The expression and properties of polyoma virus middle-T antigen in simian cells

157

Virus Research, 4 ( 1986) 151- 171 Elsevier

VRR 00233

The expression and properties of polyoma middle-T antigen in simian cells Graham

J. Belsham

Biochemistry Diaisron, NatIonal

*, Barry

K. Ely and Alan

E. Smith

virus

**

Institutefor Medical Reseaxh, Mill Hill, London N W7 IAA, L’.K.

(Accepted

for publication

15 October

1985)

SV40 late replacement vectors containing the polyoma middle-T coding sequences have been constructed. Mixed hybrid virus stocks have been obtained through ~omplementation with a defective SV40 helper genome (dl 1055) following DNA transfection into CV-1 cells. Middle-T antigen is expressed in the infected simian cells at about 5-10 fold higher levels than in polyoma virus-infected mouse cells and has the pp60’+‘“-associated tyrosine-specific protein kinase activity in vitro. However, the ‘specific activity’ of the kinase in extracts of the infected CV-1 cells is lower than that observed in polyoma infected 3T6 cell extracts. The half-life of middle-T antigen in the CV-1 cells is about 4 h but the in vitro kinase activity associated with middle-T has a haif-life of at least 8 h and hence appears to be stabilized. The in vivo phosphorylated species of middle-T has been shown by sucrose gradient analyses to be largely distinct from the middle-T with associated protein kinase activity in vitro. protein

phosphorylation,

SV40 expression

vector,

tyrosine-specific

protein

kinase

Introduction The expression of polyoma virus middle-T antigen within established rodent cell lines is sufficient to initiate and maintain a transformed phenotype (Treisman et al.,

* To whom reprint requests should be sent. Present address: Department of Genetics, The Animal Research Institute, Pirbright. Woking, Surrey GU24 ONF. U.K. ** Pre.yent address: integrated Genetics. 31 New York Avenue, Framingham. MA 01701. U.S.A.

016%1702/86/$03.50

C 1986 Elsevier Science Publishers

B.V. (Biomedical

Division)

Virus

158 1981). The biochemical basis underlying this property of middle-T is unknown (see Smith and Ely, 1983; Tooze, 1980). It has been shown that middle-T antigen exhibits a tyrosine-specific protein kinase activity in vitro (Eckhart et al., 1979; Schaffhausen and Benjamin, 1979; Smith et al.. 1979) which appears to reflect an association with the cellular tyrosine kinase pp60c+r’ (Courtneidge and Smith. 1983, 1984). Analysis of mutant middle-T antigens has revealed that those which maintain the ability to transform aiso retain the in vitro kinase activity (Smith and Ely, 1983) whereas non-transforming mutants are partially (e.g. dl 23 and dl 2208) or totally (e.g. NC-59) deficient in this activity. Biochemical characterization of middle-l’ is hampered by the low expression level of this protein, both in virus-infected cells and in polyoma-transformed cells. Analysis of non-transforming mutant middle-T antigens is often further complicated since cell lines expressing the protein cannot he obtained simply. Furthermore. polyoma virus stocks may be difficult to produce if the mutation in middle-T also causes changes in the large-T antigen amino acid sequence. resulting in a defective protein. Hence an alternative expression system would be useful if it easily allows the examination of the properties of mutant middle-T antigens and allows wild-type middle-T to display the known characteristics of this protein from poly~~ma-infected or trai~sfornled cells. In this report we describe the use of SV40 late replacement vectors to express middle-T antigen in simian celIs and describe the properties of the protein produced in this system. Recently Zhu et al. (1984) have described the use of a similar system to express individually the polyoma early region proteins. Their studies on the properties of middle-T expressed in simian cells are confirmed and extended in this report.

Materials

and Methods

Construction of SV40 lute replacement vectors designed to express po(~omu middle-T unligen Two separate constructions were carried out to obtain the plasmids shown in Fig. la. Restriction enzyme digests were carried out as recommended by the manufacturers and other procedures as described by Maniatis et al, (1982). pPSV51 was prepared by ligating the Hhal (SV343)-EcoRI (SV1782) large fragment (ca. 3800 bp) from SV40 viral DNA and the HhaI (Py96)-Hind111 (Py1656) (ca. 1550 hp) fragment from polyoma viral DNA between the HindIII-EcoRI large fragment (ca. 3700 bp) of pBR328 (Soberon et al., 1980). The ligation mixture was used for transfection of Escherichia co/i ED8767 and ampicillin-resistant colonies obtained. The structure of pPSV51 has been confirmed by restriction enzyme analysis with at least six separate restriction enzymes. To obtain the pPSVXE family of plasmids, initial modifications to fragments containing the middle-T gene and the SV40 promoter sequences were carried out. The BamHI (Py4632)-SacI (Py569) fragment of pdl 2025 (a kind gift from C. Tyndall), which contains a deleted polyoma origin with an XhoI linker inserted at PylOO. was ligated to the Sac1 (partial digest, Py569)-EcoRI (Py1560) fragment (928 bp) of pASlO0 (Rostra et al., 1983) between the BamHILEcoRI fragment of pBR322

159

(a) Early

(iI

Late

(ii)

bf--+

Barn HI (SV12533)

(b)

pPSVXE

100

TATA m

ATG S(4)

106 106 CGCGGCCGAGCTC 1

Hpall

107

S5(25)

Fig. 1. (a) Structures of SV40 late replacement vector plasmids constructed for this study. In each case the junctions of fragments used in the constructions are shown. Abbreviations used are: RI, EcoRI; HIII. HindIlL Polyoma (Py) and SV40 (SV) nucleotide numbering systems are those used in Tooze (1980). (b) The junctions of the SV40 late promoter to the 5’ end of the middle-T gene sequences in the pPSVXE family of plasmids. The residual polyoma sequences were determined by G + A single-track (Maxam and Gilbert. 1980) sequencing from the “P-1abelled Xhol site. The position of the SV40 major late transcription start (6). the polyoma early transcription start (&), its TATA box and the initiating methionine codon (Py 173) are indicated

(Bolivar et al., 1977) to yield pXIM1 (not shown). This plasmid contains the entire middle-T sequence but lacks the middle-T intron (Treisman et al., 1981). pXIM1 was digested with XhoI and treated with the exonuclease Ba131 for l-10 min. The fragments were blunt-ended with the Klenow fragment of DNA polymerase 1 (in the presence of all four dNTPs) and an XhoI linker added again before recircularization. A collection of similar plasmids with different lengths of polyoma sequence up-

160 stream of the initiation codon (Py173) was obtained following transfection into ED 8767. The position of the XhoI linker in each construct was determined by single track (G + A) sequencing (Maxam and Gilbert, 1980). SV40 viral DNA was cut at the unique HpaII site, blunt-ended as above and an XhoI linker added. The Xhol-EcoRI (SV1782) large fragment (ca. 3800 bp) was cloned between the Xhol and EcoRI sites of pXIM1 to give pSVXE (not shown). The pPSVXE family of vectors was prepared by ligating the Xhol-EcoRI (ca. 1450 bp) fragments from the pXIM1 and its Bal31-generated derivatives, with the XhoI (HpaII site)-BamHI (SV2533) fragment (ca. 3050 bp) and EcoRI (SVl7~2)-Hind111 (SV3476) fragment {ea. 1700 bp) from the pSVXE plasmid between the BamHI-Hind111 sites within pPVU-0 (Kalderon et al., 1982) (this contains the pBR328 replicon). Plasmids with the correct structure were identified and then characterized by restriction mapping with more than six different enzymes. Prepurut~~3n of ~~,brid cirus stocks The hybrid viral genomes were obtained from pPSV51 (by EcoRI digestion) and from the pPSVXE family (by BamHI digestion) and purified by agarose gel electrophoresis. The helper SV40 DNA (dl 1055) was similarly obtained following BamHI digestion of the plasmid pdl 1055. In each case the linear molecules were circularized by ligation at low concentration (approx. 5 pg/ml) and the circular form purified by agarose gel electrophoresis. The viral DNA (approx. 100 ng of the hybrid DNA and 100 ng of the dl 1055) was mixed with 1 ml of E4 medium (no serum) containing DEAE-dextran, 400 pg/ml, and added to a 50 mm dish of CV-1 cells (about 75% confluent). After 45 min the cells were washed twice in E4 medium and then incubated in E4 with 10% foetal calf serum for 7 days. At this time the cells and media were frozen, thawed and harvested, the freeze-thaw procedure was repeated twice more and 0.5 ml of this virus stock was used to infect fresh CV-1 cells (50 mm dish). A third passage was initiated 7 days later following the same procedure except that 1 ml of the virus stock was used on a 90 mm dish of CV-1 cells. When severe cytopathic effect (CPE) was observed (within 7 days) the virus was harvested. No virus-induced CPE was ever observed in cells treated only with dl 1055. However, in some cases we have only obtained apparently wild-type SV40 virus stocks rather than mixed virus stocks from cells transfected with both the hybrid virus genome and the helper DNA. Lahelling und extruction of cells Conditions used for cell growth, infection with virus, labelling with (“‘Slmethionine and 32Pi. and preparation of cell lysates, have all been described previously (Smith et al., 1978, 1979; Paucha et al., 1984). All cell lysates were rapidly frozen and stored in liquid Nz. Immunoprecipitation und kinase assuys Conditions for the in vitro kinase assay have been described previously (Smith et al., 1979; Courtneidge and Smith, 1983). Metabolically labelled cell extracts (up to 100 ~1) were immunoprecipitated by mixing with 0.5 ml of a buffer containing 20

161 mM Tris-HCl (pH 7.5) 500 mM NaCI, 1 mM EDTA, 0.2% NP40 and S-10 ~1 of antiserum and then incubated for 1 h at 2O’C followed by a further 20 min with lysed and fixed protein A-bearing Staph&coccus aureus. The bacterial pellets were washed twice and absorbed proteins were eluted with SDS-sample buffer prior to analysis on a 10% polyacrylamide gel (Laemmli, 1970). Antisera recognizing the three polyoma T antigens (anti-PyT) were obtained from tumour-bearing rats or hamsters; similarly, antisera recognizing the SV40 T antigens (anti-SVT) were obtained from tumour-bearing hamsters. Sucrose grudient analysis Cell extracts (0.4 ml) were loaded on to 5 ml continuous gradients of 5-20% sucrose over a 0.2 ml 60% sucrose cushion in a buffer containing 10 mM Tris (pH 8.0) 140 mM NaCl, 1 mM DTT, 0.1% NP40 and 1% Trasylol. Centrifugation was at 25000 rpm for 16 h at 4°C in a Beckman SW55 rotor. Fractions (20) were collected from each gradient and diluted five-fold into the immunoprecipitation buffer (as above) for analysis. Determination of the half-life of middle-T Infected cells (at 24 or 48 h post-infection) were incubated for lacking methionine in the presence of 100 PCi [“Slmethionine. then replaced by E4 (containing methionine) with 10% foetal incubation continued for the indicated times. Aliquots containing trichloroacetic acid-insoluble [ “Slmethionine were analysed by tion. To determine the half-life of middle-T-associated protein kinase protocol was followed, except that the ‘cold chase’ was performed emetine (12 pg/ml) (Sigma).

1 h in E4 medium The medium was calf serum and equal amounts of immunoprecipitaactivity a similar in the presence of

Prepurution and analysis of viral DNA Viral DNA was prepared by the method of Hirt (1967). The analysis of viral DNA replication from Hirt extracts was performed by cutting the recovered DNA with TaqI and PstI, separating the fragments of a 1% agarose gel and transferring them to a nitrocellulose filter (Southern, 1975), and probing with nick-translated (Rigby et al., 1977) pPSVXE 107.

Results Construction of SV40 lute replucement vectors to express polyoma middle-T untigen In an attempt to obtain higher level expression of polyoma middle-T antigen than is obtained in polyoma virus-infected cells, we have constructed vectors in which the sequences encoding middle-T have been inserted into the SV40 genome downstream from the SV40 late promoter. The middle-T antigen should be expressed in the same manner as the SV40 capsid proteins. The replacement of the capsid protein gene sequences in these vectors means that the genomes are non-viable. However, they

162 can be packaged into virus particles if a helper SV40 genome. able to produce the capsid proteins, is present. It is necessary for the helper SV40 also to be non-viable, hence we used the mutant dl 1055 (Pipas et al., 1983) which had previously been employed for the same purpose (Gething and Sambrook, 1981). This mutant contains a deletion within the SV40 large-T coding sequence resulting in the production of a severely truncated large-T antigen which fails to allow DNA replication. Thus dl 1055 and the polyoma SV40 hybrid genomes are capable of complementing each other to produce a mixed virus stock. Two separate constructions have been employed to build SV40 late replacement vectors and the structures obtained are shown in Fig. la. The simplest plasmid (pPSV51) was made using convenient pre-existing restriction sites within SV40 and polyoma viral DNA to insert a fragment encoding middle-T from the polyoma early region into the late region of the SV40 genome. The viral fragments (restriction fragment junctions are indicated in the Fig. la) were cloned into the EcoRI-Hind111 large fragment of pBR328. Digestion of the resultant plasmid pPSV51 with EcoRI releases a linear hybrid viral molecule which, on ligation, gives a circular viral genome. Since it is almost identical in size to SV40 the hybrid genome should be packaged into virions without difficulty. The genomic polyoma fragment (HhaI-EcoRI) used in this construction encodes not only middle-T antigen but also the small-t antigen and a truncated large-T antigen, each resulting from different splicing patterns of the initial transcript. It is difficult to manipulate this vector further for optimising expression of middle-T due to the presence of multiple HhaI sites within the pBR328 sequences. Thus, for convenience in altering the extent of sequences at the 5’-terminus of the gene (which potentially could affect the expression level obtained) and for ease in replacing the middle-T gene, a vector with unique XhoI and EcoRI restriction sites at each end of the insertion was constructed (Fig. la(ii)). For these vectors we also made use of the polyoma plasmids lacking the middle-T intron (derived from those described by Treisman et al., 1981) which are only capable of encoding middle-T antigen. The pPSVXE plasmids, when cut with BamHI, release linear SV40 polyoma hybrid molecules which, on ligation, also give rise to circular DNA of very similar size to the SV40 genome. A series of similar plasmids was generated by digesting with exonuclease Ba131, from an inserted XhoI site at polyoma nucleotide 100, towards the initiation codon at nucleotide 173. The fragments generated were blunt-ended with the Klenow fragment of DNA polymerase 1 and XhoI linkers added again to the molecules which were then inserted into the pPSVXE vector. The deletion end points (determined by Maxam and Gilbert G + A single-track sequencing) of plasmids used in this study are shown in Fig. lb. Expression of polyoma middle-T antigen in simian cells The polyoma-SV40 hybrid viral sequences were separated from their plasmid sequences and treated with T4 DNA ligase. The circular DNA formed was purified away from unligated material and then transfected into CV-1 cells together with the SV40 mutant dl 1055 DNA which had been prepared in a similar way from the plasmid pdl 1055. Following three passages through CV-1 cells, virus stocks which

163 123456

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LT

ST

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123456 Ig

MT

Fig. 2. (A) lmmunoprecipitation of middle-T and SV40 large-T from the hybrid virus-infected CV-1 cells. Equal amounts of [ “Slmethionine-labelled extracts prepared at 48 h post-infection were immunoprecipitated with anti-PyT serum (lanes 1-6) or anti-SVT serum (lanes 7-12) and analysed on SDS-containing 10% polyacrylamide gels. Lanes are: 1, 6 virus PSV51; 2. 8 virus Sl(4); 3, 9 virus 106; 4. 10 virus 108: 5, 11 virus 107; 6, 12 virus S5(25). M indicates marker proteins with molecular weights in thousands. (B) In vitro kinase assay of middle-T from the hybrid virus-infected CV-1 cells. Unlabelled cell extracts. prepared in parallel to the extracts used in A, were immunoprecipitated with hamster anti-PyT serum (lanes l-6) or rat anti-PyT serum (lanes 7-12) and incubated with [y-‘*P]ATP. Samples were prepared for gel analysis under non-reducing conditions. The order of lanes is the same as in A.

severe cytopathic effects within 3 days were obtained from all six plasmids. These stocks were used to infect fresh CV-1 cells and 48 h later the cells were labelled with [‘5S]methionine and cell lysates made. Fig. 2A shows the results obtained from immunoprecipitation of such lysates using rat anti-PyT or hamster anti-SVT sera. Each virus stock produces similar amounts of SV40 large-T antigen and also in each case produces middle-T antigen. As judged by the level of incorporation of [ “Slmethionine into middle-T compared to SV40 large-T, the virus stocks can be ordered in their relative efficiency of producing middle-T. Densitometric scans of fluorographs from three separate experiments were used for this analysis. If the [“Slmethionine incorporation into large-T is set at loo%, then the relative produced

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.alues for the production of middle-T by PSV51 is about 25%. The 106, 108 and Sl(4) stocks are similar to each other at about 50% relative efficiency and 107 and S5(25) values are about 100%. The ordering of these viral stocks in their efficiency of middle-T production parallels the order of proximity of the polyoma 5’-junction to the middle-T initiation codon (Fig. lb). The presence of the intron in PSV51 does Indeed, the lower level of protein not appear to enhance middle-T expression. expression obtained may be due to the three alternative splicing events which can take place on the initial RNA transcript effectively reducing the amount of middleT-specific mRNA synthesised. A band corresponding in mobility to polyoma small-t (M, 22000) is precipitated from the PSV51 extracts and. as expected. not from any other; no convincing band corresponding to a truncated large-T molecule was observed. In a recent report by Zhu et al. (1984) a middle-T-related peptide of M, 58000 was observed from a virus of very similar construction to PSV51 and was attributed to a novel splicing event to a normally cryptic splice site. We have not observed such a protein. In order to examine middle-T-associated protein kinase activity, unlabelled extracts made from the hybrid virus-infected cells were also immunoprecipitated with either hamster (see Fig. 2B, lanes l-6) or rat (lanes 7712) anti-PyT serum and incubated with [y-‘2P]ATP. In each case, phosphate is incorporated into middle-T antigen and the rat immunoglobulin heavy chain as observed with extracts from polyoma virus-infected cells. We have shown that the phosphorylated amino acid in the in vitro phosphorylated middle-T is phosphotyrosine (Fig. 3C) in the CV-1 material as well as in the mouse 3T6 product. Furthermore, partial proteolysis fingerprints obtained using Staphylococcus uweus V8 protease (Fig. 3A) or chymotrypsin (Fig. 3B) of the labelled middle-T from both sources results in almost identical maps which strongly suggests that the same tyrosine phosphorylation sites

Fig. 3. Cleveland analysis of in vitro phosphorylated middle-T from CV-I and 3T6 cells. (A) Unlahelled extracts from CV-1 cells infected with PSV 107 (lanes l-3) or PSV SS(25) (lanes 7-9) or from 3T6 cells infected with polyoma virus (lanes 4-6) were immunoprecipitated with hamster anti-PyT serum and incubated with [y-“P]ATP. The portion of a 10% polyacrylamide gel containing the phosphorylated middle-T was homogenized and either left untreated (lanes 1.4.7) or incubated for 2 h at 37°C with 250 ng (lanes 2, 5, 8) or 10 pg (lanes 3.6.9) Staph_plococcus mreus VX protease. Samples were then analysed on a SDS-containing 14% polyacrylamide gel. (B) Samples were obtained and analysed as in A except that chymotrypsin was used in place of S. aureus V8 protease. Please note order of lanes. M = marker proteins. (C) Phosphoamino acid analysis of the in vitro phosphorylated middle-T from polyoma A2-infected 3T6 cells (lane 1) or from CV-1 cells infected with PSV 107 (lane 2) or PSV S5(25) (lane 3). The labelled middle-T was eluted from a polyacrylamide gel and coprecipitated with bovine serum albumin (100 pg) using 10% trichloroacetic aud. The washed precipitate was hydrolysed. under vacuum. in 6 M HCI for 2 h at 110°C and analysed by electrophoresis at pH 3.5 on cellulose thin-layer plates. The position of phosphotyrosine. phosphothreonine and phosphoserine markers stained with ninhydrin is indicated. ori indicates the sample application point. (D) Unlahelled extracts from mock- or S5(25)-infected CV-1 cells were immunoprecipitated with normal rat serum (lanes 1 and 5) or with the rat anti-T serum 1.1 (lanes 2-4 and 6-8). then incubated with NBR (lanes 3 and 7) or TBR (lanes 4 and 8). washed and incubated with [ y-” P]ATP. Samples were analysed on a 10% polyacrylamide gel in the presence of SDS under non-reducing conditions.

are used in each case. We and others have previously characterized several tyrosine residues (Tyr 250, 297, 315 and 322) which are phosphorylated in this reaction (Harvey et al.. 1984; Hunter et al., 1984). We have also sought evidence that the tyrosine kinase activity is a result of association with pp60”*‘” as has been dem~~nstrated in polyoma virus infected or transformed cells (Courtneidge and Smith. 1984). Middle-T was immunoprecipitated from cell extracts using a specific rat anti-T serum and incubated with normal baby rabbit serum (NBR) or serum containing antibodies specific for ~~60’.“” (tumourbearing rabbit serum (TBR)), washed and then incubated with [y-“PjATP. Fig. 30 shows that the middle-T antigen expressed in simian cells has associated protein kinase activity which specifically phosphorylates the TBR immunoglobulin: this indicates the presence of pp60’+“.

To examine the characteristics of middle-T expression within CV-1 cells infected with the polyoma-SV40 hybrid viruses we monitored its production in comparison with other markers of the virus infection. Middle-T expression over a 72 h infection period was examined by [ “Slmethionine incorporation. by in vitro kinase assay and by radioimmunoassay. The synthesis of SV40 large-T, the major capsid protein VP1 and the replication of the viral DNA. were also investigated. These studies used 107 and SS(25) virus-infected cells since their level of middle-T expression w-as highest. A wild-type SV40 infection was carried out for comparative purposes. The incorporation of [7SS]methionine into SV40 large-T antigen in CV-1 cells infected with virus stocks is shown in Fig. 4A and indicates that in the hybrid virus-infected cells. the rate of synthesis of SV40 large-T peaks at about 24 h and then remains relatively constant. The apparent pattern of middle-T (Fig. 4B) and SV40 VP1 (Fig. 4C) expression is rather similar even though these should be expressed as ‘late’ proteins. However. examination of viral DNA in Hirt extracts by Southern hiotting (Fig. 4D) indicates that by 24 h viral DNA synthesis has occurred and hence the ‘late’ phase of infection has commenced. It is possible to estimate from this blot that the amounts of helper DNA (d! IOSS) and PySV40 hybrid DNA are approximately equal. It is noteworthy that despite the considerable later increase in the level of viral DNA within the cells, no increase in the rate of synthesis of early or late proteins apparently occurs. The wild-type SV40 infection shows a similar relationship between large-T and VP1 synthesis relative to DNA replication although events are delayed. probably reflecting a lower multiplicity of infection. It should be noted that the [ “Sjmethionine data shown does not necessarily give a true guide to the level of the protein in the cells, since this also depends on the relative stability of the protein and the time of onset of synthesis. Radioimmunoassay of middle-T (Fig. 4E) in these extracts, using the method devised by Dilworth (1982) shows that the level of middle-T is considerably lower at 24 h than at 48 h. although, as shown in Fig. 4B. the rate of synthesis is higher at 24 h. The decline in the level of middle-T by 72 h probably reflects the significant cytopathic effect observed by this time. The appearance of middle-T-associated protein kinase activity is shown in Fig. 4F. The phosphorylation of both middle-T (see also Fig. 5B) and the

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Time

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Fig. 4. Time course of infection of CV-1 cells by the hybrid PSV viruses. (A-C) CV-‘I cells infected with SV4O (lanes 1.4,7. l(I), PSV 107 (lanes 2. 5, 8.11) or PSV S5(25) (lanes 3.6.9. 12) were labelled with [‘SS]methionine at 6 h (lanes I, 2. Ii), 24 h (lanes 4. 5.6). 48 h (lanes 7,s. 9) and 72 h (lanes 10. Il. 12) post-infection and cell lysates made. Samples cvntaining equal amounts of trichioroacetic acid-insoluble ~~~S]meth~on~ne were i~~munoprecip~tated with anti-SVT serum (A), anti-PyT serum (3) or anti-SV40 capsid serum (C) and analysed on a SDS-containing 10% polyacrylamide gel. (D) Hirt extracts were prepared from parallel infections to those described above (A-C) and the viral DNA digested wrth Taql and Pstl prior tv analysis on a 1% agarose gel, transferred to nitrocellulose and probed with “P-labelled pPSVXE 107. All SV40 and PSV DNA fragments are detected. SV40- (lanes l-4). PSV 707- (lanes 5-g) and PSV S5(25)- (lanes 9-12) infected cells were snalysed at 4 h (lanes I. 5.91. 24 h (lanes 2,6. 10). 48 h ffanes 3,7. II) and 7? h (lanes 4, 8,fz). Approximate fragment sizes (base pairs) are indicated. (F.) ~adio~mrnunoa~sa~ of middle-T in uniabelled infected CV-l extracts prepared in parallel to those described above. Ahquots (100 ~1) of SV40- (+I. PSV lo’?- (A.) and PSV S5(25)- (I) infected cells were incubated for 16 h at 4°C with 10 ~1 of the anti-carboxyterminus monoclonal anttbody KF (a kmd gift from Dr. G. Walter) and IO ~1 of a 1:45 dilutian of the [%]methionine-labelled supernatant from MT7 monvclonai antibody-producing cells (a kind gift from Dr. B.E. Griffin). Immune complexes were collected on protein A bearing fixed .%aphykwuccrrs MW~~LScells and washed twice prior to analysis by scintillation counting. (F) Middle-T associated in vitro kinase activity in unlabeIled extracts used in E. Samples were immunv~r~~jpitated with rat-anti-PyT serum and incubated with (y-“PJAPP prior to analysis on a 19% poiyacrylamide gel containing SDS under non-reducing conditions. Lanes are as m A-C.

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0

Fig. 5. Middle-T

expression

Radmlmmunoassay

and in vitro

of middle-T

kinaae activity

III 3T6 cells (0.

in mouse 3T6

mock-infected:

(0. mock Infected: A. SS(25) infected: n . 107 infected) at 4X h post-infection. Fig. 4E. (B) Cell extracts middle-T anti-PyT

as determined

contaming

serum and incubated

mlde gel. Lane 107.infected

CV-I

PSV 107.infected

equal amounts

by radioammunoaaaay

1. uninfected

with CV-I

[ y-“P]ATP

serum and Incubated

cell extracts with [y-“P]ATP

h-8)

were

cells. lanes

these extracts containing

(lanes l-5)

or equal amount\

CV-I

with

prmr 10 analysis on a SDS-containing

cell protein

as quantified

10% polyacryla3T6 cells. (C)

with hamster anti-PyT 10% polyacrylamide

by radioimmunoassay

(by the addition of SV40-Infected

of

hamster

cells. lanes 3 and X.

5 and 6. polyoma A2-infected

volumes) were immunoprecipitated

equal amounts of middle-T

crlls

Samples were analysed as III

F.xtracts were made at 6 h (lane 1). 24 h (lane 2). 48 h (lane 3) and 72 h (lane 4) post-infectlon. containing equal amounts of CV-1

cells. (A)

and CV-I

prior to analysis on a SDS-containing

3T6

(equal

CV-I

immunoprecipltated

cells. lanes 2 and 7. S5(25)-infected

cells, lane 4, uninfected CV-I

of cell protein

(lanes

and slmtan

0. polyoma AZ-Infected)

gel.

Aliquots of

(see Fig. 4F.) and

cell lysate) were simllsrly

analyaed: extracts were 24 h (lane 5). 48 h (lane 6) and 72 h (lane 7) post-infection.

rat immunoglobulin heavy chain from extracts prepared at 24 h was very low, but much higher at 48 h into the infection. Similar results have been found with the other PySV40 hybrid viruses. It appears that the level of kinase activity expressed correlates with the net accumulation of middle-T within the cell. This result was unexpected since it appears that in both polyoma infection of mouse 3T6 cells and in Py transformed cells, only a fraction of the middle-T is associated with kinase activity. It seemed to us likely that the level of middle-T accumulated by 24 h should have been in excess of that normally found to be associated with kinase activity. Hence we investigated the relationship between the level of middle-T and the expression of in vitro kinase activity more closely. Middle-T expression und in vitro tyrosine kinuse actiuitl Extracts (prepared at 48 h post-infection) from polyoma-infected

mouse 3T6 cells

169 and the hybrid 107, SS(25) virus-infected CV-1 cells were analysed with respect to the level of middle-T by radioimmunoassay (see Fig. 5A). The CV-1 extracts contain up to ten times more middle-T than the polyoma-infected 3T6 extracts. The kinase activity expressed from these extracts is shown in Fig. 5B using equal amounts of total protein or middle-T as quantified by radioimmunoassay. In each case it is apparent that considerably more phosphate is incorporated into middle-T in extracts from 3T6 cells suggesting a lower ‘specific activity’ of the middle-T in the CV-1 cells, in agreement with the observation of Zhu et al. (1984). It may be expected that if the kinase activity reflects association with cellular tyrosine kinase activity. then once saturation of the kinase is achieved, no increase in middle-T-associated kinase would fall. activity would occur and hence the ‘specific activity’ of middle-T However, the apparent ‘specific activity’ of middle-T kinase activity appears to remain fairly constant throughout a 72 h infection (Fig. 5C) (note equal amount of middle-T, by radioimmunoassay, was present in each reaction and the same amount of cell protein was used by the addition of SV40-infected cell extract prepared in parallel). Furthermore, as noted above (Fig. 4E) at 24 h after infection by the hybrid virus stocks, the middle-T kinase activity is low (and sub-maximal) while the radioimmunoassay data suggest that the middle-T has accumulated in these cells to the same level as is seen maximally in polyoma-infected cells. We are hence led to the conclusion either that the molecular interaction between middle-T and the cellular tyrosine kinase activity (~~60’.“‘) is less efficient in CV-1 cells (presumably reflecting a difference in ~~60’~“’ between the two organisms) or that some processing event is required before middle-T can obtain kinase activity which must occur differently between the two systems. If some relatively slow processing event is involved, then the stability of middle-T within the two systems becomes important. Stcrhility of middle-T in CV-I cells We determined the half-life of middle-T antigen by pulse labelling experiments on 107- or S5(25)-infected CV-1 cells at 24 h and 48 h post-infection and obtained a value of about 4 h (data not shown) which is in good agreement with that reported by Zhu et al. (1984). In parallel experiments in polyoma virus-infected 3T6 cells little loss of labelled middle-T antigen occurred during a 6 h chase period. Hence middle-T would appear to be significantly more stable in 3T6 cells than in CV-1 cells. We also wanted to determine whether the half-life of the middle-T-associated kinase activity was similar to that of the protein. In order to do this it is necessary to inhibit cellular protein synthesis during the chase period so that new middle-T cannot be produced. CV-1 cells infected for 48 h with the S5(25) virus were labelled with [35S]methionine and then incubated in medium containing excess unlabelled methionine and emetine. Lysates were prepared at the indicated times and immunoprecipitated with anti-PyT serum for direct analysis (Fig. 6A) or following incubation with [y-32P]ATP to assay middle-T-associated kinase activity (Fig. 6B). The half-life of middle-T protein in the emetine-treated CV-1 cells is about 4 h. Hence the inhibition by emetine of cellular protein synthesis appears not to affect the turnover of the middle-T polypeptide. The half-life of the protein kinase activity

170

A 12345 81-

B 12345 81-

6053-

40Fig 6. Stability of middle-T-associated protein kinase activity. (A) CV-1 cells infected 48 h previously ulth PSV S5(25) virus were labelled with [ “Slmethionine and then extracted immediately (lane 1) or incubated in the presence of excess unlabelled methionine and emetine (12 pg/ml) for 2 h (lane 2). 4 h (lane 3). 6 h (lane 4) or 8 h (lane 5). Cell lysates were immunoprecipitated with anti-PyT serum and analysed on a 10% polyacrylamide gel containing SDS. (B) Cell extracts used in A were immunoprecipitated with hamster anti-PyT serum and incubated with [y-“P]ATP prior to analysis on a SDS-containing 109 polyacrylamide gel. The order of lanes is the same as in A.

associated with middle-T is greater than that determined for the protein itself, and in all experiments is greater than 8 h (Fig. 6B). The combined effects of the toxicity of emetine and the virus infection prevent the turnover of the kinase activity from being followed significantly further. It appears that the middle-T associated with kinase activity is significantly more stable than the bulk fraction of the middle-T. We wished to investigate the nature of the kinase active fraction of middle-T in further detail. It has been shown previously that the kinase active form of middle-T in extracts from polyoma-infected or transformed cells runs on sucrose gradients with an apparent molecular weight of approximately 200000 (Schaffhausen and Benjamin. 1981b; Walter et al., 1982; Courtneidge and Smith, 1983). It has also been demonstrated that middle-T is phosphorylated at a low level in vivo (Schaff-

171 hausen and Benjamin, 1979. 1981a; Smith et al.. 1980; wished to determine whether the in vivo phosphorylation its acquisition of kinase activity.

Segawa and Ito. 1982). We of middle-T was related to

In uivo phosphoq-lution of middle-T antigerl CV-1 cells were infected for 48 h with 107 or S5(25) hybrid virus stocks and 3T6 cells were similarly infected with polyoma virus. Extracts were made from the infected ceils and immunoprecipitated with anti PyT or anti-SVT serum either following in vivo labelling with [“5S]methionine or “Pi. Fig. 7A shows the r7’S]methionine-labelled T antigens produced in these systems and clearly shows the

12345

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B

M

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M

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81

67

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Fig. 7. Immunoprecipitatlon Uninfected

(lanes

1 and

of

[ “5S]methionin~-labrlled

6) or polyoma-infected

and in viva

[ “P]phosphorylated

and 8) or PSV S5(25)- (lanes 4 and 9) or PSV 107- (lanes 5 and 10) infected CV-I with [35S]methionine serum (lanes I-5)

and cell extracts prepared.

or anti-SVT

serum (lanes 6-10)

Immunoprecipitated

and

kinase assay (lanes 7-9).

analysed

for

Phosphoamino

A.

(C)

acid analysis

and

then

analysed

on a 147

of in viva phohphorylated

from “‘P-lahelled

CV-1

analysla

of

middle-T

antigen

or phosphorylatcd

from

in the 111\ilro

the lahelled protein here left untrrlted

and analysed as in Fig. 2C.

polyacrylamide

middle-T

antigen.

gel containtng ” P-labelled

SIX.

middle-T

(1)) ~\a5

cells infected for 4X h with PSV 107 (lane 1) or PSV S5(25)

(lane 2). Lane 3 is a parallel analysis of SV40 large-T were prepared

Cleveland

” P,-lahelled cells (lanes 4-6)

containing

in viva and cell estract\~rrpared.

with 250 ng (lanes 2. 5. 8) or 10 pg (lanes 3, 6. 9) SIN~/~~~/O~.O~.~II.\ ~,WYII.~ VX

2 h at 37°C

immunoprecipitated

in

v.ith anti-P!T

and analysed on a 1OY polqacrylamick~l

Samples of gel slurry (50 ~1) containing

(lanes 1. 4. 7) or incubated protease

as

cells (lanes l-3).

(lanes 3

cells uere incubated

Equal aliquots were immunoprecipit~lted

SDS. (B) Parallel cell cultures to those used In A were lahelled with “Pi

[ ‘SS]methionine-lahelled

T antigens. (A)

(lanes 2 and 7) mouse 3T6 cells and uninfected

antigen prepared

from the umt‘

extract.

Samples

172 higher level of middle-T synthesis achieved within the CV-1 cells compared to the polyoma-infected cells. A striking feature of the experiment is the relatively high level of “P, incorporation into middle-T within CV-1 cells (Fig. 7B). Although the phosphate incorporation into middle-T is much less than that into SV40 large-T, it is elevated compared to the middle-T in 3T6 cells since, at this exposure, no phosphorylation of this protein is apparent in these cells. Previous studies, making use of deletion mutants (Segawa and Ito, 1982; Schaffhausen and Benjamin, 1979. 1981b), have been able to demonstrate in vivo phosphorylation of middle-T in polyoma-infected cells. Only partial mapping of the site(s) of phosphorylation has been carried out and the phosphorylation has been shown to be principally on serine residues. We have attempted Cleveland mapping with Stuphylococcus uureus V8 protease of the in vivo phosphorylated middle-T from CV-1 cells (see Fig. 7C). It is clear from the Cleveland maps that the in vivo phosphorylation sites are quite distinct from the major in vitro tyrosine phosphorylation sites. The in vivo phosphorylation sites are also separate from the two major stable [“5S]methionine peptides (A and B) which are derived from the N and C termini of the protein respectively (see Courtneidge et al., 1984). This narrows the potential major phosphorylated region to be between residues approximately 200 and 314. From further Cleveland analysis of various mutant middle-T antigens (A.E. Smith, unpublished observations) it is possible to refine this mapping to approximately residues 200-250 which is in accord with the previous observations that dl 8 and dl 45 retain in vivo phosphorylation sites (Segawa and Ito. 1982; Schaffhausen and Benjamin, 1981b). These mutants have deletions covering the region between approximately 250 and 300. The major amino acid phosphorylated in vivo in middle-T in the CV-1 (see Fig. 7D) and 3T6 cells is serine: however. a remarkably large number of serine residues (in fact 12 of 29 in the molecule) occur within the relevant region (serines 213, 218, 220. 227. 228, 230. 236, 243. 246. 251, 253 and 257). Further mapping will require obtaining specific mutants within the 200-250 region of middle-T and ultimately peptide sequence analysis. Relationship between the in uir?ophosphorylution of middle-T and kinase actic7itJ Sucrose gradient analysis of middle-T-containing extracts has allowed the discrimination of the fraction of middle-T with associated kinase activity away from the kinase inactive fraction. We have carried out this analysis of material from CV-1 cells and also examined how the in vivo phosphorylated form of middle-T sediments in such a gradient. The large bulk of the [‘5S]methionine-labelled middle-T in CV-1 extracts (see Fig. 8A) runs as a very high molecular weight complex (larger than the SV40 large-T-p53 complex) and the peak of middle-T-associated kinase activity runs at a size intermediate between the light and heavy forms of SV40 large-T (Fig. XD) which seems in accord with previous estimates of a molecular weight of about 200 000 for this form. Only a small fraction of the [ 35S]methionine-labelled middle-T co-sediments with the kinase activity. In contrast the in vivo phosphorylated fraction of middle-T appears to co-migrate with the bulk of the [‘5S]methionine-labelled middle-T (see Fig. 8C) and thus is largely distinct from the kinase activity. The data shown are using virus S5(25)-infected cell extracts but almost identical results were also obtained with virus 107.

A

1 3

5 7 9 11 131517

19 PS

M

B

1 3 5 7 9 11 13 15 17 19 P S

M

D

C

MT

Fig.

8. Immunoprecipitation

of sucrose gradient analyses of hybrid virus-infected CV-1 cells. (A) cell extracts prepared from CV-1 cells infected 48 h previously with the S5(25) virus stock were fractionated on linear 5--20% sucrose gradients as described in Materials and Methods. Aliquots (100 al) of every other fraction were co-immunoprecipitated with anti-PyT and anti-SVT sera (except for P and S tracks when only the appropriate single serum was used) and analysed on SDS-poiyac~lamide gels. Fraction 1 is at the bottom of the gradient. (Bt Same as A, exposed approximately 10 times longer. (C) fn vivo ‘2Pi-lahelied extract analysed as for A except that immunoprecipitation was with anti-PyT serum alone. (D) In vitro middle-T associated kinase activity. unlabelled extracts were fractionated and then immunoprecipitated with hamster anti-PyT serum and incubated with (y-‘*P]ATP prior to gel analysis. P and S indicate the initial extract immunoprecipitated with anti-PyT serum and anti-SVT serum respectively. M denotes the molecular weight marker track.

[ s5S]Methionine-lahelled

The expression level of middle-T in the CV-1 cells allows it to be observed readily in this gradient fraction following a simple immunoprecipitation procedure (i.e. no denaturing step is involved; (cf. Courtneidge and Smith, 1983). However, in the gradient fraction co-migrating with kinase activity, no major ~35S~methionine-labelled band could be seen to be co-immunoprecipitating with middle-T. A minor protein of subunit M, 20000 which does closely co-migrate with the kinase activity can be discerned on a long exposure of the [“5S]methionine-labelled samples (see Fig. 8B) and is immunoprecipitated by other middle-T-specific sera. This feature was also observed with virus 1074nfected cell extract. The relevance of this protein to the kinase activity is unknown.

174 Discussion Using SV40 late replacement vectors in association with defective helper SV40 genomes, we have obtained mixed virus stocks which express polyoma middle-T antigen in a lytic infection of CV-1 cells. In general terms it appears that the vectors containing the shortest extent of polyoma sequences preceding the initiation codon express the highest level of middle-T. As judged by both [j5S]methionine labelling and radioimmunoassay we obtained approximately 5-10 fold higher expression of middle-T in the CV-1 cells than in comparable polyoma infections of mouse 3T6 cells. The level of middle-T-associated kinase activity is not elevated in these cells; indeed, the ‘specific activity’ of the middle-T-associated kinase in CV-1 cells is substantially reduced compared to polyoma-infected 3T6 cells. These observations are in good general agreement with and extend those recently reported by Zhu et al. (1984). It seems likely to us that for middle-T to exhibit tyrosine kinase activity, it is not merely sufficient for middle-T and pp60’+” to co-exist within cells but that some further processing must occur. The low specific activity of the middle-T-associated protein kinase activity in CV-1 cells may reflect a difference in ~~60’~” between CV-1 cells and that found in rodent cells or may reflect a difference in a processing event of the two proteins to form a kinase active complex. It is of interest in this regard that Bolen et al. (1984) have recently reported that the interaction between middle-T and ~~60’.” in mouse cells leads to the stimulation of ~~60’.‘” tyrosyl kinase activity. It may be that a smaller stimulation of simian ~~60’.“’ activity occurs when bound to middle-T than occurs with mouse ~~60’.“‘. We have also explored certain other properties of the middle-T antigen expressed in CV-1 cells relative to those found in polyoma-infected mouse cells in order to characterise the expressed protein further and to attempt to explain the difference in kinase activity observed. Stability of middle-T protein und ussociated kinme uctiuit? Pulse-chase experiments suggested that the middle-T protein in CV-1 cells is less stable than in mouse 3T6 cells. We have also estimated the half-life of the protein kinase activity associated with middle-T by observing the decay of the activity in infected CV-1 cells incubated in the presence of an inhibitor of protein synthesis. The half-life of the protein kinase activity in these experiments is at least two-fold greater than that determined for the polypeptide by pulse-chase analysis both in the presence and absence of the inhibition of protein synthesis. This suggests that a stable subpopulation of middle-T molecules is associated with the kinase activity. Alternatively, it is possible that the residual middle-T is able to exchange rapidly into the pool possessing kinase activity since this appears to represent a minor portion of the total middle-T. However. this seems unlikely to us as we have shown that a significant level of middle-T can exist in CV-1 ceils (as high as is achieved in polyoma-infected 3T6 cells) without maximal expression of kinase activity. We are unaware of any previous studies showing a difference in the stability of middle-T protein and its associated kinase activity but in polyoma-infected cells such a study would be difficult due to the greater stability of the middle-T.

175 We do not know the reason for the relative instability of middle-T antigen in CV-1 cells compared to 3T6 cells. It may be due to the cell difference or reflect differences in the middle-T as a result of different associations with other proteins or different chemical modification or different subcellular location. The latter does not appear to be the case, at least in gross terms since the localization of middle-T in CV-1 cells that we (data not shown) and others (Zhu et al., 1984) have observed by indirect immunofluorescence is in good agreement with that observed by Dilworth (1982) by immunoelectronmicroscopic examination of polyoma-infected mouse cells. In oiuo phosphorylation of middle-T It appears that middle-T may be more highly phosphorylated in CV-1 cells compared to mouse 3T6 cells and, as a result of the higher level of expression, we have been able to characterize properties of the in vivo phosphorylated form further. Phospho-amino acid analysis shows that phosphoserine is the principal phosphorylated amino acid present. No phosphotyrosine was detected. Cleveland mapping data show that the major in vivo phosphorylation sites are upstream of the in vitro phosphorylation sites at tyrosine 315 and 322 since the C-terminal Staphylococcus aweus V8 peptide designated by us as fragment B (Harvey et al., 1984) is not phosphorylated in vivo. The N-terminal V8 peptide (M, = 24000) is also not phosphorylated in vivo (or in vitro). Although it is possible that different sites are phosphorylated in simian cells than in mouse cells these mapping studies are consistent with the previous studies using polyoma mutants dl 8, dl 45 and SD15 which are also phosphorylated in vivo. A large number of serine residues are present in the region of middle-T which is phosphorylated in vivo. Hence further mapping will be simplified by obtaining point and deletion mutants within the region covered by residues 200&250. It is interesting to note that the phosphorylation of SV40 large-T occurs in clusters of serine residues (Scheidtmann et al., 1982). Since the level of phosphorylation is low and the number of possible sites high, it is quite possible that the phosphorylation is adventitious. However, some specificity does appear to exist as to which middle-T molecules are phosphorylated. Sucrose gradient analysis of the in vivo phosphorylated form showed that it migrated with the bulk fraction of the [ ‘SS]methionine-labelled middle-T, principally as a very high molecular weight species. In contrast, the kinase active fraction of middle-T runs with an apparent molecular weight of approximately 200000 in a manner indistinguishable from that observed with polyoma-infected 3T6 cell extracts analysed in parallel. These data indicate that the in vivo phosphorylated form and the kinase active form of middle-T are both minor but are apparently unrelated. The role of the phosphorylation of middle-T remains to be determined. The expression of middle-T antigen using the SV40 late replacement vector represents a useful means for studying the phenotypes of mutant middle-T sequences produced by in vitro mutagenesis, which are non-transforming (in rodent cells) and non-viable when reconstructed back into polyoma virus. This system has been exploited for the expression and characterization of two sets of mutants in the vicinity of the NG-59 mutation and of the hydrophobic sequence towards the C-terminus of the protein. Results from these studies will be presented elsewhere.

176 Acknowledgements We thank Mr. J. Brock and the Photography Department at N.I.M.R. and Mrs. P. Thomas at A.V.R.I. for their assistance in the preparation of this manuscript. We also acknowledge the gifts of monoclonal antibodies from Dr. B.E. Griffin and Dr. G. Walter and plasmids from Dr. R. Kamen and Dr. C. Tyndall. G.J.B. gratefully acknowledges the support of a Medical Research Council Training Fellowship.

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177 Righy. P.W.J., Dieckmann. M., Rhodes, C. and Berg, P. (1977) Lahelling deoxyrthonucleic acid to high specific activity in vitro by nick translation with DNA polymerase 1. J. Mol. Biol. 113. 237-251. Schaffhausen. B.S. and Benjamin, T.L. (1979) Phosphorylation of polyoma T antigens. Cell 18, 9355946. Schaffhausen, B. and Benjamin. T.L. (1981a) Comparison of phosphorylation of two polyoma virus middle-T antigens in viva and in vitro. J. Viral. 40. 184-196. Schaffhausen. B. and Benjamin. T.L. (1981h) Protein kinase activity associated with polyoma middle-T antigen. In: Protein Phosphorylation (Rosen, O.M. and Krehs, E.G., eds.), Vol. 2. pp. 1281-129X. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. Scheidtmann. K.-H.. Echle. B. and Walter, G. (1982) Simian virus 40 large T antigen is phosphorylated at multiple sites clustered in two separate regions. J. Viral. 44. 116-133. Segawa, K. and Ito. Y. (1982) Differential subcellular localization of in viva-phosphorylated and non-phosphorylated middle-sized tumour antigen of polyoma virus and its relationship to middle-sized tumour antigen phosphorylating activity in vitro. Proc. Natl. Acad. Sci. U.S.A. 79. 6812-6816. Smith, A.E. and Ely. B.K. (1983) The biochemical basis of transformation by polyoma virus. Adv. Viral Oncol. 3, 3-30. Smith, A.E.. Smtth. R. and Paucha. E. (1978) Extraction and fingerprint analysis of simian virus 40 large and small T antigens. J. Viral. 2X. 140-153. Smith, A.E., Smith. R.. Griffin. B.E. and Fried. M. (1979) Protein kinase activity associated with polyoma virus middle T antigen in vitro. Cell 18. 915-924. Smith. A.E.. Fried, M.. Ito. Y.. Spurr. N. and Smith. R. (1980) Is middle-T of polyoma virus a protein kinase? Cold Spring Harbor Symp. Quant. Biol. 44, 141-147. Soberon. X.. Covarrubias, L. and Boliver, F. (1980) Construction and characterization of new cloning vehicles. iv. Deletion derivatives of pBR322 and pBR325. Gene 9, 2877305. Southern. E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 5033517. Tooze. J. (1980) DNA Tumour Viruses. Part 2, 2nd Edn. Cold Spring Harbor Laboratory, Cold Spring Harbor. NY. Treisman. R.H., Novak. U.. Favaloro, J. and Kamen, R. (1981) Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature (London) 292. 5955600. Walter. G., Hutchinson. M.A., Hunter. T. and Eckhart, W. (1982) Purification of polyoma virus middle-size tumor antigen by immunoaffinity chromatography. Proc. Natl. Acad. Sci. U.S.A. 79. 4025-4029. Zhu. Z.. Veldman, G.M.. Cowie, A., Carr, A., Schaffhausen, B. and Kamen. R. (1984) Construction and functional characterization of polyomavirus genomes that separately encode the three early proteins. J. Virol. 51. 170-180. (Manuscript

received

10 July 1985)