Nucleic Acids Research, Vol. 19, No. 16 4531-4535
The constitutively expressed octamer binding protein OTF-1 and a novel octamer binding protein expressed specifically in cervical cells bind to an octamer-related sequence in the human papillomavirus 16 enhancer C.L.Dent, G.A.J.Mcindoel and D.S.Latchman* Medical Molecular Biology Unit, Department of Biochemistry and 'ICRF Human Tumour Immunology Unit, University College and Middlesex School of Medicine, London, UK Received March 26, 1991; Revised and Accepted July 17, 1991
ABSTRACT A novel octamer binding protein expressed specifically in cervical cells but not in other cell types has been identified. This protein differs in size and sequence specificity from the constitutively expressed octamer binding protein OTF-1. In particular it binds with higher affinity to a sequence in the human papillomavirus 16 (HPV) upstream regulatory region which has a seven out of eight base pair match compared to the consensus octamer motif. This is the first example of a tissue specific protein which has been observed to bind to the papillomavirus enhancer. The possible role of this protein in producing the observed tissue specific activity of the enhancer and in cervical carcinogenesis induced by HPV is discussed. INTRODUCTION Octamer transcription factors (OTFs) have been implicated in the expression of both cellular and viral genes containing the 8 nucleotide octamer recognition consensus sequence ATGCAAAT within their promoters (for review see 1). The ubiquitous octamer binding protein, OTF-1, is present in virtually all cell types, and is required for the correct expression of the histone H2B and small nuclear RNA genes (2). In addition many cell types contain other OTFs that may be involved in cell type specific gene expression. The best characterized of these is OTF-2, which is necessary for B cell specific expression of immunoglobulin genes (3). Other octamer binding proteins expressed specifically in neuronal cells (4,5) embryonal carcinoma cells (6) and in the testis (7) have recently been described. It is likely that these proteins play a critical role in the regulation of cellular gene expression in the tissues which contain them. In addition however, octamer binding proteins also play a critical role in the regulation of viral gene expression notably in the case of herpes simplex virus (HSV). Thus trans-activation of the immediate-early genes of HSV types 1 and 2 is dependent upon the formation of a complex between the HSV virion protein *
To whom correspondence should be addressed at Medical Molecular
Vmw65 and the octamer binding protein OTF- 1. This complex binds to the octamer related sequence TAATGARAT (R=purine) in the viral immediate-early promoters and activates transcription (8-11). Moreover, the inhibition of HSV immediate-early gene expression which occurs in neuronal cell lines is dependent on the presence of a cell type specific octamer binding protein which inhibits binding of the OTF-1/Vmw65 complex to the TAATGARAT motif (12). Thus the OTF protein content of a cell has important repercussions on the virulence of HSV, an effect that could well apply to other viruses containing octamer-related sequences in their promoters. In this study we therefore investigated the nature of the octamer binding proteins present in human cells of cervical origin. Such a study is of particular importance in view of the known interactions of viruses with cells of this type. Thus the human papillomaviruses HPV-16 and HPV-18 are likely to play a central role in the aetiology of cervical carcinoma (for reviews see 13,14). Although these viruses are found in the great majority of women with the disease (15,16) they are also found in some women with no cervical abnormality (17,18). This has led to the suggestion that the regulation of papillomavirus gene expression by cellular factors (19) or the presence of co-factors notably infection with other viruses such as HSV-2 or cigarette smoking (20) may play a critical role in the development of cervical cancers. Hence the processes which regulate HPV gene expression in the cervix are likely to be of critical importance in the development of cervical carcinoma.
MATERIALS AND METHODS Oligonucleotides Complementary pairs of oligonucleotides with the sequences indicated in Figure 3 were synthesized on an Applied Biosystems model 381A oligonucleotide synthesizer. All oligonucleotides were synthesized so that following annealing, they would have a single stranded 5' GATC overhang at either end to facilitate any subsequent cloning procedures. Following annealing, the
Biology Unit, The Windeyer Building, Cleveland Street, London, WIP 6DB, UK
4532 Nucleic Acids Research, Vol. 19, No. 16
oligonucleotides were labelled by phosphorylation with gamma 32P ATP and T4 polynucleotide kinase. DNA mobility shift assay Nuclear extracts were made from about 5 x 107 cells according to the method of Dignam et al (21). For the binding assay, 10 fmol of 32P ATP labelled oligonucleotide probe was mixed wit 1 micro-litre of nuclear extract containing 2Lg of protein in the presence of 20 mM Hepes, 5 mM MgCl2, 50 mM KCl, 0.5 mM DTIT, 4% ficoll and 2 micro-grams poly (dIdC) per 20 ;d reaction volume. Competitor DNA was added at lOx or 100 xmolar excess at this stage as required. The binding reaction was incubated for 40 minutes on ice, before separation of DNA/protein complexes by electrophoresis on a 4% polyacrylamide gel in 0.25 xTBE. Gels were run for 2-3 hrs at 150 V and 4°C, following pre-electrophoresis for about 2 hrs before use. Complexes were visualized by autoradiography of the dried gel. Autoradiographs were scanned on a Bio-rad model 620 video densitometer.
Transfection A total of 106 BHK-21 cells (22) were transfected with 2pg of pBL2 Cat vector or the same vector containing the HPV octamer oligonucleotide (pap.oct:- Figure 3) cloned into the Bam HI site at -105 (23) by the calcium phosphate method (24). After transfection cells were harvested and following assay of protein concentration (25) equal amounts of extract were assayed for chloramphenicol acetyl transferase activity (24).
RESULTS To demonstrate the presence of octamer binding proteins in cervical material we carried out DNA mobility shift assays using as a probe an overlapping octamer/TAATGARAT motif (ATGCTAATGAGAT). An oligonucleotide containing this sequence has previously been demonstrated to bind both OTF-1 and OTF-2 23
A
5
with high affinity in DNA mobility shift assays (26). As exeted, OTF-1 binding was observed in all cell extracts tested (Figure 1). However, in addition to the OTF-1 bands, all the extracts of cervical orgin tested formed a second faster migrating, complex on the octamer oligonucleotide (arrowed in Figure 1). This complex was of higher mobility than the OTF-2 complex formed in B cell extracts and the similarly sized complex formed by neuronal cell extrcts (data not shown). All extracts tested showed similar binding patterns to other probes such as Spl, indicating that increased protein degradation in the cervical extracts was not responsible for the additional band. In competition analysis (Figure 2) both the OTF-1 band (I) and the cervical-specific bad (c) could be removed only by cometition with octamer oligonucleotides (tracks 2 and 3) and not by the binding sites for unrelated transcription factors such as SpI (track 4). An additional band of low intensity between OTF-l and the cervical specific band was also specifically completed by octamer oligonucleotides. The identify of this band is uncertain but it is of similar mobility to dth produced by OTF-2 which was originally thought to be B cell-specific but which has recently been detected in other cell tpes such as neuronal cells (4,5) and the testis (7). This competition behaviour was distinct from that of a high mobility band (ns) which is present in all extracts. This band is not specifically removed by competition with octamer oligonucleotides and represents a non sequence specific DNA binding protein which we have previously observed in other cell types (12). The cervical cell protein, therefore represents a novel octmer binding protein whose expression is likely to be specific to cervical cell types being absent in a range of other celltpes including, 3T3 cells, BHK-21 cells, T cells, neuronal cells and the epithelial mam carcinoma cell line Si15. The cervical cells which contained the novel protein included cell lines derived r
4
7 (
0 I4 *
io
.0 S
S
h11
Figure 2. Competition analysis of the sequence specificity ofthe cervical specific band. A DNA mobility shift assay was carried out using 310A cell extract and
Figure 1. DNA nwbility shift assay using an overlapping octamer/TAATGARAT oligonucleotide (ATGCTAATGAGAT) from the HSV-l IEl gene promoter (40) and extracts from 3T3 cells (track 1), BHK-21 cells (track 2) J2 cells (track 3), S115 mammaly epithelal cells (see reference 41:- track 4), 310 primary cervical cells with no evidence of papillomavirus infection (see reference 27:- track 5), 310 A cells (310 cells tnsfomed with HPV-16 DNA, track 6), and SiHa cervical carcinoma cells (track 7). The arrow indicates the cervical-specific band.
the labelled octamer/TAATGARAT oligonucleotide, in the absence of competition (track 1) or in the presence of a one hundred fold excess of competing octamer/TAATGARAT oligonucleotide (track 2), or of a one hundred fold excess of a consensus octamer oligonucleotide (ATGCAAATAA:- track 3) or a one hundred fold excess of an unrelated Spl oligonucleotide (:- AAGGGCGGGC track 4). The arrows indcate the positions of Oct-I (1), the cervical specific octamer binding protein (C) and the non specific band which forms on all probes tested (ns). Note the removal of the cervical-specific band (C) using unlabelled octamer competitors but not by the unrelated competitor.
I.
Nucleic Acids Research, Vol. 19, No. 16 4533 from HPV infected cervical carcinomas (CaSki, SiHa and HeLa), a cervical carcinoma cell line lacking any HPV DNA (C33) cells of limited life span derived from normal cervical epithelium and with no detectable HPV DNA (310:- see 27), and the same cells transformed with HPV (310A). This protein is therefore present in several different cervical cell samples and is not a papillomavirus gene product since it is present also in both primary and transformed cervical cells lacking HPV sequences. Our ability to detect this protein in HeLa cells in which others have detected only OTF-1 (see for example 2,28) is likely to reflect our use of a very high affinity binding site for octamer binding proteins (26) and the different sequence specificity of the cervical protein (see below). We note also however, that one of three HeLa cell lines we tested from different sources contained only very low levels of this protein suggesting that its expression may vary between different HeLa cells. Having established the existence of this novel octamer binding protein we wished to identify its possible role. In particular we examined the upstream regulatory region (URR) of HPV 16 and 18 for the presence of any octamer-like sequences. This region is known to act as a tissue specific enhancer element which regulates expression of the papillomavirus genome (29,30) and contains binding sites for a number of trans-acting transcription factors (31-33). One such sequence which matches the octamer in seven out of eight positions (Figure 3) was observed at positions 7731 to 7738 within the URR. This sequence is exactly adjacent to one of the five binding sites for nuclear factor I which are found in the URR and play a critical role in its expression Octamer
consensus
HPV16 HPV18 HPV6 HPV1I
ATTTGCAT
[
NFI
I
CT AATTGCATIAT TT GGCAT CT AATTGCATACTT GGCTT TT AAAAGCATTT TT GGCT T TT AAAAGCATTTTT GGCTT
Figure 3. Relationship of the consensus octamer sequence and the octamer like sequences in the HPV enhancers. The adjacent binding site for nuclear factor I (NFI) is indicated and the oligonucleotide used in subsequent studies (pap-oct) is boxed. In order to conform to the conventional direction of the papillomavirus genome the octamer sequence has been written as ATTTGCAT rather than the complementary sequence ATGCAAAT which is more often presented. b
a
(33). Interestingly Chong et al., (34) noted an extension of the NFI footprint across this octamer like motif. They did not note however, the relationship of this sequence to the octamer motif and hence termed the protein which bound to this sequence NFA for NF-1 associated factor. Subsequently Cripe et al., (35) suggested that this site might bind Oct-I but did not directly test whether this was the case. To determine whether this octamer-like sequence was able to bind cervical octamer binding proteins we synthesized an oligonucleotide containing it (pap-oct:- Figure 3) and used it as a competitor in a DNA mobility shift assay with labelled octamer/TAATGARAT oligonucleotide and cervical cell extract. As shown in Figure 4, the papilloma oligonucleotide competed specifically for both the OTF-1 band and the cervical specific band to a much greater extent than did the non-specific Spl oligonucleotide. This indicates therefore that the papilloma oligonucleotide can bind both these octamer binding proteins in a sequence specific manner. Interestingly in these experiments, the papillomavirus sequence exhibited some difference in sequence specificity compared to the homologous octamer competitor. Thus the papillomavirus sequence competed relatively poorly for OTF-l compared to the control octamer (panel a) but competed much better for the cervical cell specific octamer binding protein (panel b). This suggested that the cervical cell protein may have a different sequence specificity compared to OTF-1, exhibiting high affinity binding to the sequence in the papillomavirus URR. This suggestion was confirmed in DNA mobility shift assays using the papillomavirus oligonucleotide as the probe. In this experiment (Figure 5a) a complex formed on this oligonucleotide which was equivalent in size to the faster migrating cervical complex formed with the control octamer sequence. Moreover, in longer exposures a weaker complex corresponding in size to the OTF-1 band was also observed on this oligonucleotide (Figure Sb). To confirm that the proteins binding the HPV octamer were the same as those binding the consensus octamer competition experiments were carried out. As shown in Table 1, an excess of unlabelled control octamer, when added during HPV octamer complex formation, completely eliminated any binding to OTF-1 but only weakly competed binding to the cervicalspecific species. As a control, an unrelated SpI binding site does not compete for either band. In contrast an excess of unlabelled b
1C
2
ic
2
:E 0o CM6
x
l34:
01
2
@"I
.. .>
~:Z
_s
i,,
.1 ..... :.
O
Xlo00x40O 0 Excess of competitor
34
3
x100
x400
Excess of competor
Figure 4. Competition analysis of the effects of various competitor oligonucleotides on the ability of the octamer/TAATGARAT oligonucleotide to bind OTF-l (panel a) or the cervical specific band (panel b) using extract prepared from 310A cells. Solid squares indicate the results with the homologous octamer
oligonucleotide competitor, open squares results with papilloma oligonucleotide competitor and solid circles results with Spl oligonucleotide competitor.
_
d
Figure 5. DNA mobility shift assay using the octamer/TAATGARAT oligonucleotide (tracks 1 and 2) or the papilloma virus oligonucleotide (tracks 3 and 4) using extract prepared from 310A cells. The positions of Oct-I (1) and the cervical specific band (c) are indicated. Panel b represents a longer exposure of the same gel shown in panel a.
4534 Nucleic Acids Research, Vol. 19, No. 16 Table 1. Effect of adding various competing oligonucleotides on the formation of specific DNA-protein complexes on the papilloma-octamer oligonucleotide Complex
Competitor None Octamer Papilloma octamer Non specific (Spl)
OTF-1
Cervical-specific complex
100 0 30 100
100 55 33 100
A.
Figures indicate the effect on the intensity of the DNA-protein complexes (as determined by scanning densitometry) which form on the papilloma-octamer oligonucleotide using 310A cervical cell extract when an excess of the indicated unlabelled competitor oligonucleotide is added and are expressed as a percentage of the binding observed in the absence of any competitor.
homologous HPV octamer added during complex formation competes, as expected, for both OTF-l and the cervical-specific complex. Hence the cervical cell specific octamer binding protein we have detected represents a protein with a novel sequence specificity distinct from OTF-1 which can bind to the octamerlike sequence in the papillomavirus URR with high affinity as well as to the consensus octamer motif In order to demonstrate that the HPV octamer bound this protein in a tissue specific manner, as did the consensus octamer, binding to the papillomavirus Oct oligonucleotide was investigated using the same panel of cell types (Figure 6). The cell type specificity was identical to that observed with the consensus octamer sequence, with binding in all the cervical cell extracts and no others. Having established that octamer binding proteins could bind to the HPV octamer, we wished to test whether this binding had any functional effect. To do this, the HPV octamer was cloned upstream of the herpes simplex virus thymidine kinase promoter in the vector pBL2Cat (23). When this construct was introduced by transfection into BHK cells which contain only Oct-i increased Cat activity compared to the parental vector was observed (figure 7) confirming that the binding of Oct-I to the HPV octamer can increase promoter activity.
DISCUSSION In the studies reported here we have shown that both the constitutively expressed OTF-1 octamer binding protein and a novel cervical specific octamer-binding protein can bind to an octamer-like sequence in the papilloma upstream regulatory region. These findings therefore confirm and extend those of Chong et al., (34) who observed a DNaseI footprint extending both over this site and the adjacent NF1 site indicating that it represents a site of protein binding, but did not define the nature and cell type specificity of the proteins which bind to it. The cervical specific octamer binding protein reported here has a sequence specificity distinct from OTF-1 which results in its binding to the octamer-like sequence in the URR with higher affinity than OTF-1. Moreover, this protein is expressed in a cell type specific manner being present in several cervical cell lines and primary cervical material but being absent in a range of other cell types. Such a finding is of particular interest in that several studies have demonstrated that the papillomavirus URR functions as a tissue specific enhancer whose activity is higher in cervical cells (29,30). To date however, only ubiquitously expressed transcription factors such as NFl (33) API (32) and the glucocorticoid receptor (31) have been shown to bind to the
Fgure 6. DNA mobility shift assay usmg the papillomavirus oligonucleotide and cell extracts from 3T3 cells (track 1), BHK-21 cells (tack 2), J2 cells (track 3), S115 cells (track 4), 310 cells (track 5), 310A cells (tack 6), SiHa cells (track 7) CaSki cells (track 8) and C33 cells (track 9). The arrow indicates the cervicalspecific band.
Fgure 7. Assay of chlomamphenicol acetyl trfase activity in BHK-21 cells followig tansfection with pBL2 Cat caining a sngle copy of the papilomavirus octamer oligonucleotide (track 1) or with pBLq Cat vector (track 2).
URR. It is possible therefore that our factor may play a role in the tissue specific activation of the papillomavirus genome. This could occur by interaction of this factor with NFl bound to adjacent sites since NFl binding has been shown to be necessary but not sufficient for gene activation by the URR (33). Moreover the cervical specific complex we observe forms more strongly on a composite oligonucleotide containing both the octamer sequence and the NF1 site than on the octamer alone, demonstrating that although the HPV octamer is capable of binding this protein, binding is enhanced by the presence of the NF1 site (data not shown). In agreement with this possibility, a recent study by Nakshatri et al., (36) reported the binding of two factors present in cervical cells to an oligonucleotide from the HPV16 enhancer containing both the NFl and octamer-like sequences, only one of these proteins being observed in fibroblast cells. However, the precise binding sites within the oligonucleotide for each of the two proteins were not determined. We are currently investigating the level of enhancer activity of the HPV octamer site when linked to the NFl binding site in driving gene expression from a heterologous promoter following transfection into cervical and non cervical cells.
Nucleic Acids Research, Vol. 19, No. 16 4535 In terms of cervical carcinogenesis by HPV, it is of importance that the URR drives expression of the E6 and E7 gene products which are thought to be responsible for virally-induced transformation (37,38). It has been speculated that changes in the levels of cellular factors binding to this region may result in activation of papillomavirus gene expression and tumour induction (19) and it is possible that the cervical-specific factor defined here may play a role in this process. In this regard it is also of interest that whilst the highly homologous octamer type sequence is found in HPV 16 and 18, the HPVs most often associated with cervical carcinomas other HPVs such as HPV6 and 11 which cause only benign warts (13,14) show a much reduced homology to the octamer, (Figure 3). We are currently investigating the binding of our octamer binding protein to the sequence found in HPV6 and 11 in order to determine whether differences in the ability to bind this factor play any role in the different activities of these viruses. It should also be noted that the protein we have identified here, in addition to any role regulating HPV expression in the cervix is likely also to be involved in cellular gene regulation in this tissue and possibly also in regulating other viruses. In particular the activity of the HSV immediate-early genes is known to be critically dependent on the nature of the octamer binding proteins in a particular cell type (8-11). In view of the suggestion that HSV-2 acts as a co-factor to papilloma-virus in cervical carcinoma, (20) it will be of interest to determine the effect of our novel octamer binding protein on HSV gene expression in cervical cells. Moreoever, a recent study (39) has shown that the HSV virion protein Vmw65 which normally interacts with OTF-1 to form a DNA binding complex that trans-activates HSV immediate-early gene transcription (10,11) can also trans-activate the HPV 18 URR, acting through a 230 base pair region which contains the octamer-binding motif we have defined. Hence the identification of octamer-binding proteins interacting with the HPV URR may have implications not only for its cell type specific expression pattern but also for its interaction with the regulatory proteins of other viruses.
ACKNOWLEDGEMENT We thank the Cancer Research Campaign for supporting this work.
REFERENCES 1. Falkner, F.G., Moickat, R. and Zachau, H.G. (1986). Nucl. Acids Res.
13, 7847-7863. 2. Fletcher, C., Heintz, N. and Roeder, R.G. (1987). Cell 51, 773-781. 3. Scheidereit, C., Hegay, A. and Roeder, R.G. (1987). Cell 51, 783-793, 4. He, X., Treacy, M.N., Simmons, D.M., Ingraham, H.A., Swanson, L.S. and Rosenfeld, M.G. (1989). Nature 340, 35-42. 5. Scholer, H.R., Hatzopoulos, A.K., Balling, R., Suzuki, N. and Gruss, P. (1989). EMBO J. 8, 2543-2550. 6. Lenardo, M.J., Staudt, L., Robbins, P., Kuang, A., Milligan, R.C. and Baltimore, D. (1989). Science 243, 544-546. 7. Goldsborough, A., Ashworth, A. and Willison, K. (1990). Nucl. Acids Res. 18, 1643. 8. Gerster, T. and Roeder, R.G. (1988). Proc. Natl. Acad. Sci., USA., 85, 6347-6351. 9. McKnight, J.L.C., Kristie, T.M. and Roizman, B. (1987). Proc. Natl. Acad. Sci., USA., 84, 7408-7412. 10. O'Hare, P. and Goding, C.R. (1988). Cell 52, 435-445. 11. Preston, C.M., Frame, M.C. and Campbell, M.E.M. (1988). Cell 52, 425-434. 12. Kemp, L.M., Dent, C.L. and Latchman, D.S. (1990). Neuron 4, 215-222.
13. Peto, R. and zur Hausen, H. (1986). Banbury Report 21. Cold Spring Harbor Laboratory New York. 14. Syrjjanen, K.J., Gissman, L. and Koss, L.G. (1987). Papilloma viruses and human disease. Springer Verlag, Berlin. 15. Boshart, M., Gissmann, L., Ikenberg, H., Kleinheinz, A., Scheurlen, W. and Zur Hausen, H. (1984). EMBO J 3, 1151-1157. 16. Durst, M., Gissmann, L., Ikenberg, H. and zur Hausen, H. (1983). Proc. Natl. Acad. Sci., USA 80. 3812-3815. 17. Cox, M.F., Meanwell, C.A., Maitland, N.J., Blackledge, G., Scully, C. and Jordan, J.A. (1986). Lancet 2, 157-158. 18. Meanwell, C.A., Cox, M.F., Blackledge, G. and Maitland, N.J. (1987). Lancet 1, 703-707. 19. zur Hausen, H. (1986). Lancet 2, 489-491. 20. zur Hausen, H. (1982). Lancet 2, 1370-1372. 21. Dignam, J.D., Lebovitz, R.M. and Roeder, R.G. (1983). Nucl. Acids. Res. 11, 1475-1489. 22. Macpherson, I. and Stoker, M. (1962). Virology 16, 147-151. 23. Luckow, B. and Schutz, G. (1987). Nucleic Acids Res. 15, 5490. 24. Gorman, C.M. (1985). In DNA cloning, a practical approach, ed. Glover, D.M., vol. 2, pp.143-190. 25. Bradford, M. (1976). Anal. Biochem. 72, 248-254. 26. Dent, C.L. and Latchman, D.S. (1991). Biochemical Journal (in press). 27. Bal, V., McIndoe, A., Denton, G., Hudson, D., Lombard, G., Lamb, J. and Lechler, R. (1990). Eur. J. Immunol 20, 1893-1897. 28. Sturm, R., Baumruker, T., Franza, R.B. and Herr, W. (1987). Genes and Development 1, 1147-1160. 29. Cripe, T.C., Haugen, T.H., Turk, J.P., Tabatabni, F., Schmid, P.G., Durst, M., Gissmann, L., Roman, A. Turek, L. (1987). EMBO Journal 6, 3745-3753. 30. Gloss, B., Bernard, H.U., Seedorf, K. and Klock, G. (1987). EMBO Journal 6, 3735-3743. 31. Chan, W-K., Klock, G. and Bernard, H-U. (1989). Journal of Virology 63, 3261 -3269. 32. Chan, W-K., Chong, T., Bernard, U. and Klock, G. (1990). Nucleic Acids Research 18, 763-769. 33. Gloss, B., Yeo-Gloss, M., Meisterenst, M., Rogge, L., Winnacker, E.L. and Bernard, H-U. (1989). Nucleic Acids Research 17, 3519-3533. 34. Chong, T., Chan, W-K. and Bernard, H-U (1990). Nucleic Acids Research 18, 465-470. 35. Cripe, T.P., Alderbourn, A., Anderson, R.D., Parkkinen, S., Bergman, P., Haugen, T.H., Petterson, U. and Turek, L.P. (1990). The New Biologist 2, 450-463. 36. Nakshatri, H., Pater, M.M. and Pater, A. (1990). Virology 178, 92-103. 37. Phelps, W.C., Yee, C.L., Muenger, K.and Howley, P.W. (1988). Cell 53, 539-547. 38. Storey, A., Pim, D., Murray, A., Osborn, K., Banks, L. and Crawford, L. (1988). EMBO Journal 7, 1815-1820. 39. Gius, D. and Laimins, L.A. (1989). J. Virol. 63, 555-563. 40. Perry, L.J., Rixon, F.J., Everett, R.D. Frame, M.C. and McGeogh, D.J. (1986). J. Gen. Virol. 67, 2365-2380. 41. Darbre, P., Page, M. and King, R.J.B. (1986). Mol. Cell. Biol. 6, 2847-2854.
The constitutively expressed octamer binding protein OTF-1 and a novel octamer binding protein expressed specifically in cervical cells bind to an octamer-related sequence in the human papillomavirus 16 enhancer C.L.Dent, G.A.J.Mcindoel and D.S.Latchman* Medical Molecular Biology Unit, Department of Biochemistry and 'ICRF Human Tumour Immunology Unit, University College and Middlesex School of Medicine, London, UK Received March 26, 1991; Revised and Accepted July 17, 1991
ABSTRACT A novel octamer binding protein expressed specifically in cervical cells but not in other cell types has been identified. This protein differs in size and sequence specificity from the constitutively expressed octamer binding protein OTF-1. In particular it binds with higher affinity to a sequence in the human papillomavirus 16 (HPV) upstream regulatory region which has a seven out of eight base pair match compared to the consensus octamer motif. This is the first example of a tissue specific protein which has been observed to bind to the papillomavirus enhancer. The possible role of this protein in producing the observed tissue specific activity of the enhancer and in cervical carcinogenesis induced by HPV is discussed. INTRODUCTION Octamer transcription factors (OTFs) have been implicated in the expression of both cellular and viral genes containing the 8 nucleotide octamer recognition consensus sequence ATGCAAAT within their promoters (for review see 1). The ubiquitous octamer binding protein, OTF-1, is present in virtually all cell types, and is required for the correct expression of the histone H2B and small nuclear RNA genes (2). In addition many cell types contain other OTFs that may be involved in cell type specific gene expression. The best characterized of these is OTF-2, which is necessary for B cell specific expression of immunoglobulin genes (3). Other octamer binding proteins expressed specifically in neuronal cells (4,5) embryonal carcinoma cells (6) and in the testis (7) have recently been described. It is likely that these proteins play a critical role in the regulation of cellular gene expression in the tissues which contain them. In addition however, octamer binding proteins also play a critical role in the regulation of viral gene expression notably in the case of herpes simplex virus (HSV). Thus trans-activation of the immediate-early genes of HSV types 1 and 2 is dependent upon the formation of a complex between the HSV virion protein *
To whom correspondence should be addressed at Medical Molecular
Vmw65 and the octamer binding protein OTF- 1. This complex binds to the octamer related sequence TAATGARAT (R=purine) in the viral immediate-early promoters and activates transcription (8-11). Moreover, the inhibition of HSV immediate-early gene expression which occurs in neuronal cell lines is dependent on the presence of a cell type specific octamer binding protein which inhibits binding of the OTF-1/Vmw65 complex to the TAATGARAT motif (12). Thus the OTF protein content of a cell has important repercussions on the virulence of HSV, an effect that could well apply to other viruses containing octamer-related sequences in their promoters. In this study we therefore investigated the nature of the octamer binding proteins present in human cells of cervical origin. Such a study is of particular importance in view of the known interactions of viruses with cells of this type. Thus the human papillomaviruses HPV-16 and HPV-18 are likely to play a central role in the aetiology of cervical carcinoma (for reviews see 13,14). Although these viruses are found in the great majority of women with the disease (15,16) they are also found in some women with no cervical abnormality (17,18). This has led to the suggestion that the regulation of papillomavirus gene expression by cellular factors (19) or the presence of co-factors notably infection with other viruses such as HSV-2 or cigarette smoking (20) may play a critical role in the development of cervical cancers. Hence the processes which regulate HPV gene expression in the cervix are likely to be of critical importance in the development of cervical carcinoma.
MATERIALS AND METHODS Oligonucleotides Complementary pairs of oligonucleotides with the sequences indicated in Figure 3 were synthesized on an Applied Biosystems model 381A oligonucleotide synthesizer. All oligonucleotides were synthesized so that following annealing, they would have a single stranded 5' GATC overhang at either end to facilitate any subsequent cloning procedures. Following annealing, the
Biology Unit, The Windeyer Building, Cleveland Street, London, WIP 6DB, UK
4532 Nucleic Acids Research, Vol. 19, No. 16
oligonucleotides were labelled by phosphorylation with gamma 32P ATP and T4 polynucleotide kinase. DNA mobility shift assay Nuclear extracts were made from about 5 x 107 cells according to the method of Dignam et al (21). For the binding assay, 10 fmol of 32P ATP labelled oligonucleotide probe was mixed wit 1 micro-litre of nuclear extract containing 2Lg of protein in the presence of 20 mM Hepes, 5 mM MgCl2, 50 mM KCl, 0.5 mM DTIT, 4% ficoll and 2 micro-grams poly (dIdC) per 20 ;d reaction volume. Competitor DNA was added at lOx or 100 xmolar excess at this stage as required. The binding reaction was incubated for 40 minutes on ice, before separation of DNA/protein complexes by electrophoresis on a 4% polyacrylamide gel in 0.25 xTBE. Gels were run for 2-3 hrs at 150 V and 4°C, following pre-electrophoresis for about 2 hrs before use. Complexes were visualized by autoradiography of the dried gel. Autoradiographs were scanned on a Bio-rad model 620 video densitometer.
Transfection A total of 106 BHK-21 cells (22) were transfected with 2pg of pBL2 Cat vector or the same vector containing the HPV octamer oligonucleotide (pap.oct:- Figure 3) cloned into the Bam HI site at -105 (23) by the calcium phosphate method (24). After transfection cells were harvested and following assay of protein concentration (25) equal amounts of extract were assayed for chloramphenicol acetyl transferase activity (24).
RESULTS To demonstrate the presence of octamer binding proteins in cervical material we carried out DNA mobility shift assays using as a probe an overlapping octamer/TAATGARAT motif (ATGCTAATGAGAT). An oligonucleotide containing this sequence has previously been demonstrated to bind both OTF-1 and OTF-2 23
A
5
with high affinity in DNA mobility shift assays (26). As exeted, OTF-1 binding was observed in all cell extracts tested (Figure 1). However, in addition to the OTF-1 bands, all the extracts of cervical orgin tested formed a second faster migrating, complex on the octamer oligonucleotide (arrowed in Figure 1). This complex was of higher mobility than the OTF-2 complex formed in B cell extracts and the similarly sized complex formed by neuronal cell extrcts (data not shown). All extracts tested showed similar binding patterns to other probes such as Spl, indicating that increased protein degradation in the cervical extracts was not responsible for the additional band. In competition analysis (Figure 2) both the OTF-1 band (I) and the cervical-specific bad (c) could be removed only by cometition with octamer oligonucleotides (tracks 2 and 3) and not by the binding sites for unrelated transcription factors such as SpI (track 4). An additional band of low intensity between OTF-l and the cervical specific band was also specifically completed by octamer oligonucleotides. The identify of this band is uncertain but it is of similar mobility to dth produced by OTF-2 which was originally thought to be B cell-specific but which has recently been detected in other cell tpes such as neuronal cells (4,5) and the testis (7). This competition behaviour was distinct from that of a high mobility band (ns) which is present in all extracts. This band is not specifically removed by competition with octamer oligonucleotides and represents a non sequence specific DNA binding protein which we have previously observed in other cell types (12). The cervical cell protein, therefore represents a novel octmer binding protein whose expression is likely to be specific to cervical cell types being absent in a range of other celltpes including, 3T3 cells, BHK-21 cells, T cells, neuronal cells and the epithelial mam carcinoma cell line Si15. The cervical cells which contained the novel protein included cell lines derived r
4
7 (
0 I4 *
io
.0 S
S
h11
Figure 2. Competition analysis of the sequence specificity ofthe cervical specific band. A DNA mobility shift assay was carried out using 310A cell extract and
Figure 1. DNA nwbility shift assay using an overlapping octamer/TAATGARAT oligonucleotide (ATGCTAATGAGAT) from the HSV-l IEl gene promoter (40) and extracts from 3T3 cells (track 1), BHK-21 cells (track 2) J2 cells (track 3), S115 mammaly epithelal cells (see reference 41:- track 4), 310 primary cervical cells with no evidence of papillomavirus infection (see reference 27:- track 5), 310 A cells (310 cells tnsfomed with HPV-16 DNA, track 6), and SiHa cervical carcinoma cells (track 7). The arrow indicates the cervical-specific band.
the labelled octamer/TAATGARAT oligonucleotide, in the absence of competition (track 1) or in the presence of a one hundred fold excess of competing octamer/TAATGARAT oligonucleotide (track 2), or of a one hundred fold excess of a consensus octamer oligonucleotide (ATGCAAATAA:- track 3) or a one hundred fold excess of an unrelated Spl oligonucleotide (:- AAGGGCGGGC track 4). The arrows indcate the positions of Oct-I (1), the cervical specific octamer binding protein (C) and the non specific band which forms on all probes tested (ns). Note the removal of the cervical-specific band (C) using unlabelled octamer competitors but not by the unrelated competitor.
I.
Nucleic Acids Research, Vol. 19, No. 16 4533 from HPV infected cervical carcinomas (CaSki, SiHa and HeLa), a cervical carcinoma cell line lacking any HPV DNA (C33) cells of limited life span derived from normal cervical epithelium and with no detectable HPV DNA (310:- see 27), and the same cells transformed with HPV (310A). This protein is therefore present in several different cervical cell samples and is not a papillomavirus gene product since it is present also in both primary and transformed cervical cells lacking HPV sequences. Our ability to detect this protein in HeLa cells in which others have detected only OTF-1 (see for example 2,28) is likely to reflect our use of a very high affinity binding site for octamer binding proteins (26) and the different sequence specificity of the cervical protein (see below). We note also however, that one of three HeLa cell lines we tested from different sources contained only very low levels of this protein suggesting that its expression may vary between different HeLa cells. Having established the existence of this novel octamer binding protein we wished to identify its possible role. In particular we examined the upstream regulatory region (URR) of HPV 16 and 18 for the presence of any octamer-like sequences. This region is known to act as a tissue specific enhancer element which regulates expression of the papillomavirus genome (29,30) and contains binding sites for a number of trans-acting transcription factors (31-33). One such sequence which matches the octamer in seven out of eight positions (Figure 3) was observed at positions 7731 to 7738 within the URR. This sequence is exactly adjacent to one of the five binding sites for nuclear factor I which are found in the URR and play a critical role in its expression Octamer
consensus
HPV16 HPV18 HPV6 HPV1I
ATTTGCAT
[
NFI
I
CT AATTGCATIAT TT GGCAT CT AATTGCATACTT GGCTT TT AAAAGCATTT TT GGCT T TT AAAAGCATTTTT GGCTT
Figure 3. Relationship of the consensus octamer sequence and the octamer like sequences in the HPV enhancers. The adjacent binding site for nuclear factor I (NFI) is indicated and the oligonucleotide used in subsequent studies (pap-oct) is boxed. In order to conform to the conventional direction of the papillomavirus genome the octamer sequence has been written as ATTTGCAT rather than the complementary sequence ATGCAAAT which is more often presented. b
a
(33). Interestingly Chong et al., (34) noted an extension of the NFI footprint across this octamer like motif. They did not note however, the relationship of this sequence to the octamer motif and hence termed the protein which bound to this sequence NFA for NF-1 associated factor. Subsequently Cripe et al., (35) suggested that this site might bind Oct-I but did not directly test whether this was the case. To determine whether this octamer-like sequence was able to bind cervical octamer binding proteins we synthesized an oligonucleotide containing it (pap-oct:- Figure 3) and used it as a competitor in a DNA mobility shift assay with labelled octamer/TAATGARAT oligonucleotide and cervical cell extract. As shown in Figure 4, the papilloma oligonucleotide competed specifically for both the OTF-1 band and the cervical specific band to a much greater extent than did the non-specific Spl oligonucleotide. This indicates therefore that the papilloma oligonucleotide can bind both these octamer binding proteins in a sequence specific manner. Interestingly in these experiments, the papillomavirus sequence exhibited some difference in sequence specificity compared to the homologous octamer competitor. Thus the papillomavirus sequence competed relatively poorly for OTF-l compared to the control octamer (panel a) but competed much better for the cervical cell specific octamer binding protein (panel b). This suggested that the cervical cell protein may have a different sequence specificity compared to OTF-1, exhibiting high affinity binding to the sequence in the papillomavirus URR. This suggestion was confirmed in DNA mobility shift assays using the papillomavirus oligonucleotide as the probe. In this experiment (Figure 5a) a complex formed on this oligonucleotide which was equivalent in size to the faster migrating cervical complex formed with the control octamer sequence. Moreover, in longer exposures a weaker complex corresponding in size to the OTF-1 band was also observed on this oligonucleotide (Figure Sb). To confirm that the proteins binding the HPV octamer were the same as those binding the consensus octamer competition experiments were carried out. As shown in Table 1, an excess of unlabelled control octamer, when added during HPV octamer complex formation, completely eliminated any binding to OTF-1 but only weakly competed binding to the cervicalspecific species. As a control, an unrelated SpI binding site does not compete for either band. In contrast an excess of unlabelled b
1C
2
ic
2
:E 0o CM6
x
l34:
01
2
@"I
.. .>
~:Z
_s
i,,
.1 ..... :.
O
Xlo00x40O 0 Excess of competitor
34
3
x100
x400
Excess of competor
Figure 4. Competition analysis of the effects of various competitor oligonucleotides on the ability of the octamer/TAATGARAT oligonucleotide to bind OTF-l (panel a) or the cervical specific band (panel b) using extract prepared from 310A cells. Solid squares indicate the results with the homologous octamer
oligonucleotide competitor, open squares results with papilloma oligonucleotide competitor and solid circles results with Spl oligonucleotide competitor.
_
d
Figure 5. DNA mobility shift assay using the octamer/TAATGARAT oligonucleotide (tracks 1 and 2) or the papilloma virus oligonucleotide (tracks 3 and 4) using extract prepared from 310A cells. The positions of Oct-I (1) and the cervical specific band (c) are indicated. Panel b represents a longer exposure of the same gel shown in panel a.
4534 Nucleic Acids Research, Vol. 19, No. 16 Table 1. Effect of adding various competing oligonucleotides on the formation of specific DNA-protein complexes on the papilloma-octamer oligonucleotide Complex
Competitor None Octamer Papilloma octamer Non specific (Spl)
OTF-1
Cervical-specific complex
100 0 30 100
100 55 33 100
A.
Figures indicate the effect on the intensity of the DNA-protein complexes (as determined by scanning densitometry) which form on the papilloma-octamer oligonucleotide using 310A cervical cell extract when an excess of the indicated unlabelled competitor oligonucleotide is added and are expressed as a percentage of the binding observed in the absence of any competitor.
homologous HPV octamer added during complex formation competes, as expected, for both OTF-l and the cervical-specific complex. Hence the cervical cell specific octamer binding protein we have detected represents a protein with a novel sequence specificity distinct from OTF-1 which can bind to the octamerlike sequence in the papillomavirus URR with high affinity as well as to the consensus octamer motif In order to demonstrate that the HPV octamer bound this protein in a tissue specific manner, as did the consensus octamer, binding to the papillomavirus Oct oligonucleotide was investigated using the same panel of cell types (Figure 6). The cell type specificity was identical to that observed with the consensus octamer sequence, with binding in all the cervical cell extracts and no others. Having established that octamer binding proteins could bind to the HPV octamer, we wished to test whether this binding had any functional effect. To do this, the HPV octamer was cloned upstream of the herpes simplex virus thymidine kinase promoter in the vector pBL2Cat (23). When this construct was introduced by transfection into BHK cells which contain only Oct-i increased Cat activity compared to the parental vector was observed (figure 7) confirming that the binding of Oct-I to the HPV octamer can increase promoter activity.
DISCUSSION In the studies reported here we have shown that both the constitutively expressed OTF-1 octamer binding protein and a novel cervical specific octamer-binding protein can bind to an octamer-like sequence in the papilloma upstream regulatory region. These findings therefore confirm and extend those of Chong et al., (34) who observed a DNaseI footprint extending both over this site and the adjacent NF1 site indicating that it represents a site of protein binding, but did not define the nature and cell type specificity of the proteins which bind to it. The cervical specific octamer binding protein reported here has a sequence specificity distinct from OTF-1 which results in its binding to the octamer-like sequence in the URR with higher affinity than OTF-1. Moreover, this protein is expressed in a cell type specific manner being present in several cervical cell lines and primary cervical material but being absent in a range of other cell types. Such a finding is of particular interest in that several studies have demonstrated that the papillomavirus URR functions as a tissue specific enhancer whose activity is higher in cervical cells (29,30). To date however, only ubiquitously expressed transcription factors such as NFl (33) API (32) and the glucocorticoid receptor (31) have been shown to bind to the
Fgure 6. DNA mobility shift assay usmg the papillomavirus oligonucleotide and cell extracts from 3T3 cells (track 1), BHK-21 cells (tack 2), J2 cells (track 3), S115 cells (track 4), 310 cells (track 5), 310A cells (tack 6), SiHa cells (track 7) CaSki cells (track 8) and C33 cells (track 9). The arrow indicates the cervicalspecific band.
Fgure 7. Assay of chlomamphenicol acetyl trfase activity in BHK-21 cells followig tansfection with pBL2 Cat caining a sngle copy of the papilomavirus octamer oligonucleotide (track 1) or with pBLq Cat vector (track 2).
URR. It is possible therefore that our factor may play a role in the tissue specific activation of the papillomavirus genome. This could occur by interaction of this factor with NFl bound to adjacent sites since NFl binding has been shown to be necessary but not sufficient for gene activation by the URR (33). Moreover the cervical specific complex we observe forms more strongly on a composite oligonucleotide containing both the octamer sequence and the NF1 site than on the octamer alone, demonstrating that although the HPV octamer is capable of binding this protein, binding is enhanced by the presence of the NF1 site (data not shown). In agreement with this possibility, a recent study by Nakshatri et al., (36) reported the binding of two factors present in cervical cells to an oligonucleotide from the HPV16 enhancer containing both the NFl and octamer-like sequences, only one of these proteins being observed in fibroblast cells. However, the precise binding sites within the oligonucleotide for each of the two proteins were not determined. We are currently investigating the level of enhancer activity of the HPV octamer site when linked to the NFl binding site in driving gene expression from a heterologous promoter following transfection into cervical and non cervical cells.
Nucleic Acids Research, Vol. 19, No. 16 4535 In terms of cervical carcinogenesis by HPV, it is of importance that the URR drives expression of the E6 and E7 gene products which are thought to be responsible for virally-induced transformation (37,38). It has been speculated that changes in the levels of cellular factors binding to this region may result in activation of papillomavirus gene expression and tumour induction (19) and it is possible that the cervical-specific factor defined here may play a role in this process. In this regard it is also of interest that whilst the highly homologous octamer type sequence is found in HPV 16 and 18, the HPVs most often associated with cervical carcinomas other HPVs such as HPV6 and 11 which cause only benign warts (13,14) show a much reduced homology to the octamer, (Figure 3). We are currently investigating the binding of our octamer binding protein to the sequence found in HPV6 and 11 in order to determine whether differences in the ability to bind this factor play any role in the different activities of these viruses. It should also be noted that the protein we have identified here, in addition to any role regulating HPV expression in the cervix is likely also to be involved in cellular gene regulation in this tissue and possibly also in regulating other viruses. In particular the activity of the HSV immediate-early genes is known to be critically dependent on the nature of the octamer binding proteins in a particular cell type (8-11). In view of the suggestion that HSV-2 acts as a co-factor to papilloma-virus in cervical carcinoma, (20) it will be of interest to determine the effect of our novel octamer binding protein on HSV gene expression in cervical cells. Moreoever, a recent study (39) has shown that the HSV virion protein Vmw65 which normally interacts with OTF-1 to form a DNA binding complex that trans-activates HSV immediate-early gene transcription (10,11) can also trans-activate the HPV 18 URR, acting through a 230 base pair region which contains the octamer-binding motif we have defined. Hence the identification of octamer-binding proteins interacting with the HPV URR may have implications not only for its cell type specific expression pattern but also for its interaction with the regulatory proteins of other viruses.
ACKNOWLEDGEMENT We thank the Cancer Research Campaign for supporting this work.
REFERENCES 1. Falkner, F.G., Moickat, R. and Zachau, H.G. (1986). Nucl. Acids Res.
13, 7847-7863. 2. Fletcher, C., Heintz, N. and Roeder, R.G. (1987). Cell 51, 773-781. 3. Scheidereit, C., Hegay, A. and Roeder, R.G. (1987). Cell 51, 783-793, 4. He, X., Treacy, M.N., Simmons, D.M., Ingraham, H.A., Swanson, L.S. and Rosenfeld, M.G. (1989). Nature 340, 35-42. 5. Scholer, H.R., Hatzopoulos, A.K., Balling, R., Suzuki, N. and Gruss, P. (1989). EMBO J. 8, 2543-2550. 6. Lenardo, M.J., Staudt, L., Robbins, P., Kuang, A., Milligan, R.C. and Baltimore, D. (1989). Science 243, 544-546. 7. Goldsborough, A., Ashworth, A. and Willison, K. (1990). Nucl. Acids Res. 18, 1643. 8. Gerster, T. and Roeder, R.G. (1988). Proc. Natl. Acad. Sci., USA., 85, 6347-6351. 9. McKnight, J.L.C., Kristie, T.M. and Roizman, B. (1987). Proc. Natl. Acad. Sci., USA., 84, 7408-7412. 10. O'Hare, P. and Goding, C.R. (1988). Cell 52, 435-445. 11. Preston, C.M., Frame, M.C. and Campbell, M.E.M. (1988). Cell 52, 425-434. 12. Kemp, L.M., Dent, C.L. and Latchman, D.S. (1990). Neuron 4, 215-222.
13. Peto, R. and zur Hausen, H. (1986). Banbury Report 21. Cold Spring Harbor Laboratory New York. 14. Syrjjanen, K.J., Gissman, L. and Koss, L.G. (1987). Papilloma viruses and human disease. Springer Verlag, Berlin. 15. Boshart, M., Gissmann, L., Ikenberg, H., Kleinheinz, A., Scheurlen, W. and Zur Hausen, H. (1984). EMBO J 3, 1151-1157. 16. Durst, M., Gissmann, L., Ikenberg, H. and zur Hausen, H. (1983). Proc. Natl. Acad. Sci., USA 80. 3812-3815. 17. Cox, M.F., Meanwell, C.A., Maitland, N.J., Blackledge, G., Scully, C. and Jordan, J.A. (1986). Lancet 2, 157-158. 18. Meanwell, C.A., Cox, M.F., Blackledge, G. and Maitland, N.J. (1987). Lancet 1, 703-707. 19. zur Hausen, H. (1986). Lancet 2, 489-491. 20. zur Hausen, H. (1982). Lancet 2, 1370-1372. 21. Dignam, J.D., Lebovitz, R.M. and Roeder, R.G. (1983). Nucl. Acids. Res. 11, 1475-1489. 22. Macpherson, I. and Stoker, M. (1962). Virology 16, 147-151. 23. Luckow, B. and Schutz, G. (1987). Nucleic Acids Res. 15, 5490. 24. Gorman, C.M. (1985). In DNA cloning, a practical approach, ed. Glover, D.M., vol. 2, pp.143-190. 25. Bradford, M. (1976). Anal. Biochem. 72, 248-254. 26. Dent, C.L. and Latchman, D.S. (1991). Biochemical Journal (in press). 27. Bal, V., McIndoe, A., Denton, G., Hudson, D., Lombard, G., Lamb, J. and Lechler, R. (1990). Eur. J. Immunol 20, 1893-1897. 28. Sturm, R., Baumruker, T., Franza, R.B. and Herr, W. (1987). Genes and Development 1, 1147-1160. 29. Cripe, T.C., Haugen, T.H., Turk, J.P., Tabatabni, F., Schmid, P.G., Durst, M., Gissmann, L., Roman, A. Turek, L. (1987). EMBO Journal 6, 3745-3753. 30. Gloss, B., Bernard, H.U., Seedorf, K. and Klock, G. (1987). EMBO Journal 6, 3735-3743. 31. Chan, W-K., Klock, G. and Bernard, H-U. (1989). Journal of Virology 63, 3261 -3269. 32. Chan, W-K., Chong, T., Bernard, U. and Klock, G. (1990). Nucleic Acids Research 18, 763-769. 33. Gloss, B., Yeo-Gloss, M., Meisterenst, M., Rogge, L., Winnacker, E.L. and Bernard, H-U. (1989). Nucleic Acids Research 17, 3519-3533. 34. Chong, T., Chan, W-K. and Bernard, H-U (1990). Nucleic Acids Research 18, 465-470. 35. Cripe, T.P., Alderbourn, A., Anderson, R.D., Parkkinen, S., Bergman, P., Haugen, T.H., Petterson, U. and Turek, L.P. (1990). The New Biologist 2, 450-463. 36. Nakshatri, H., Pater, M.M. and Pater, A. (1990). Virology 178, 92-103. 37. Phelps, W.C., Yee, C.L., Muenger, K.and Howley, P.W. (1988). Cell 53, 539-547. 38. Storey, A., Pim, D., Murray, A., Osborn, K., Banks, L. and Crawford, L. (1988). EMBO Journal 7, 1815-1820. 39. Gius, D. and Laimins, L.A. (1989). J. Virol. 63, 555-563. 40. Perry, L.J., Rixon, F.J., Everett, R.D. Frame, M.C. and McGeogh, D.J. (1986). J. Gen. Virol. 67, 2365-2380. 41. Darbre, P., Page, M. and King, R.J.B. (1986). Mol. Cell. Biol. 6, 2847-2854.
