins in roughly comparable amounts. By immunofluorescence analysis, large T, the 147 amino acid fragment of large T, and the l7kT protein were all localized exclusively to the cell nucleus (data not shown). The phenotypic properties of these cells are summarized in Table I. These data reveal that the 17kT protein was able to establish transformed cells with the phenotype of minimal transformants according to the nomenclature of Risser and Pollack (1974) regarding morphology, actin cable staining and growth in soft agar (Zerrahn and Deppert, 1993). Thus in this assay the 17kT protein exhibited transforming properties similar to those of large T expressed in limiting concentrations by vector pZIPTEX (Zerrahn and Deppert, 1993), and the 147 amino acid large T fragment expressed by vector pRDD3. Generation of the 17kT protein might allow independent regulation of amino-terminal large T functions The amino-terminal domain of large T, as expressed by the 17kT protein, contains several regulatory elements, e.g. the large T specific transactivator domain (Zhu et al., 1991), the domain for Rb binding (DeCaprio et al., 1988) and a postulated binding site for protein 'X', most likely corresponding to the 300 kDa protein associated with the adenovirus ElA protein (Yaciuk et al., 1991). One can assume that the various interactions of this domain with cellular targets are tightly regulated. Very little is known about such regulatory processes. However, this domain contains several phosphorylation sites which are important for large T functions in lytic infection and cellular transformation (Deppert et al., 1991; Knippschild et al., 1991; Fanning and Knippers, 1992). It is therefore conceivable that differential phosphorylation of these sites could be one means of regulating the various functions of this domain. However, there is strong biological and biochemical evidence that the phosphorylation of aminoterminal phosphorylation sites of large T, and hence large T function, is greatly influenced by phosphorylation of carboxy-terminal phosphorylation sites of large T, especially by Ser677 (Schneider and Fanning, 1988; Scheidtmann et al., 1991). Thus regulation of amino-terminal functions on full-length large T seems to be subject to the control of the large T carboxy-terminus. Therefore it is conceivable that, due to the interdependence of amino-terminal and carboxy-terminal phosphorylation, individual regulation of amino-terminal functions by phosphorylation is limited. The

strong influence exerted by carboxy-terminal sequences of large T on the large T amino-terminus could be verified by analyzing the properties of large T mutant Ac containing a 144 amino acid deletion in the carboxy-terminus of large T (amino acids 539-682), including the carboxy-terminal phosphorylation sites Ser639, Ser677 and Ser679. Table I shows that mutant Ac large T is completely inactive in transforming rat Fill cells, despite the presence of a genetically unaltered large T amino-terminal transforming domain. This domain, though 'intact' according to sequence parameters, must thus have become inactivated by carboxyterminal sequences on the mutant Ac large T. This assumption could be verified by protein chemical analyses. Mutant Ac large T in FTPB.3 cells is expressed at least as well as the 17kT protein in FR(wt 648) cells (Figure 7A) and is metabolically stable. However, it fails to complex p53 to any significant extent, and Figure 7B reveals that mutant Ac large T in FTPB.3 cells is severely underphosphorylated relative to full-length large T, and to the 17kT protein in FR(wt 648) cells, although it accumulates to at least the level of the 17kT protein in FR(wt648) cells (data not shown). This drastic underphosphorylation reflects a severe defect in the phosphorylation of mutant Ac large T amino-terminal phosphorylation sites, which is most probably induced by the lack of any phosphorylation site in the Ac mutant large T carboxy-terminus. Having demonstrated the strong interdependence of aminoterminal and carboxy-terminal phosphorylation on large T, we asked whether the independent expression of the large T amino-terminus as a 17kT protein might result in an altered phosphorylation of large T amino-terminal phosphorylation sites, as compared with phosphorylation of these sites in fulllength large T. Comparison of the phosphorylation patterns of amino-terminal phosphorylation sites in full-length large T (Figure 2A) with those in the 17kT protein (Figure 2B) indeed demonstrated significant alterations. In 17kT, phosphorylation of site 6a and (even more drastically) site 4 was reduced as compared with phosphorylation of these sites in full-length large T. Peptide 6a represents a so far unidentified phosphorylation site, 'x', in the vicinity of Sern12 (Deppert et al., 1991; Knippschild et al., 1991). Peptide 4 contains the large T phosphorylation sites Serl20, Serl23 and Thr124 (Knippschild etal., 1991). Underphosphorylation of this peptide mainly reflects an underphosphorylation of Serl20 and Serl23, as the Thrl24 specific phosphorylation sites in peptides 12 and 12a remained

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largely unaltered (Deppert et al., 1991; Knippschild et al., 1991). However, sites 7 and d were phosphorylated like in full-length large T, emphasizing a specifically altered phosphorylation of amino-terminal phosphorylation sites in the l7kT protein. These results suggests that the independent expression of the large T amino-terminus in the l7kT protein indeed allowed for an independent phosphorylation of certain amino-terminal large T phosphorylation sites, which could possibly allow an independent regulation of the biological functions of this domain.

Discussion Understanding the regulation of the multitude of activities exerted by large T in lytic infection and cellular transformation will constitute the next level in our understanding of the complex interactions of this viral protein with its host cells. Previous studies have revealed that large T is made up of several functionally distinct domains (reviewed in Pipas, 1992), which, when separated from each other, may act independently in various transformation assays (Pipas et al., 1983; Srinivasan et al., 1989; Sompayrac and Danna, 1992; Kierstead and Tevethia, 1993). In this report we provide data suggesting that expression of the functionally important amino-terminal domain of large T as an independent protein may be part of the viral strategy for independently regulating distinct activities of this domain, which might constitute a novel mechanism for the regulation of large T functions. The amino-terminus of large T has previously been identified as harboring a transforming domain of large T (Clayton et al., 1982; Colby and Shenk, 1982; Sompayrac and Danna, 1983, 1988, 1991). It contains the binding sites for the tumor suppressor pRb (DeCaprio et al., 1988), a postulated binding site for protein 'X', most likely corresponding to the 300 kDa protein associated with the adenovirus EIA protein (Yaciuk et al., 1991), and the large T specific transactivator domain (Zhu et al., 1991). The large T amino-terminus thus is in itself a multifunctional domain. It is therefore plausible that separate expression of this domain can augment large T functions, either by simply providing more copies of this domain, or possibly more interesting, by allowing a different regulation of its various activities within the cell. In this regard, it is interesting that SV40 transformed cells in culture often express a 'super' T antigen (super T) of higher molecular weight than authentic large T. These super T proteins are fusion proteins generated by tandem duplications of large T amino-terminal regions (Clayton and Rigby, 1981; Lovett et al., 1982; May et al., 1982; Levitt et al., 1985). As the occurrence of super T has been linked to progression to a more transformed phenotype (Butel et al., 1986), one can conclude that duplication of the large T amino-terminus provides a selective advantage to these cells. In this study, we presented data showing that all SV40 transformed cells analyzed, and, at a lower level, also lytically infected monkey cells, independently expressed an amino-terminal domain of large T in the form of a 17 kDa protein (17kT protein). We provide evidence that this l7kT protein is generated by alternative splicing of early SV40 RNA. Starting from early SV40 mRNA containing the large T specific splice, a second splice donor site at position 4425, and a splice acceptor site at position 3679 on the SV40 genome is used to generate a doubly spliced early SV40 4744

mRNA encoding the l7kT protein. Our sequence analyses of PCR amplification products using two different pairs of primers (see Figure 4A) of SV40 specific cDNA from all cells analyzed demonstrated that in all cases the second splice sites used were identical, thereby excluding the possibility of a PCR artefact or of a peculiar property of a certain cell line with regard to splicing. The presence of a l7kT mRNA in all SV40 transformed cells analyzed, as well as its presence in SV40 lytically infected cells, further stresses the biological relevance of generating this protein. The l7kT protein is colinear with large T up to amino acid 131, but has a unique carboxy-terminus of four amino acids (AlaLeu-Leu-Thr) encoded by a different reading frame. Our finding that the l7kT protein, at least in certain transformed cells, is expressed up to the level of small t, which has a proven supportive role in SV40 infection and transformation (Zerrahn and Deppert, 1993 and references therein), strongly argues for a similarly important biological role of this

protein. The l7kT protein exhibited transforming properties when expressed in normal rat F1 1 fibroblasts. This result is in line with previous analyses of the transforming properties of the large T amino-terminal region (Clayton et al., 1982; Colby and Shenk, 1982; Sompayrac and Danna, 1983, 1988, 1991; Srinivasan et al., 1989) and might suggest a role for the 17kT protein in SV40 mediated cellular transformation. However, as the l7kT protein is also expressed in lytically infected cells, we expect that this protein, like small t, exerts a biological function both in lytic infection and cellular transformation by SV40. These functions have not yet been defined and require further analyses of the l7kT protein itself, as well as of its concerted action in the context of the other SV40 early protein large T and small t. An interesting hypothesis regarding the functional aspect of 17kT expression in lytic infection and cellular transformation might be developed from our observation that certain amino-terminal phosphorylation sites in the l7kT protein are differently phosphorylated than the same sites in full-length large T (see Figure 2A and B). As phosphorylation of large T and of the 17kT protein was analyzed from the same cells [FR(wt648)] and, as large T and the l7kT protein both localize predominantly to the cell nucleus, these differences are not attributable to differences in the kinase or phosphatase status of the cell, but rather argue that the amino-terminus in full-length large T is in a different conformation from the l7kT protein. Previous data (Scheidtmann et al., 1991), which were substantiated in this paper, provided evidence that phosphorylation and function of the large T amino-terminus are greatly influenced by carboxyterminal large T sequences. Amino-terminal functions of large T thus seem to be tightly controlled by a carboxyterminal domain(s) of large T. Our demonstration that mutant Ac large T, containing an altered carboxy-terminus, was unable to exert any transforming effect on F 11 cells, and showed an almost complete lack of phosphorylation of amino-terminal large T phosphorylation sites exemplified this statement. Thus the intriguing possibility exists that the separate expression of the large T amino-terminus as a l7kT protein would allow this domain to escape any cooperative regulation by the large T carboxy-terminus. Clearly, this hypothesis has to be substantiated by further experiments which will demonstrate whether the l7kT protein will display different properties regarding its interactions with

17kT: a third SV40 early protein

cellular targets than large T itself. Such studies will require a tool to analyze the l7kT protein separately (i.e. an antibody specific for the unique carboxy-terminus of the l7kT protein), and then should lead to the elucidation of the biological role of the l7kT protein in SV40 infection and transformation.

Materials and methods Cells Normal Fischer rat fibroblast FIll cells (Freeman et al., 1975), the SV40 transformed Fl 11 cells FR(wt648) (Pintel et al., 1981), SV40 large T minimally transformed Fl 11 cells, e.g. F5 cells (Zerrahn and Deppert, 1993), Fl 11 cells transformed with an amino-terminal fragment of large T (amino acids 1-147), e.g. FR3 cells, Ac large T (amino acids 539-682 deleted large T) expressing Fl 11 cells, e.g. FTPB.3 cells, SV40 l7kT transformed Fl 11 cells, e.g. FT4.5, mKSA cells (Kit et al., 1969), SV52 cells (Bauer et al., 1987), COS cells (Gluzman, 1981), TC7 cells (Robb and Hubner, 1973) and the SV40 transformed BALB/c mouse embryo fibroblast cells SVMEF cl.3 were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS).

Establishment of cell lines The expression vector pUC18SV40T4 contains an SV40 l7kT cDNA, driven from the authentic SV40 early promotor. phyg mediates resistance to hygromycin B (Sugden et al., 1985). The packaging cell line pRDD3, transfected with a retroviral vector encoding a 147 amino acid amino-terminal fragment of large T, was kindly provided by L.Sompayrac. The vector pZIPTEXTPB expresses a large T with amino acids 539-682 deleted (Ac large T) cloned into the vector pZIP-NeoSV(X)l (Cepko et al., 1984). A pZIPTEXTPB packaging cell line was established after transfection of GP+E86 cells (Markowitz et al., 1988). Transfection or retroviral infection of Fl 11 cells for establishment of cell lines expressing SV40 l7kT, amino acids 1-147 comprising large T, or large T with amino acids 539-682 deleted were as described previously (Zerrahn and Deppert, 1993). mRNA and cDNA analysis Total RNA isolation and first strand cDNA synthesis of 1 Ag of total RNA were according to standard protocols (Sambrook et al., 1989). PCR was performed in 50 1A 20 mM Tris, pH 8.3, 50 mM KCI, 2.5 mM MgCl2, 0.01% gelatine. Primers (T3E: 5'-CGCGCGGAATTCTGATGAATGGGAGCAGTGGTGGAA-3'; T3B: 5'-CGGCCGGGATCCCAGGGGGAGGTGTGGGAGGTTTTT-3', see Figure 2) were used at 4 yg per ml, each dNTP at 200 jtM and Taq DNA polymerase at 25 U per ml. One-twentieth of the cDNA synthesis reaction was used as template. Samples were subjected to 30 cycles of amplification, each consisting of denaturation at 94'C for 30 s, annealing at 54'C for 30 s and polymerization at 72'C for 120 s. DNA fragments were isolated, purified, cleaved with EcoRI and BamHI and cloned into pUC18. DNA sequencing was performed using the T7 sequencing kit (Phamacia) according to the manufacturer's instuctions using primer T3E. For semi-quantitative mRNA analysis primers used were EN5 (5'-TGCAAGGAGT'TCATCCTGATAAAGG-3') and HM2 (5'-TTT-

TAGAATTCAGGCCTACAGTGTTTTAGGCACACTGTACTCATTC) (see Figure 2). SDS-PAGE analysis of amplified DNA and silver staining were as described previously (Hinzpeter et al., 1986). For Northern blot analysis -10 gg of purified RNA were electrophoresed on a denaturing 1.2% agarose gel containing 3% formaldehyde and transferred to Gene Screen Plus (Dupont, NEN Research Products) as suggested by the suppliers. The filter was hybridized with an SV40 large T cDNA probe (nt 5162-4558) labeled with [a-32P]dCTP by random primer synthesis (Feinberg and Vogelstein, 1983). Hybridization of the filter and washing procedures were as recommended by the manufacturer.

Radiolabeling, extraction, immunoprecipitation and SDS - PAGE For immunoprecipitation of large T and amino-terminal large T derivatives, cells were metabolically labeled either for 2 h in Pi-free medium supplemented with 250 ttCi/ml Pi (Amersham) or for 1 h in methioninefree medium supplemented with 50 ltCi/ml [35S]methionine (Translabel, ICN). Extation of cells, immunoprecipitations with PAb419 (Harlow et al., 1981), recognizing an amino-tenninal epitope on large T, and SDS-PAGE analysis were performed as described previously (Zerrahn and Deppert, 1993). To prevent proteolytic degradation of large T, the lysis buffer was supplemented with 20% immunoglobulin-depleted FCS (Schirmbeck and Deppert, 1987).

Analysis of the cellular phenotype For actin cable staining cells were seeded on coverslips (12 mm) and grown for 2 days. Coverslips were rinsed in phosphate buffered saline (PBS; 140 mM NaCl, 3 mM KCI, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) and fixed for 15 min at 2°C in 3.7% paraformaldehyde dissolved in PBS, containing 1 mM CaCl2 and 0.5 mM MgCl2. Actin cables were stained with TRITC-phalloidin (0.05 mg/ml; Sigma) according to Wulf et ad (1979). For analysis of growth in soft agar cells were plated in duplicate at 1 x 104, 5 x 103 and 1 x 103 per 35 mm diameter culture dish in DMEM containing 10% FCS and 0.3% (w/v) agar (Difco Bacto-agar, Difco laboratories) onto a bottom layer of 0.5 % (w/v) agar in DMEM. Colonies were scored 14 days after plaing. Colonies were termed positive in reference to F 111 cells, which never exceeded a two cell stage in several independent experiments.

Phosphopeptide analysis Two-dimensional analysis of phosphorylated peptides of SV40 large T was performed as described previously (Deppert et ad, 1991; Knippschild et ad, 1991). Briefly, actively growing cells were pulse labeled for 4 h with 32p, (carrier-free; Amersham), 0.7-1.0 mCi 32P, per ml, in Pi-free DMEM supplemented with 5% Pi-free FCS (2 ml per 90 mm dish). Whole-cell extracts were then prepared followed by immunoprecipitation of large T with the monoclonal antibody PAb419. Imnmunoprecipitated large T specific proteins were purified by SDS-PAGE on 1 mm thick, 7.5 or 11% acrylamide gels, extracted from the gels and oxidized with performic acid as cited above. Phosphopeptdes of SV40 large T were prepared by sequential digestion with trypsin-TPCK (Wordtington) and pronase E (Sigma) and then analyzed on cellulose thin-layer plates by electrophoresis at pH 1.9 followed by ascending chromatography in isobutyric acid buffer. Phosphopeptides were visualized by autoradiography.

Acknowleduements We thank L.Sompayrac for a retroviral vector coding for the TI-147 fragment and for stimulating discussion. This work was supported by grant De 212/9-2 from the Deutsche Forschungsgemeinschaft and by the Fond der Chemischen Industrie. The Heinrich-Pette-Institut is supported by the Freie und Hansestadt Hamburg and by the Bundesministerium fir Gesundheit. This work is part of the Ph.D. thesis of J.Z.

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