Gone, 93 (1990) 265-270 Elsevier
265
G E N E 03654
Synthesis, phosphorylation, and nuclear IocaHzafian of human papillomaviros E7 protein in ScA/zo-
saccharomyces pombe (Recombinant DNA; fission yeast; molecular cloning; yeast expression vector; viral protein; antiserum)
M. Tommasino', M. Contorni% V. Searlato% M. Buguoli% K. Maundrell b and F. Cavalieri" ° Molecular Virology and b Molecular Biology, Sclavo Research Centre, 53100 Siena (Italy) Received by J.K.C...Knowles: 8 January 1990 Revised: 19 March 1990 Accepted: 25 March 1990
SUMMARY
The complete E7 protein-encoding open reading frame of human papillomavims type 16 (HPV-16) was expressed in the fission yeast Schi:osaccharovnycespombe, under the control of a cloned yeast promoter. The HPV-16 E7 protein synthesized in $. pombe is a 17-kDa phosphoprotein which is recognized by anti-E7 antibodies (raised in rabbits against E7 fusion protein produced in EscherichiacolO.The mobility during sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of native E7 phosphoprotein synthesized in $. pombe is identical to that ofthe E7 phosphoprotein immunoprecipitated from human CaSki cells. Immunofluorescence staining showed that HPV-16 E7 phosphoprotein is localized in the nuclei of transformed $. ,vombe. These results indicate that E7 protein synthesized by S. pombe is apparently indistinguishable from HPV-16 E7 protein synthesized in higher eukaryotic cells expressing genes of HPV-16, and also that the phosphorylated, nuclear HPV-16 E7 protein is synthesized in S. pombe in a form compatible with its biological activity.
INTRODUCTION
Recent evidence implies a major role for human papillomavirus HPV-16 E7 gone product in the tumorigenic conversion of HPV-16 infected cells. The E7 ORF, together with the E6 ORF, is commonly retained and expressed in cervical carcinomas (Baker et al., 1987; Schwartz et al., 1985), and has been shown to immortalize and transform rodent fibroblasts (Kanda et al., 1988a,b) and extend the Correspondence to: Dr. F. Cavaiieri, Sclavo Research Centre, via Fiorentina 1, 53100 Siena (Italy) Tel. 39577-293235; Fax 39577-293493.
Abbreviations: aa, amino acid(s); ars, autonomous replication sequence; bp, base pair(s); cDNA, DNA complementary to mRNA; E7, gone (DNA) encoding protein E7 of HPV-16; HPV-16, human papillomavirus type 16; IgG, immunoglobulin G; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame; p, plasmid; PAGE, polyacrylamide-gel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SV40, simian virus 40; S., $¢ht:osaccharomyces; [ ], denotes plasmid-carrier state. 0378-1119/90/$03.50 © 1990ElsevierScience Publishers B.V.(BiomedicalDivision)
lifospan of human primary fibroblasts (Watanabe ctal., 1989). E7 gone expression stimulates DNA synthesis in rat 3YI cells (Sate ot al., 1989b) and cooperates with the activated Ha-ras gone product in the transformation of secondary rat fibroblasts (Chesters and McCance, 1989; Matlashewsky etal., 1987; Storey et al., 1988). This line of evidence has led to the notion of functional similarity between HPV-16 E7 gene product on the one hand, and two known nuclear viral oncoproteins: adenovirus Ela and SV40 large T antigen, on the other (Phelps et al., 1988; Voudsen and Parmjit, 1989). The HPV-16 E7 gene product is related to both E la and SV40 large T antigen at the aa level, and the region of homology has been implicated as necessary for binding to the RBI gene product (Dyson et al., 1989). Both structural and functional relatedness to Ela and SV40 largeT antigen argue that the HPV-16 E7 gone product can interferewith the controlof cellproliferation. Yeast has provided a valuable model system for the study
266
of higher eukaryotic gene function (Russell and Nurse, 1986b). Despite the great evolutionary distance between yeasts and higher eukaryotes, several instances have been reported in which homologous proteins or domains of proreins, can function interchangeably in yeast or mammalian cells. Such is the case for human ras and jun oncogene products in Saccharomyces cerevisiae(Kataoka et al., 1985; Struhl, 1987),and for the human pJ4CDC2 gene, which was cloned from a human eDNA library by complementation of $.pombe cell division control mutant, cdc2- (Lee and Nurse, 1987). The ability of the human p34CDC2 gene product to restore the prolife:ative functions of cdc2$. pombe suggests that at least some ofthe cell cycle control mechanisms of $. pombe and of higher eukaryotes have been remarkably conserved (Lee and Nurse, 1987). Interestingiy HPV-16 E? gene product shares a significant structural motif with another protein implicated in S. pombe cell division control, the cdc25 + gene product (Figge and Smith, 1988), which has been shown to affect the rate of entry of 8rowing yeast cells into mitosis by modulating the activity of the cdc2 + gene product (Russell and Nurse, 1986a). These considerations prompted us to establish the experimental conditions in which to study HPV-16 E7 gene function in 5. pombe. The purpose of this study was to establish conditions for correct transcription and translation of cloned HPV-16 E7 ORF in fission yeast, and to determine whether the E7 protein synthesized in S. pombe is similar to that produced in human cells (Smotkin and Wettstein, 1986; 1987). In addition, immunofluorescence has been used to see whether the HPV-16 E7 in S. pombe is targeted to the nucleus as it is in mammalian cells (Sate etal,, 1989a).
RESULTS AND DISCUSSION
(a) Correct transcription of HPV.16 E70RF in Schizo. sacchawm¥ces pomb¢ The fragment of the viral genome encoding the E7 protein was cloned into a high copy number, extrachromosomally replicating expression vector (pMBS21L; Fig. 1), and the resulting plasmid, pMBS21L/ET, was used to transform S. pombe leul-32 to Leu prototrophy. Transformant colonies contained approx. 75 ~o Leu ÷ cells, at an average copy number of approx. 30 plasmids per ceil. The pattern of HPV- 16 E7 gene transcription in 8. pombe was analyzed by Northern blot, to estimate the overall size of the transcript, and by S 1 nuclease protection, to map its 5' end (Fig. 2,A and B). Northern-blot analysis of transformed $. pombe RNAs was performed using E 7 0 R F [ 32P]DNA as probe. The E7 probe detected a single transcript of approx. 700 nt in RNA from pMBS21L/E7 trans-
A Ultlt
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HPVI6 GENOME
El
B ~ ~"'pBLUEecNPI' s~ ~a
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I I S! PROBE
Fig. I. Schematic representation of cloning strategies and final constructs. HPV-16 E? ORF was directly amplifiedby PCR from the HPV-16 •genome cloned in pBR322 (kindly provided by Dr. H. zur Hausen). Smal and Hindlll sites were added, respectively, upstream and downstream from the ORF via the following oligos (synthesized on Appfied Biosystems DNA Synthesizer): A = 5 ' - T ~ A T G C A T G . GAGATACACCTACATrG and B = 5 ' - A T A A ~ A T G G T r F CTGAGAACAGATG. Standard PCR reactions were performed with reagents of the Perkin-Elmer Cetus Gene-Amp kit. The amplified E7 DNA fragment was cloned into pBluescript (Strategene, La Jolla, CA) and the nt sequence was verified by dldeoxy chein-termination methods (Sanger etal., 19'/7). For expression in E. coil, the E 7 0 R F was cloned into the pEX34A expression vector, in frame with the N terminus of coliphage MS2 RNA polymerase (Klinkert et el., 1985; Nicosia et 81., 1987). For expression in 8. pombe, the E? ORF was cloned into the •$. pomb¢ expression vector pMBS21L. Thi~ vector contains the LEU2 selectable marker, the a•! replication origin from $. pombe (lesson and Lacroute, 1983) and the $. pombe nmtl promoter (Manndrell, 1990) which drives the transcription ofthe exosenous ORF. S I probe indicates the location of the 494-bp Hindlll fi'agment which was used as probe in SI protection experiments.
formants, while it did not react with any transcript in RNA from control cells transformed with pMBS21L alone (Fig. 2A, lanes a and b). S I nuclease protection analysis was performed using the 494-bp probe from pMBS21L/E7 which covers the 3' extremity of the yeast nmtl promoter and the entire E70RF. The fragment was excised from pMBS21L/E7 (Fig. 1) by H/ndlll digestion. [?-32P]ATP end labelled at the 5' extremities, and hybridized with total RNA from pMBS21L/ET-transformed S.pombe before digestion with S 1 nuclease. Analysis of the sizes of the protected fragments (Fig. 2B, lanes d, e, O shows that the E7 transcripts are initiated approx. 74 nt upstream from the start codon of the E70RF, at the expected site within the yeast promoter. Taken together these results indicate that transcription is initiated correctly, traverses the ET-coding region and terminates within the downstream ars sequence.
(b) Native HPV-16 E7 protein synthesis In Scbizosae' dmromyees pembe Having established that the E7 gene was correctly transcribed, we analyzed E7 protein synthesis in pMBS21L/E7 transformants by Western blot. The E7 ORF corresponds to the only possible gene product
Fig. 2.
a
267
b
a
bc
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kDa
4,46 4,30 4,21
kDa @200 4"92 4"69 4,46
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u
A
B
Fig. 2. Detectionand characterization of exogenous HPV-16E7 transcripts in S. pombe.The/eat.32 auxotrophic mutant of'$. pombewas transformed with the construct shown in Fig. i as described (Beach and Nurse, 1981). RNA was prepared from mid log.phase $.pombe cultures as described (Maundrell etal., 1985a,b) and analyzed by standard Northern (Maniatis et al., 1982,and Dupont Gane Screen Handbook)blot techniques and Sl nuclease digestion protection analysis(Scarlato etal., 1989; Maniatis et al., 1982).(Panel A) Formaldehyde(2.2 M) alptrose(I.2%) gel ~ i s and Northern-blot analysis. Lanes: a, 10pg total RNA from $.pombe[pMBS21L]; b, 10#8 total RNA born $.pombe[pMBS21L/E7]. Arrowheads specif~ the positions of 28S and 18S ribosomal RNA. The $mal-HImdlll E7 frs8mant purified born pBluescript/E7(see Fig. !)was nP-labeiled using random primers(Feinbergand Vogelstein,1984).(Panel B) S I protectionanalysis. Increasingamountsof'total $. pomfe RNA werehybridizedto 20 finol of 32p-end-labelled S l probe (see Fig. I). Lanes a and e, control samples without RNA. Control samples were processed identicallyto experimental samples (lanes d, e, f), without (lane a) or with (lane e) Sl nuclcase. Lane b, end-labelledMr markers; f/tnfl digest OfpBR32ZLanesd, e, f, Sl nuclease protection analysis performed in the presence of respectively 2, 4 and 6 ~8 RNA from $. pombe[pMBS21L/E7]. Smallarrow indicates the position of sell"annealed, fisll.langthprobe. Large arrow indicates the position of the approx. 374-nt-lon8p r o t e c t e d f r a g m e n t . Fill. 3. Detection and phosphorylationof HPV. 16 E7 protein synthesizedin $. pombe.Antibody preparation:pEX34A/E7 (Fill. i) was transformed into £, co//K-12dHldtrp (Kiinkert et al., 1985; Strebel et al., 1986).The fission protein was purified and used to immunize rabbits (NicoW et al., 1987). Antiserum specificity was verified by its ability to immunoprecipitetethe HPV-16 E7 protein from CaSki cell lysate, (Smotkin and Wemtetn, 1987). (Panel A)Western.blot analysis.Total cellular protein was recovered I~om the phenolic phaseof the samples processedfor RNA purification by cold acetoneprecipitation. Protein concentrationwas estimatedwith BCA Protein Assay Reagent(Pierce)and equal amounts(100~8) of total proteinswere analysedby 0.1% SDS-12% PAGE (Laemmli, 1970).Separatedproteins were screenedby Western.blot technique(Burnette, 1981)with either rabbit anti.MS2/E'/fusion protein (specificantiserum)or preimmunerabbit serum(negativecontrol) and peroxidaseconjulpttedgoat anti.rabbit lgO antiserum (cuppel). M0 prestainedMr markers (Rainbow, Amersham); their size in kDa is indicated on the right. Lanes: a, total cellular protein extracted $.pombe[pMBS21L]; b, total ceUular protein from $.pombt|pMBS21L/E7]. (Panel B)lmmunoprecipitation. Cultures of $.pombe[pMBS21L] or 5. pombe[pMBS21L/E7] and of human CaSki cells were radiolabelledwith npo43", and total protein extracts were prepared as described(Simanis and Nurse, 1986;Smotkin and Wettstein, 1986;1987).Total cellularprotein extractswereimmunoprecipitatedwith anti-MS21E7fissionprotein polyclonal antiserum0.1% SDS-12% PAGE was performed usingequal amountsof incorporated radiolabeland the gel was dried and autoradiographed.The size of M, markers is indicatedon the right margin(kDa). Lanes:a, 5. pombe[pMBS21L]protein extract; b, $. pombe[pMBS21LIE7] protein extract; e, CaSki protein extract.
encoded by the cloned fragment. For Western blotting we employed antisera raised in rabbits against coliphage MS2 RNA polymerase/E7 fusion protein previously produced in E. coli (see legend Fig. 3). Total protein extracts were prepared from pMBS21L or pMBS21L/E7 transformed $. pombe cultures and equal amounts were resolved on SDS-polyacrylamide gels, electrotransferred to nitrocellulose filters and reacted with immune serum from rabbits
immunized with MS2/E7 fusion protein produced in E. coli. Consistent with the RNA analysis (Fig. 2), a single, approx. 17-kDa protein band was detected in the pMBS21L/ E7-transformed colonies (Fig. 3A, lane b). This protein was not detected by anti-MS2/E7 antiserum in negative control S.pom~[pMBS21L] 0ane a), nor was it detected by preimmune rabbit serum in either transformants (not shown).
268 (¢) Pheslp~lafleu and nuclear Ioc811nflon of HPV.16 E7 p r o ~ synthesized in SeAizosacclunemyeespombe To assess how closely the HPV-16 E7 gene product expressed in S, pombe resembles the same protein expressed in higher eukaryotes, we analyzed its phos-
Fi~4. Nuclear localization of E7 protein in $.pombe. $.pom. be[pMBS21L] or $. pombe[pMBS21L/E?] cultures were grown to mid Io8 phase, fixed with formaldehyde.slutaraldehyde, and permeabilized with mutenase (Nero-Enzyme Products, U.K.) and zymolase (Seikagaku Kogyo Company, Ltd.) exactly as described (Hagan and Hyams, 1988)~ Permeabilized protoplasts were allowed to adhere to po|ylysine-©oated slats ceverslips for approx. 30 rain. The coverslips were extensively washed with PBS (130 ram Na~/6.4 ram Na=HPO4.12H=O/2.6 ram K~/I.4 ram KH=PO4 pH 7.4) and then incubated for I h at 25°C with rabbit anU-MS2/E7 fusion protein.spe¢ii|c antiserum or with rabbit prelmraune control serum. Treated coversHps w.-re extensively washed with PBS and incubated with rhodamine-©onjugated8oat anti-rabbit 18t3 antiserum (Cappel) for 20 rain at 25°C. The stained coversHps were examined on a Con-focal Fluorescence lmagi~ System MRC-$00 BioRad microscope. (Panels a and b) $. pombe[pMBS21L/E7], two separate experiments; (panel ¢) $. pombe[pMBS21L], The bar represents 10 #m.
phorylation and subeellular localization. When expressed in human cells, the HPV-16 E7 protein is phosphorylated on Ser residues (Smotkin and Wettstein, 1987). Moreover, immunofluorescence staining of monkey COS-I cells expressing HPV-16 E7 protein from an SV40 promoter demonstrated the nuclear localization of the protein (Sate et al., 1989a). The association of the HPV-16 E7 protein with cell nuclei appears to be rather weak, since attempts at cell frac~onation prior to immunoprecipitation were reported to result in the release of the protein into the cytoplasmic fraction (Sate et al., 1989a; Smotkin and Wettstein, 1987). To assay the phosphorylation state of HPV- 16 E7 protein in fission yeast, $. pombe transformants were labelled for I h with 32p and cell lysates were prepared and immuneprecipitated using polyclonal antiserum to E7 fusion protein (Fig. 3B). As control, human CaSki cells harbouring HPV-16 were 32P-labelled and processed in parallel. Specific anti E7 fusion protein serum detected phosphoprotein bands of identical mobility (approx. 17 kDa) in both S. pombe[pMBS2 IL/ET] and CaSki cells (Fig. 3B, lanes b, c). This phosphoprotein was not detectable in immuneprecipitates from S. pombe[pMBS21L] (lane a). A second phosphoprotein of slightly higher mobility was also detected by anti MS2/E7 antiserum in S.pombe[pMBS21L/ET]. The absence of this protein in cells transformed with pMBS21L and the presence of full-length E7 messenger RNA only in $.pombe[pMBS21L/ET] (Fig. 2B) suggest that the lower band is due either to processing of E7 or to the presence of a protein coprecipitared with E7. Both hypotheses are being investigated. We determined the subeellular localization of HPV.16 E7 protein in S.pombe[pMBS21LIE7] colonies by immunofluorescence staining, which showed strong nuclear staining, contrasting with a much weaker cytoplasmic staining (Fig. 4) which appeared to be somewhat more intense at the extremities of the cells. No staining was detectable in either S. pombe[pMBS21L] reacted with the specific antiserum (Fig. 4) or in either transformants reacted with rabbit preimmune serum (data not shown). In addition, preliminary immunoblot analysis of nuclear and cytoplasmic fractions of $. pombe[pMBS21L/ET] showed roughly equal distribution of E7 protein between the two cellular compartments (not shown). In conclusion HPV-16 E7 phosphoprotein synthesized in S. pombe, on the bases of size, phosphorylation and subcellular localization, correlates well with the properties of the HPV-16 E7 phosphoprotein produced in higher eukaryotes.
(d) Conclusions Expression of foreign genes in yeast is especially useful when applied to genes whose expression in the natural host cell is either very poor or difficult to reproduce in tissue culture, as is the case with HPV-16 genes in inf~ted human
269
"keratinocytes. We report the expression of HPV-16 E7 O R F from a yeast promoter in $. pombe. Our data demonstrate that the native HPV-16 E7 phosphoprotein synthesized in $. pombe is a 17-kDa phosphoprotein which has the same electrophorefic mobility in S D S - P A G E as the E7 phosphoprotein produced in mammalian cells. Immunofluorescence shows that the E7 gene product in fission yeast is targeted to the nucleus consistent with current data concerning the subcellular localization of E7 in higher eukaryotes (Barbosa et al., 1989; Dyson et al., 1989; Sato etal., 1989a). We therefore conclude that $. pombe correctly produces HPV-16 E7 protein which displays characteristic features of the protein produced in mammalian cells, at higher levels.
ACKNOWLEDGEMENTS The authors wish to thank A. Covacci for providing reagents and useful discussion, M. Melli and R. Rappuoli for critical evaluation of the data, G. Corsi for artwork and A. Mori for typing.
REFERENCES
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Kataoka,T.,Powers,S.,Cameron, S.,Famum, O.oOoldfmb, M., J.and Wiglet,M.: Funmimml Immolo~ nfmmmalion mul y e a nu genes.Cell40 (1985) 19-26~ Klinkert, M., Hmuuum, R. and Scballer, H.: Surfaceiwoteins o t ' ~ ~ ~ identiSed from an ~ ,=a ~ plasmid Ubrary. Infect. hnmun. 49 (1985) 329-335. Laemmli, U.K.: Cleavap of' structural proteins dmh~ the acmnldy ot the head of b a c t e ~ T4. Nature 227 (1970) 680-685. Lee, M.G. and Nurse, P.: C o m l ~ t a t i o n used to clone • human homolosueofthe fissionyeast cell cyclecootrolIpme~1~.2.Nature 327 (1987) 31-35. Louon, R. and Laeroute, F.: Piasmid citr~in8 the yeast OMP decarboxylase structural and regulatory Sines: ~ re8ulation in a foreignenvironment.Cell 32 (1983) 371-377. Man]axis, 1"., Fritsch, F..F. and Sambronk, J.: Moleudar Clonins. A Laboratory Manual. Cold Sprin8 Harbor Laboratory, Cold Sprbt8 Harbor, NY, 1982. Madashewski, G., Schneider,J., Banks, L., Jones~N., Murray, A. and Crawford, L: Human pepillomavirus t3q~ 16 DNA cooperateswith activated ~s in transforming p ~ cells. EMBO J. 6 (1987) 1741-1746. Maundrell, K.: rim/! offission yeast:a highlytranscribedlleneconJpletdy repressed in thiamine. J. Biol. Chem. (1990) in press. Maunclrell, K., Wight, A.P.H., Piper, M. and Shall, S.: Evaluation of heterologousARS activity in S. ~ e usingcloned DNA ftmn $. pomP. Nucleic Acids Res. 13 (1985a) 3711-3722. Maondrell, K., Nurse, P., Sch6nholzer, F. and Schweinsruber, M.13.: Cloning and characterization of'twoipmesrestorins acid phosphatsse activity in pAol- mutants of' $cTd.-omcc~romyc~pombe. Gem 39 (1985b) 223-230. Nicosia, A., Bartoloni, A., Perusini, M. and Rsppuoli, it.: Expressionand immunolo8ical properties of the five subonits of the penu,is toxin. Infect. Immon. $5 (1987) 963-967. Phelps, W.C., Yeo, C.L., Munipr, C.. and Howley, P.M.: The human pepillomavirus type 16 137ipme encod0s transa¢tivation and transFormingFunctionssimilar to those nf adcnoviru8 131a.Cell 53 (1988) 539-547. Russell, P. and Nurse, P.: ¢dc2.5Functions u an inducer in the mytotic control of fission yeast. Cell 45 (1986a) 145-153. Russell, P. and Nurse, P.: $¢Alro.m¢¢Aa~spombe and Sacc~romy~s cere~iae: a look at yeast divided. Cell 45 (1986b) 781-782. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencingwith chldn. terminating inhibitors. Proc, Natl. Acsd. Sci. USA 74 (1977)
5463-$467. Sate, H., Watanabe, S., Furuno, A. and Yoshiike,K.: Human papillomavirus type 16 E7 protein expressed in £. co~and monkeyCOS-I cells: immunofiuorescence detection of' the nuclear 137 protein. Virology 170 (1989a) 311-315. Sate, H., Furuno, A. and Yoshiike, K.: Expression nfhuman papilloma~ virus type 161378oneinduces DNA synthesisof'rat 3'/I cells.Virology 168 (1989b) 195-199. Scarlato, V., Storlazzi, A., Garsano, S. and Cascino, A.: Bacteriophqle 1"4 late gone expression: ovedappin8 promoters direct diveqpnt transcription of the base plate gone cluster. ViroloiD' 171 (1989) 4"/$-483. Schwarz, E., Fresco, U.K., Gissmaan, L, Mayer, W., Roggenbuck,B., Stremlau, A. and zur Hanson, H.: Structure and transcription of human pepillomaviruses sequences in cervical carcinoma cells. Nature 314 (1985) 111-114. Simanis, V. and Nurse, P.: The cell cyclecontrol 8eriecdc2of'fissionyeast encode~ a protein kinase potentially regulated by phosphor~tation. Cell 45 (1986) 261-268. Smotkin, D. and Wettstein, P.O.: Transcription of'humanpapillomavirus type 16 early genes in a cervical cancer and a cancer.derived cell line
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265
G E N E 03654
Synthesis, phosphorylation, and nuclear IocaHzafian of human papillomaviros E7 protein in ScA/zo-
saccharomyces pombe (Recombinant DNA; fission yeast; molecular cloning; yeast expression vector; viral protein; antiserum)
M. Tommasino', M. Contorni% V. Searlato% M. Buguoli% K. Maundrell b and F. Cavalieri" ° Molecular Virology and b Molecular Biology, Sclavo Research Centre, 53100 Siena (Italy) Received by J.K.C...Knowles: 8 January 1990 Revised: 19 March 1990 Accepted: 25 March 1990
SUMMARY
The complete E7 protein-encoding open reading frame of human papillomavims type 16 (HPV-16) was expressed in the fission yeast Schi:osaccharovnycespombe, under the control of a cloned yeast promoter. The HPV-16 E7 protein synthesized in $. pombe is a 17-kDa phosphoprotein which is recognized by anti-E7 antibodies (raised in rabbits against E7 fusion protein produced in EscherichiacolO.The mobility during sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of native E7 phosphoprotein synthesized in $. pombe is identical to that ofthe E7 phosphoprotein immunoprecipitated from human CaSki cells. Immunofluorescence staining showed that HPV-16 E7 phosphoprotein is localized in the nuclei of transformed $. ,vombe. These results indicate that E7 protein synthesized by S. pombe is apparently indistinguishable from HPV-16 E7 protein synthesized in higher eukaryotic cells expressing genes of HPV-16, and also that the phosphorylated, nuclear HPV-16 E7 protein is synthesized in S. pombe in a form compatible with its biological activity.
INTRODUCTION
Recent evidence implies a major role for human papillomavirus HPV-16 E7 gone product in the tumorigenic conversion of HPV-16 infected cells. The E7 ORF, together with the E6 ORF, is commonly retained and expressed in cervical carcinomas (Baker et al., 1987; Schwartz et al., 1985), and has been shown to immortalize and transform rodent fibroblasts (Kanda et al., 1988a,b) and extend the Correspondence to: Dr. F. Cavaiieri, Sclavo Research Centre, via Fiorentina 1, 53100 Siena (Italy) Tel. 39577-293235; Fax 39577-293493.
Abbreviations: aa, amino acid(s); ars, autonomous replication sequence; bp, base pair(s); cDNA, DNA complementary to mRNA; E7, gone (DNA) encoding protein E7 of HPV-16; HPV-16, human papillomavirus type 16; IgG, immunoglobulin G; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame; p, plasmid; PAGE, polyacrylamide-gel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SV40, simian virus 40; S., $¢ht:osaccharomyces; [ ], denotes plasmid-carrier state. 0378-1119/90/$03.50 © 1990ElsevierScience Publishers B.V.(BiomedicalDivision)
lifospan of human primary fibroblasts (Watanabe ctal., 1989). E7 gone expression stimulates DNA synthesis in rat 3YI cells (Sate ot al., 1989b) and cooperates with the activated Ha-ras gone product in the transformation of secondary rat fibroblasts (Chesters and McCance, 1989; Matlashewsky etal., 1987; Storey et al., 1988). This line of evidence has led to the notion of functional similarity between HPV-16 E7 gene product on the one hand, and two known nuclear viral oncoproteins: adenovirus Ela and SV40 large T antigen, on the other (Phelps et al., 1988; Voudsen and Parmjit, 1989). The HPV-16 E7 gene product is related to both E la and SV40 large T antigen at the aa level, and the region of homology has been implicated as necessary for binding to the RBI gene product (Dyson et al., 1989). Both structural and functional relatedness to Ela and SV40 largeT antigen argue that the HPV-16 E7 gone product can interferewith the controlof cellproliferation. Yeast has provided a valuable model system for the study
266
of higher eukaryotic gene function (Russell and Nurse, 1986b). Despite the great evolutionary distance between yeasts and higher eukaryotes, several instances have been reported in which homologous proteins or domains of proreins, can function interchangeably in yeast or mammalian cells. Such is the case for human ras and jun oncogene products in Saccharomyces cerevisiae(Kataoka et al., 1985; Struhl, 1987),and for the human pJ4CDC2 gene, which was cloned from a human eDNA library by complementation of $.pombe cell division control mutant, cdc2- (Lee and Nurse, 1987). The ability of the human p34CDC2 gene product to restore the prolife:ative functions of cdc2$. pombe suggests that at least some ofthe cell cycle control mechanisms of $. pombe and of higher eukaryotes have been remarkably conserved (Lee and Nurse, 1987). Interestingiy HPV-16 E? gene product shares a significant structural motif with another protein implicated in S. pombe cell division control, the cdc25 + gene product (Figge and Smith, 1988), which has been shown to affect the rate of entry of 8rowing yeast cells into mitosis by modulating the activity of the cdc2 + gene product (Russell and Nurse, 1986a). These considerations prompted us to establish the experimental conditions in which to study HPV-16 E7 gene function in 5. pombe. The purpose of this study was to establish conditions for correct transcription and translation of cloned HPV-16 E7 ORF in fission yeast, and to determine whether the E7 protein synthesized in S. pombe is similar to that produced in human cells (Smotkin and Wettstein, 1986; 1987). In addition, immunofluorescence has been used to see whether the HPV-16 E7 in S. pombe is targeted to the nucleus as it is in mammalian cells (Sate etal,, 1989a).
RESULTS AND DISCUSSION
(a) Correct transcription of HPV.16 E70RF in Schizo. sacchawm¥ces pomb¢ The fragment of the viral genome encoding the E7 protein was cloned into a high copy number, extrachromosomally replicating expression vector (pMBS21L; Fig. 1), and the resulting plasmid, pMBS21L/ET, was used to transform S. pombe leul-32 to Leu prototrophy. Transformant colonies contained approx. 75 ~o Leu ÷ cells, at an average copy number of approx. 30 plasmids per ceil. The pattern of HPV- 16 E7 gene transcription in 8. pombe was analyzed by Northern blot, to estimate the overall size of the transcript, and by S 1 nuclease protection, to map its 5' end (Fig. 2,A and B). Northern-blot analysis of transformed $. pombe RNAs was performed using E 7 0 R F [ 32P]DNA as probe. The E7 probe detected a single transcript of approx. 700 nt in RNA from pMBS21L/E7 trans-
A Ultlt
I
IM
ET I
HPVI6 GENOME
El
B ~ ~"'pBLUEecNPI' s~ ~a
~e~L.e7
~1
Idl~
~X~
I I S! PROBE
Fig. I. Schematic representation of cloning strategies and final constructs. HPV-16 E? ORF was directly amplifiedby PCR from the HPV-16 •genome cloned in pBR322 (kindly provided by Dr. H. zur Hausen). Smal and Hindlll sites were added, respectively, upstream and downstream from the ORF via the following oligos (synthesized on Appfied Biosystems DNA Synthesizer): A = 5 ' - T ~ A T G C A T G . GAGATACACCTACATrG and B = 5 ' - A T A A ~ A T G G T r F CTGAGAACAGATG. Standard PCR reactions were performed with reagents of the Perkin-Elmer Cetus Gene-Amp kit. The amplified E7 DNA fragment was cloned into pBluescript (Strategene, La Jolla, CA) and the nt sequence was verified by dldeoxy chein-termination methods (Sanger etal., 19'/7). For expression in E. coil, the E 7 0 R F was cloned into the pEX34A expression vector, in frame with the N terminus of coliphage MS2 RNA polymerase (Klinkert et el., 1985; Nicosia et 81., 1987). For expression in 8. pombe, the E? ORF was cloned into the •$. pomb¢ expression vector pMBS21L. Thi~ vector contains the LEU2 selectable marker, the a•! replication origin from $. pombe (lesson and Lacroute, 1983) and the $. pombe nmtl promoter (Manndrell, 1990) which drives the transcription ofthe exosenous ORF. S I probe indicates the location of the 494-bp Hindlll fi'agment which was used as probe in SI protection experiments.
formants, while it did not react with any transcript in RNA from control cells transformed with pMBS21L alone (Fig. 2A, lanes a and b). S I nuclease protection analysis was performed using the 494-bp probe from pMBS21L/E7 which covers the 3' extremity of the yeast nmtl promoter and the entire E70RF. The fragment was excised from pMBS21L/E7 (Fig. 1) by H/ndlll digestion. [?-32P]ATP end labelled at the 5' extremities, and hybridized with total RNA from pMBS21L/ET-transformed S.pombe before digestion with S 1 nuclease. Analysis of the sizes of the protected fragments (Fig. 2B, lanes d, e, O shows that the E7 transcripts are initiated approx. 74 nt upstream from the start codon of the E70RF, at the expected site within the yeast promoter. Taken together these results indicate that transcription is initiated correctly, traverses the ET-coding region and terminates within the downstream ars sequence.
(b) Native HPV-16 E7 protein synthesis In Scbizosae' dmromyees pembe Having established that the E7 gene was correctly transcribed, we analyzed E7 protein synthesis in pMBS21L/E7 transformants by Western blot. The E7 ORF corresponds to the only possible gene product
Fig. 2.
a
267
b
a
bc
de
f
M
kDa
4,46 4,30 4,21
kDa @200 4"92 4"69 4,46
4,30
4,14 4, 21 4, 14 ~i~i¸ : •~ .~'•
A
u
A
B
Fig. 2. Detectionand characterization of exogenous HPV-16E7 transcripts in S. pombe.The/eat.32 auxotrophic mutant of'$. pombewas transformed with the construct shown in Fig. i as described (Beach and Nurse, 1981). RNA was prepared from mid log.phase $.pombe cultures as described (Maundrell etal., 1985a,b) and analyzed by standard Northern (Maniatis et al., 1982,and Dupont Gane Screen Handbook)blot techniques and Sl nuclease digestion protection analysis(Scarlato etal., 1989; Maniatis et al., 1982).(Panel A) Formaldehyde(2.2 M) alptrose(I.2%) gel ~ i s and Northern-blot analysis. Lanes: a, 10pg total RNA from $.pombe[pMBS21L]; b, 10#8 total RNA born $.pombe[pMBS21L/E7]. Arrowheads specif~ the positions of 28S and 18S ribosomal RNA. The $mal-HImdlll E7 frs8mant purified born pBluescript/E7(see Fig. !)was nP-labeiled using random primers(Feinbergand Vogelstein,1984).(Panel B) S I protectionanalysis. Increasingamountsof'total $. pomfe RNA werehybridizedto 20 finol of 32p-end-labelled S l probe (see Fig. I). Lanes a and e, control samples without RNA. Control samples were processed identicallyto experimental samples (lanes d, e, f), without (lane a) or with (lane e) Sl nuclcase. Lane b, end-labelledMr markers; f/tnfl digest OfpBR32ZLanesd, e, f, Sl nuclease protection analysis performed in the presence of respectively 2, 4 and 6 ~8 RNA from $. pombe[pMBS21L/E7]. Smallarrow indicates the position of sell"annealed, fisll.langthprobe. Large arrow indicates the position of the approx. 374-nt-lon8p r o t e c t e d f r a g m e n t . Fill. 3. Detection and phosphorylationof HPV. 16 E7 protein synthesizedin $. pombe.Antibody preparation:pEX34A/E7 (Fill. i) was transformed into £, co//K-12dHldtrp (Kiinkert et al., 1985; Strebel et al., 1986).The fission protein was purified and used to immunize rabbits (NicoW et al., 1987). Antiserum specificity was verified by its ability to immunoprecipitetethe HPV-16 E7 protein from CaSki cell lysate, (Smotkin and Wemtetn, 1987). (Panel A)Western.blot analysis.Total cellular protein was recovered I~om the phenolic phaseof the samples processedfor RNA purification by cold acetoneprecipitation. Protein concentrationwas estimatedwith BCA Protein Assay Reagent(Pierce)and equal amounts(100~8) of total proteinswere analysedby 0.1% SDS-12% PAGE (Laemmli, 1970).Separatedproteins were screenedby Western.blot technique(Burnette, 1981)with either rabbit anti.MS2/E'/fusion protein (specificantiserum)or preimmunerabbit serum(negativecontrol) and peroxidaseconjulpttedgoat anti.rabbit lgO antiserum (cuppel). M0 prestainedMr markers (Rainbow, Amersham); their size in kDa is indicated on the right. Lanes: a, total cellular protein extracted $.pombe[pMBS21L]; b, total ceUular protein from $.pombt|pMBS21L/E7]. (Panel B)lmmunoprecipitation. Cultures of $.pombe[pMBS21L] or 5. pombe[pMBS21L/E7] and of human CaSki cells were radiolabelledwith npo43", and total protein extracts were prepared as described(Simanis and Nurse, 1986;Smotkin and Wettstein, 1986;1987).Total cellularprotein extractswereimmunoprecipitatedwith anti-MS21E7fissionprotein polyclonal antiserum0.1% SDS-12% PAGE was performed usingequal amountsof incorporated radiolabeland the gel was dried and autoradiographed.The size of M, markers is indicatedon the right margin(kDa). Lanes:a, 5. pombe[pMBS21L]protein extract; b, $. pombe[pMBS21LIE7] protein extract; e, CaSki protein extract.
encoded by the cloned fragment. For Western blotting we employed antisera raised in rabbits against coliphage MS2 RNA polymerase/E7 fusion protein previously produced in E. coli (see legend Fig. 3). Total protein extracts were prepared from pMBS21L or pMBS21L/E7 transformed $. pombe cultures and equal amounts were resolved on SDS-polyacrylamide gels, electrotransferred to nitrocellulose filters and reacted with immune serum from rabbits
immunized with MS2/E7 fusion protein produced in E. coli. Consistent with the RNA analysis (Fig. 2), a single, approx. 17-kDa protein band was detected in the pMBS21L/ E7-transformed colonies (Fig. 3A, lane b). This protein was not detected by anti-MS2/E7 antiserum in negative control S.pom~[pMBS21L] 0ane a), nor was it detected by preimmune rabbit serum in either transformants (not shown).
268 (¢) Pheslp~lafleu and nuclear Ioc811nflon of HPV.16 E7 p r o ~ synthesized in SeAizosacclunemyeespombe To assess how closely the HPV-16 E7 gene product expressed in S, pombe resembles the same protein expressed in higher eukaryotes, we analyzed its phos-
Fi~4. Nuclear localization of E7 protein in $.pombe. $.pom. be[pMBS21L] or $. pombe[pMBS21L/E?] cultures were grown to mid Io8 phase, fixed with formaldehyde.slutaraldehyde, and permeabilized with mutenase (Nero-Enzyme Products, U.K.) and zymolase (Seikagaku Kogyo Company, Ltd.) exactly as described (Hagan and Hyams, 1988)~ Permeabilized protoplasts were allowed to adhere to po|ylysine-©oated slats ceverslips for approx. 30 rain. The coverslips were extensively washed with PBS (130 ram Na~/6.4 ram Na=HPO4.12H=O/2.6 ram K~/I.4 ram KH=PO4 pH 7.4) and then incubated for I h at 25°C with rabbit anU-MS2/E7 fusion protein.spe¢ii|c antiserum or with rabbit prelmraune control serum. Treated coversHps w.-re extensively washed with PBS and incubated with rhodamine-©onjugated8oat anti-rabbit 18t3 antiserum (Cappel) for 20 rain at 25°C. The stained coversHps were examined on a Con-focal Fluorescence lmagi~ System MRC-$00 BioRad microscope. (Panels a and b) $. pombe[pMBS21L/E7], two separate experiments; (panel ¢) $. pombe[pMBS21L], The bar represents 10 #m.
phorylation and subeellular localization. When expressed in human cells, the HPV-16 E7 protein is phosphorylated on Ser residues (Smotkin and Wettstein, 1987). Moreover, immunofluorescence staining of monkey COS-I cells expressing HPV-16 E7 protein from an SV40 promoter demonstrated the nuclear localization of the protein (Sate et al., 1989a). The association of the HPV-16 E7 protein with cell nuclei appears to be rather weak, since attempts at cell frac~onation prior to immunoprecipitation were reported to result in the release of the protein into the cytoplasmic fraction (Sate et al., 1989a; Smotkin and Wettstein, 1987). To assay the phosphorylation state of HPV- 16 E7 protein in fission yeast, $. pombe transformants were labelled for I h with 32p and cell lysates were prepared and immuneprecipitated using polyclonal antiserum to E7 fusion protein (Fig. 3B). As control, human CaSki cells harbouring HPV-16 were 32P-labelled and processed in parallel. Specific anti E7 fusion protein serum detected phosphoprotein bands of identical mobility (approx. 17 kDa) in both S. pombe[pMBS2 IL/ET] and CaSki cells (Fig. 3B, lanes b, c). This phosphoprotein was not detectable in immuneprecipitates from S. pombe[pMBS21L] (lane a). A second phosphoprotein of slightly higher mobility was also detected by anti MS2/E7 antiserum in S.pombe[pMBS21L/ET]. The absence of this protein in cells transformed with pMBS21L and the presence of full-length E7 messenger RNA only in $.pombe[pMBS21L/ET] (Fig. 2B) suggest that the lower band is due either to processing of E7 or to the presence of a protein coprecipitared with E7. Both hypotheses are being investigated. We determined the subeellular localization of HPV.16 E7 protein in S.pombe[pMBS21LIE7] colonies by immunofluorescence staining, which showed strong nuclear staining, contrasting with a much weaker cytoplasmic staining (Fig. 4) which appeared to be somewhat more intense at the extremities of the cells. No staining was detectable in either S. pombe[pMBS21L] reacted with the specific antiserum (Fig. 4) or in either transformants reacted with rabbit preimmune serum (data not shown). In addition, preliminary immunoblot analysis of nuclear and cytoplasmic fractions of $. pombe[pMBS21L/ET] showed roughly equal distribution of E7 protein between the two cellular compartments (not shown). In conclusion HPV-16 E7 phosphoprotein synthesized in S. pombe, on the bases of size, phosphorylation and subcellular localization, correlates well with the properties of the HPV-16 E7 phosphoprotein produced in higher eukaryotes.
(d) Conclusions Expression of foreign genes in yeast is especially useful when applied to genes whose expression in the natural host cell is either very poor or difficult to reproduce in tissue culture, as is the case with HPV-16 genes in inf~ted human
269
"keratinocytes. We report the expression of HPV-16 E7 O R F from a yeast promoter in $. pombe. Our data demonstrate that the native HPV-16 E7 phosphoprotein synthesized in $. pombe is a 17-kDa phosphoprotein which has the same electrophorefic mobility in S D S - P A G E as the E7 phosphoprotein produced in mammalian cells. Immunofluorescence shows that the E7 gene product in fission yeast is targeted to the nucleus consistent with current data concerning the subcellular localization of E7 in higher eukaryotes (Barbosa et al., 1989; Dyson et al., 1989; Sato etal., 1989a). We therefore conclude that $. pombe correctly produces HPV-16 E7 protein which displays characteristic features of the protein produced in mammalian cells, at higher levels.
ACKNOWLEDGEMENTS The authors wish to thank A. Covacci for providing reagents and useful discussion, M. Melli and R. Rappuoli for critical evaluation of the data, G. Corsi for artwork and A. Mori for typing.
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