Journal of General Virology (1990), 71, 2457 2462. Printed in Great Britain

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Human papillomavirus type 8 contains cis-active positive and negative transcriptional control sequences Holger Reh and Herbert Pfister* Institut f ~ r Klinische und Molekulare Virologie, Friedr&h-Alexander-UniversitiJt,

D-8520 Erlangen, F.R.G.

Human papillomavirus type 8 (HPV-8) is one aetiological agent of macular and flat wart-like lesions in patients with epidermodysplasia verruciformis and appears to be closely linked to skin carcinogenesis. A 1-2 kb region of the genome, which was previously shown to contain a viral E2-dependent enhancer, was progressively shortened from both ends with Bal 31. The resulting fragments were tested for their ability to stimulate chloramphenicol acetyltransferase (CAT) expression from the simian virus 40 (SV40) promoter. This analysis showed a complex interaction between cis-active, positive and negative control elements

located throughout the non-coding region and the flanking reading frames. Two separate positively acting sequences significantly stimulated expression only in cooperation with a third region, which led to 12-fold, E2-dependent enhancement on its own. A major negative element was not only active in the context of HPV-8 sequences, but also down-regulated SV40 enhancer-promoter-driven CAT expression when cloned downstream of the transcription unit. It acted at the transcriptional level as shown by RNase protection assays and can therefore be regarded as a cis-acting silencer of transcription.

Human papillomaviruses (HPV) induce benign tumours of the skin and mucosa, some of which may progress to carcinomas. HPV DNA can also frequently be detected in healthy epithelium, which indicates that many HPV infections remain clinically inapparent (Pfister, 1988). The establishment of a latent persistent infection will depend on sophisticated regulatory mechanisms as exemplified by herpesviruses or human immunodeficiency virus. Most cis-active control sequences of HPVs were mapped within non-coding regions, so-called long control regions (LCRs) between the late gene L1 encoding the major capsid protein and the early transforming gene E6 (Iftner, 1990). Nothing is known so far about the origins of HPV D N A replication, but both constitutive (Cripe et al., 1987; Swift et al., 1987; Gius et al., 1988; Hirochika et al., 1988 ; Chin et al., 1989; Steinberg et al., 1989) and inducible (Gloss et al., 1987; Hirochika et al., 1987, 1988; Phelps & Howley, 1987; Seeberger et al., 1987) enhancers as elements of R N A polymerase II promoters have been identified. The major trans-activating protein is the viral E2 gene product which binds to the palindrome ACCN6GGT which occurs several times within the LCRs of all HPVs (Iftner, 1990). When present in two or more copies, the E2-binding palindrome is sufficient to constitute an E2-

dependent enhancer (Hawley-Nelson et al., 1988). A negative control of transcription was described for amino-terminally truncated E2 proteins, which compete with the activating full-length E2 for the common palindromic binding site (Giri & Yaniv, 1988). In genital HPVs, even the full-length E2 protein can function as a transcriptional repressor, presumably by sterically hindering initiation when binding to palindromes between the viral CAAT and TATA elements (Thierry & Yaniv, 1987; Chin et al., 1988). A cis-active negative regulatory element was identified in the LCR of an HPV-6 subtype (Wu & Mounts, 1988). HPV-8 induces flat warts and macular lesions in patients with the hereditary disease epidermodysplasia verruciformis (EV) and persists in skin cancers of these patients (Pfister et al., 1981 ; Orth, 1987). In the normal population there are no clinical manifestations of HPV-8 infection, although antibody screening suggests a prevalence of at least 10 ~ (Pfister et al., 1981). HPV-8 (Fuchs e t a l . , 1986) and related EVassociated HPVs (Krubke et al., 1987) are distinguished from all other HPVs by their short control regions (400 bp compared to 1000 bp). The HPV-8 LCR contains four ACCN6GGT palindromes and constitutes an E2dependent enhancer (Seeberger et al., 1987). No constitutive enhancer activity could be detected in C127 mouse fibroblasts or human HeLa cells.

0000-9647 © 1990SGM

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I n view of the c a r c i n o g e n i c activity of H P V - 8 in the context of a specific genetic b a c k g r o u n d of the host, it is i m p o r t a n t to u n d e r s t a n d the control o f viral t r a n s c r i p t i o n in more detail. T o dissect the H P V - 8 e n h a n c e r region,

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Fig. 1. Generation of Bal 31 deletion mutants within the HPV-8 NCR and cloning into a enhancer-less eukaryotic expression vector for the bacterial CAT gene (pRZ2). Linearized plasmid (35 p.g)was incubated in a total volume of 50 ~1 containing 12 mM-CaC12, 12 mM-MgC12, 0-4 M-NaC1, 40 mM-Tris HC1 pH 8.0, 2 mM-EDTA and 4 units of Bal 31 (New England Biolabs) at 30 °C. Aliquots of 5 ~tl were taken each min. To inactivate the exonuclease, EGTA was added to a final concentration of 20 mM prior to extraction with phenol/chloroform (1:1) and ethanol precipitation. After a. second digestion with an appropriate restriction endonuclease, fragments of interest were separated on 1% agarose gels and recovered by electroelution. After phenol treatment and ethanol precipitation the fragments were ready for ligation according to standard protocols (Maniatis et al., 1982). P, SV40 promoter; grey boxes, polylinker sequences; ampr, ampicillin resistance locus; ori, origin of replication.

tt~

tr~

pHR5 + - 20.4 pHRI5 -- 19-9 pHRII --4.0 pHRI3 --2"3 pHR14 -- 3"9

p H R 5 - - 19.7 pHR26 - 8.4 pHR22 - 20-1 pHR23 - 12.1 pHR24 --7-9 pHR25 - I 1-5

Fig. 2. CAT expression under the control of HPV-8 DNA fragments. A partial physical map of the HPV-8 genome is presented on top, showing the position of the ORFs L 1 (3' end) and E6 (5' end) and the non-coding sequences between. Arrows point to the E2-binding sites and grey boxes indicate 33 bp and 29 bp motifs conserved among EV-associated HPVs. The position and size of the fragments tested is given beneath. Nucleotides are numbered according to Fuchs et al. (1986). For transient expression assays, cells were plated 24 h prior to transfection (3 x 106 cells per 10 cm dish) and incubated in Dulbecco's modified Eagle's medium (Gibco) supplemented with 5~ foetal calf serum (Gibco), penicillin (120 ~tg/ml)and streptomycin (120 ~tg/ml).Calcium phosphate DNA precipitates were prepared according to the method of Graham & van der Eb (1973). A glycerolshock was performed 4 h after adding the precipitate using 15% glycerolin phosphate-buffered saline. Cells were harvested 48 h after transfection. Protein extracts were prepared and assayed for CAT activity according to the method of Gorman et al. (1982). Extracts were adjusted for different protein concentrations using a commercial protein assay (Bio-Rad). Chloramphenicol and its acetylated forms were separated by thin-layer chromatography, visualized by autoradiography and measured by liquid scintillation. The relative enzyme activity induced by the various constructs is given to the right.

two series of Bal 31 deletion m u t a n t s were generated. The p l a s m i d p P F 4 3 c o n t a i n s the H P V - 8 E c o R I - E c o R V f r a g m e n t s p a n n i n g the entire n o n - c o d i n g region ( N C R ) a n d parts of the L1 a n d E6 o p e n r e a d i n g frames ( O R F s ) cloned into the c o r r e s p o n d i n g cleavage sites of p I C 1 9 H (Marsh et al., 1984). W e linearized p P F 4 3 with E c o R I or E c o R V a n d then digested with Bal 31. T h e shortened H P V - 8 f r a g m e n t s were cut free using B g l I I or B a m H I , a n d ligated to the e n h a n c e r - d e p e n d e n t c h l o r a m p h e n i c o l acetyltransferase ( C A T ) vector p R Z 2 , w h i c h was cleaved w i t h i n the polylinker u p s t r e a m of the s i m i a n virus 40 (SV40) p r o m o t e r (Fig. 1). T h e vector p R Z 2 is a derivative of pSV2cat ( G o r m a n et al., 1982) c o n t a i n i n g the SV40 early promoter, the bacterial C A T gene, the SV40 small t i n t r o n a n d the SV40 p o l y a d e n y l a t i o n site cloned into p I C 2 0 H (Marsh et al., 1984). R e c o m b i n a n t plasmids with H P V - 8 inserts o f different length were selected a n d the exact m a p positions of the deletions (Fig. 2) were confirmed by s e q u e n c i n g by the m e t h o d of M a x a m & G i l b e r t (1980). T h e deletions from the E c o R I

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(a) (c) 1 2

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Fig. 3. CAT expression from pHR31 and pRR52 (a) determined by enzyme assay (b) and RNase protection (c). Riboprobes were synthesized from 1 ~tg linearized plasmid DNA under standard conditions (Green et al., 1983) in the presence of 5 units RNAguard (Pharmacia), 50 ~tCi [~-32p]UTP (400 Ci per retool) and 10 units T7 or SP6 polymerase. The template was removed by digestion with 40 units of RNase-free DNase (Boehringer Mannheim). After phenol treatment and ethanol precipitation the pellet was dissolved in a hybridization buffer containing 80~ formamide, 40 mM-PIPES pH 6.4, 400 mMNaCI and 1 mM-EDTA(sp. act. 250000 c.p.m, per 10 rtl). Before use in RNase protection assays (Zinn et al., 1983),an aliquot was run on a 7 Murea, 6~ polyacrylamide gel to ascertain that the probes contained at least 90% full-length transcripts. (b). pRR52, lanes I and 2; pHR3I, lanes 3 and 4. Cellular RNA (10lag) was dissolved in 10~tl of the adjusted riboprobe. After denaturing at 70 °C for 10 min, the probes were incubated in a water bath at 37 °C for at least 12 h. Then 300 ~tl of freshly prepared, ice-cold RNase mixture was added, containing RNase A at 6 ~tg per ml and RNase T1 at 12 units per ml in 300 mMNaCI, 5 mM-EDTA, 10 mM-Tris-HC1 pH 7-5. After a second incubation at 37 °C for 1 h, SDS and proteinase K were added to denature RNases, which were finally removed by the following phenol treatment. After precipitation, probes were run on a 6~ polyacrylamide urea gel and the protected RNAs were visualized by autoradiography (c). The upper arrow points to bands corresponding to

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and E c o R V sites have been cloned in the opposite orientation relative to the promoter. Only deletions with the same orientation will be directly c o m p a r e d in the present report. T h e entire E c o R I - E c o R V f r a g m e n t was cut from the multiple cloning site o f p P F 4 3 and inserted in both sense and antisense orientations into p R Z 2 to create p H R 5 + and p H R 5 - . To test for transient expression of the C A T gene, the D N A s were transfected into C82 cells, w h i c h are derivatives of C127 mouse fibroblasts expressing the H P V - 8 E2 gene under control o f a retroviral large terminal repeat (Iftner et al., 1988). T h e C A T activities shown in Fig. 2 are m e a n values of seven independently performed transfections normalized to the baseline activity o f p R Z 2 . Care was taken that the chloramphenicol conversion was in the range of 1 ~ to 6 0 ~ to guarantee a linear correlation between chloramphenicol acetylation and C A T e n z y m e activity. T h e entire E c o R I E c o R V H P V - 8 D N A f r a g m e n t present in p H R 5 enh a n c e d C A T expression about 20-fold w h e n transactivated by the viral E2 protein. The deletion o f the E2binding site at position 7327 ( p H R 1 5 ) had almost no effect, but the loss o f two more palindromes (pHR11) led to a d r a m a t i c decrease in e n h a n c e r activity. The low activity o f p H R 1 1 was surprising in view o f the presence o f two E2-binding sites. T h e palindromes at positions 7649 and 22, present in p H R 1 1 , showed no e n h a n c e r activity at all when tested in the S a u 3 A f r a g m e n t f r o m position 7604 to position 108, which is in contrast to the findings of Hawley-Nelson et al. (1988), who demonstrated that two palindromes are sufficient to create an E2-dependent enhancer. This difference m a y be due to a low E2 affinity o f the palindromes at positions 7649 and 22 or to surrounding repressor sequences. A deletion o f nucleotides 243 to 555 (compare p H R 5 to p H R 2 6 ) and 5 to 163 (compare p H R 2 2 to p H R 2 3 ) led to a m a r k e d decrease of e n h a n c e r activity, which indicates that additional positive regulatory elements exist in these regions. There are three pieces of evidence for an increase of C A T expression due to the deletion of H P V - 8 sequences, which is consistent with the existence of cis-active negative control elements. These are defined by the slightly lower activity o f p H R 2 4 in c o m p a r i s o n to p H R 2 5 (70~o) and the lower activities o f p H R 1 3 c o m p a r e d to p H R 1 4 ( 6 0 ~ ) and p H R 2 6 c o m p a r e d to p H R 2 2 (42~), which point to a silencer around the 5' terminus of O R F E6. This is adjacent to the S a u 3 A fragment which is supposed to contain sequences responsible for the repression o f its two E2-binding CAT mRNA fragments initiated at the SV40 promoter and the lower arrow to the band corresponding to/~-globin mRNA transcribed from the cotransfected SVOVEC vector. Lane 1 pRR52; lane 2, pHR31.

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palindromes. To obtain further experimental evidence for silencer activity in this region of the HPV-8 genome, we cloned the HpaI-AluI fragment (positions 1 to 243) downstream of a SV40 promoter-enhancer-driven CAT gene (Fig. 3). Restriction endonuclease digests and Southern blot analysis confirmed that the fragment was inserted in the sense orientation. This plasmid was compared in transfection experiments with the parent vector, pRR52, which expresses the CAT gene under the control of the SV40 promoter-enhancer. Extracts from pHR31-transfected cells showed only 30 ~o CAT activity in comparison to pRR52. RNase protection experiments were performed to assess whether the reduction in CAT expression is due to reduced levels of correctly initiated CAT mRNAs. For this purpose, a fragment composed of the entire SV40 early promoter and the 5' part of the cat gene was cloned into pGEM1. Using the T7 RNA polymerase promoter, a 570 bp antisense riboprobe could be transcribed in vitro after appropriate linearization of the plasmid. The length of the protected RNA fragments proved the correct initiation of the CAT messengers at the SV40 early start sites and the different intensities of the signals obtained with RNA from pHR31- and pRR52-transfected cells, respectively, were in agreement with the data from the CAT assays. The cells were cotransfected in these experiments with SVOVEC (Westin etal., 1987) as an internal control of transfection efficiency. SVOVEC is an SV40 enhancer-driven expression vector for the rabbit fl-globin gene and its transcript could be detected in the protection reactions together with CAT mRNA using an SP6 riboprobe transcribed from SP6ffI'S (Westin et al., 1987). The analysis of fl-globin expression revealed almost identical transfection efficiencies in all assays. The data on CAT gene expression from pHR31 in comparison to pRR52 confirmed the existence of a transcriptional silencer within the HpaI-AluI (positions 1 to 243) fragment of HPV-8. A cis-active, negative control element in papillomaviruses has been so far described for only HPV-6 (Wu & Mounts, 1988). The 413 bp fragment with silencer activity differed from the homologous non-silencing DNA of a closely related subtype by a 3 bp deletion and the exchange of one nucleotide. The ACTGT core defined by these mutations was not detectable in the HPV-8 silencer. This HPV-8 silencer did have, however, some similarity to other silencer elements (Brand et al., 1985, 1987; Laimins et al., 1986; Wu & Mounts, 1988; Burt et al., 1989; Cao et al., 1989; Pech et al., 1989) as summarized in Fig. 4. A highly similar AT-rich region was also observed in the silencer of the yeast silent mating type locus HMR (Brand et al., 1985, 1987) and was shown to represent an origin of yeast DNA replication (Brand et al., 1987). This

aac~taa~TTT~AT~AGTGT~A~Gtgcggt~tgAAAATTT~tTaAtCAtAAGTT~TTATTGC~A~a~C~aT~t~t~ta~cATGtT~WG~CTgTA TTTCcTCAGTGT -2.8 kb B -361 CATgCCTGTgfiCA~ ~347 T

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Fig. 4. Sequence similarity between the HPV-8 H p a I - A l u I fragment (positions 1 to 243) and silencer elements of different genes (Grindlay et al., 1984; Brand et al., 1985, 1987; Lakshmikumaran et al., 1985; Laimins et al., 1986; Wu & Mounts, 1988; Burt et al., 1989; Cao et al., 1989; Pech et al., 1989). Uppercase letters designate identical nucleotides. REV indicates reverse orientation relative to the direction of mRNA transcription. The line above the nucleotides marks the conserved 29 bp (see text). ~ stands for the upstream sequences of the human renin gene (Burt et al., 1989); fl for rat insulin 1 gene upstream sequences (Lakshmikumaran et al., 1985; Laimins et al., 1986); z for the PDGF-2 promoter silencer element (Pech et al., 1989); e for the human e-globin upstream sequences (Grindlay et al., 1984; Cao et al., 1989) a n d / t for the yeast HMR silencer (Brand et al., 1985, 1987).

could indicate an interdependence of transcription control and replication. The similarity with other silencers was found throughout the whole HpaI-AluI fragment of HPV-8, including the 29 bp motif which appears to be highly conserved among HPV-8-related, EV-associated papillomaviruses (Krubke et al., 1987). In conclusion, there are both positive and negative transcriptional control elements in HPV-8 which appear to be connected and interact in a complex way. One enhancer resides within the Ll-proximal non-coding sequences and extends into ORF L1. This genome segment contains three E2-binding palindromes (positions 7327, 7401 and 7491) and a sequence of 33 nucleotides (positions 7425 to 7457), which was shown to be highly conserved among and specific for EVassociated HPVs (Krubke et al., 1987). At least two additional positive control elements (positions 7573 to 163 and 243 to 555) exert marked stimulatory effects in cooperation with the major enhancer. Both were only marginally active when tested in the sense orientation on their own (pHR14 and pHR11). It is therefore possible that they counteract the silencer sequences rather than truly activate transcription. The overall enhancer activity seems to be considerably weakened by the silencer between positions 1 and 243, which is able to downregulate even the strong SV40 enhancer by 70~. A second negative control sequence may be interspersed between positions 7474 and 7573. It remains to be established whether this is related to a putative PEA2 binding site overlapping the E2 palindrome 7491, as PEA2 was identified as a labile repressor of the polyomavirus enhancer (Waslyk et al., 1988). It is noteworthy that the control elements of HPV-8 extend

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into ORFs L1 and E6 and are not confined to the N C R , which should be considered in the context of its small size relative to HPVs not associated with EV. The arrangement of enhancers and silencers in HPV-8 will allow a fine adjustment of gene expression, which may be relevant to its usually inapparent persistence in the normal population in contrast to its pathogenicity in EV patients. To achieve a better understanding of the role of these elements it will be essential to evaluate their effects on authentic HPV-8 transcription and their activity in different host cells. We thank P. G. Fuchs, R. Riiger, W. Schaffner, G. Westin and R. Zimmermann for providing pPF43, pRR52, SVOVEC, pSP6ffI'S and pRZ2. We are indebted to S. Iguchi-Ariga and H. Fickenscher for helpful discussion. The devoted technical assistance of A. Bergauer is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft (Pf 123/3-2).

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GLOSS, B., BERNARD, H. U., SEEDORF, K. & KLOCK, G. (1987). The upstream regulatory region of the human papillomavirus 16 contains an E2 protein-independent enhancer which is specific for cervical carcinoma cells and regulated by glucocorticoid hormones. EMBO Journal 6, 3735 3743. GORMAN, C. M., MOFFAT, L. F. & HOWARD, B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Molecular and Cellular Biology 2, 1044-1051. GRAHAM, F. L. & VAN DER EB, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456467. GREEN, M. R., MANIATIS, T. & MELTON, D. A. (1983). Human flglobin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell 32, 681-694. GRINDLAY, G. J., LANYON, W. G., ALLAN, M. & PAUL, J. (1984). Alternative sites of transcription initiation upstream of the canonical cap-site in human gamma-globin and beta-globin genes. Nucleic Acids Research 12, 1811 1821. HAWLEY-NELSON, P., ANDROPHY, E. J., LOWY, D. R. & SCHILLER,J. T. (I988)_ The specific DNA recognition sequence of the bovine papillomavirus E2 protein is an E2-dependent enhancer. EMBO Journal 7, 525-531. HIROCH1KA, H., BROKER, T. R. & CHOW, L. T. (1987). Enhancers and transacting E2 transcriptional factors of papillomaviruses. Journal of Virology 61, 2599 2606. H1ROCHIKA, H., HIROCHIKA, R., BROKER, T. R. & CHOW, L. T. (1988). Functional mapping of the human papillomavirus type 11 transcriptional enhancer and its interaction with the trans-acting E2 proteins. Genes and Development 2, 54-67. lrTNER, T. (1990). Papillomavirus genomes: sequence analysis related to functional aspects. In Papillomaviruses and Human Cancer, pp. 181-202. Edited by H. Pfister. Boca Raton: CRC Press. IFTNER, T., BIERFELDER, S., CSAPO, Z. & PFISTER, H. (1988). Involvement of human papillomavirus type 8 genes E6 and E7 in transformation and replication. Journal of Virology 62, 3655-3661. KRUBKE, J., KRAUS, J., DELIUS, H., CHOW, L., BROKER, T., ]FTNER, T. & PFISTER, H. (1987). Genetic relationship among human papillomaviruses associated with benign and malignant tumours of patients with epidermodysplasia verruciformis. Journal of General Virology 68, 3091-3103. LAKSHMIKUMARAN,M. S., D'AMBROSIO, E., LAIMINS,L. A., LIN, D. T. & FURANO, A. V. (1985). Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin locus. Molecular and Cellular Biology 5, 2197-2203. LAIMINS, L., HOLMGREN-K6NIG, M. & KHOURY, G. (1986). Transcriptional "silencer" element in rat insulin 1 gene locus. Proceedings of the National Academy of Sciences, U.S.A. 83, 3151-3155. MANIATIS, T., FRITSCH, E. F. & SAMBROOK, J. (1982). Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory. MARSH, L. J., ERFILE, M. & WYKES, E. J. (1984). The pIC-plasmid and phage vectors with versatile cloning sites for recombinant selection by insertion inactivation. Gene 32, 481-485. MAXAM, A. M. & GILBERT, W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. Methods in Enzymology 65, 499-560. ORTH, G. (1987). Epidermodysplasia verruciformis. In The Papovaviridae, pp. 199-244. Edited by N. P. Salzman & P. M. Howley. New York: Plenum Press. PECH, M., RAO, C. D., ROBBINS, K. C. & AARONSON, S. A. (1989). Functional identification of regulatory elements within the promoter region of platelet-derived growth factor 2. Molecular and Cellular Biology 9, 396-405. PFtSTER, H. (1988). The papillomaviruses. In Laboratory Diagnosis of Infectious Diseases. Principles and Practice, vol. II, pp. 301-316. Edited by A. Balows, W. J. Hausler Jr & E. H. Lennette. New York: Springer-Verlag. PFISTER, H., NURNBERGER, F., GISSMANN, L. & ZUR HAUSEN, H. (1981). Characterization of a human papillomavirus from epidermodysplasia verruciformis lesions of a patient from Upper-Volta. International Journal of Cancer 27, 645 650.

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Short communication

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(Received 30 April 1990; Accepted 4 July 1990)