Journal of General Virology (1992), 73, 1833-1838. Printed in Great Britain
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Sense and antisense transcripts of human papiHomavirus type 16 in cervical cancers Vera Vormwaid-Dogan, 1 Bertram Fischer, 2 Hubertus Bludau, 1 Ulrich Karl Freese, 1 Lutz Gissmann, 1 Dagmar Glitz, 1 Elisabeth Schwarz 1 and Matthias Diirst 1. Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-6900 Heidelberg and 2Department of Biochemistry, Boehringer/Mannheim, D-6800 Mannheim, Germany
Human papillomavirus type 16 (HPV-16) transcription was analysed in one squamous cervical carcinoma by cDNA cloning and DNA sequencing, and in eight additional squamous cervical carcinomas and 11 precancerous lesions by R N A - R N A in situ hybridization. The nucleotide sequences of the cDNA clones revealed structures of early HPV-16 mRNAs (E6*E7-E1 E4-E5) in agreement with data reported for other premalignant and malignant tumours, cDNA clones possibly representing viral RNA of antisense orientation were also detected. These RNAs included sequences of the upstream regulatory region, part of
the early and the late region of the genome. In three of eight squamous cervical carcinomas examined by in situ hybridization, signals specific for viral antisense RNA were also found. The antisense RNAs had a predominantly nuclear localization. Viral antisense RNA could not be detected in any of 11 HPV-16positive premalignant lesions. The expression of HPV antisense RNA is likely to be linked to viral integration into the host genome. The possible effects of viral antisense transcription with regard to tumour progression remain to be determined.
Human papillomavirus type 16 (HPV-16) together with a subset of other genital types are thought to play a key role in the genesis of cervical cancer. Evidence in support of this is largely of an experimental nature (for review see zur Hausen, 1989). The DNA of HPV types associated with anogenital carcinomas, particularly that of HPV-16 and HPV-18, but also that of some other cancer-linked HPV types, is able to immortalize normal human foreskin and cervical keratinocytes, and to extend the lifespan of fibroblasts (Diirst et al., 1987; Pecoraro et al., 1989; Pirisi et al., 1987; Schlegel et al., 1988; Woodworth et al., 1989). The viral genes E6 and E7 have been specifically identified as viral oncogenes responsible for an altered proliferation of the target cell (Barbosa et al., 1991; Hawley-Nelson et al., 1989; Mfinger et al., 1989a; Watanabe et at., 1989). Recent experiments suggest that an interaction of these viral proteins with cellular regulatory proteins could at least in part be responsible for these phenotypic alterations (Barbosa et al., 1990; Dyson et al., 1989; Gage et al., 1990; M/inger et al., 1989b; Werness et al., 1990). Maps of HPV-16-specific transcripts in cell lines derived from premalignant and malignant lesions have been produced but differences which might correlate with progression of cervical neoplasia were not apparent (Baker et al., 1987; Doorbar et al., 1990; Schneider-Maunoury et al., 1990; Schwarz et
al., 1985). Irrespective of the malignant potential of the cell line involved, the majority of transcripts encoding E6 and E7 were initiated from the viral promoter, P97, just downstream of the upstream regulatory region (URR). Three types of E6-E7 mRNAs were identified. Of these, two mRNA species (E6*I and E6*II) are spliced within the E6 open reading frame (ORF) and are presumed to encode the E7 protein which is abundantly expressed in HPV-16-positive cell lines (Smotkin & Wettstein, 1986; Smotkin et al., 1989). As HPV transcription is strongly dependent on epithelial cell differentiation (Bedell et al., 1991 ; Crum et al., 1988, 1990; Diirst et al., 1991 ; Stoler et al., 1990) some investigators have also analysed the viral transcriptional pattern in precursor lesions and cervical carcinomas (Cornelissen et al., 1990; Dilts et al., 1990; Johnson et al., 1990; Sherman et al., 1992; Shirasawa et al., 1988). However most of these studies have concentrated on the early region of the viral genome and were performed by reverse transcription and polymerase chain reaction. We have studied a cervical carcinoma which harboured both episomal and integrated HPV-16 DNA viral transcript structures by cDNA cloning and sequence analysis. For this purpose a cDNA library was constructed in 2 NM1149 using poly(A)÷ RNA according to standard procedures (Sambrook et al., 1989). The library
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Fig. 1. Schematic representation of the eDNA clones of an HPV-16-positive cervical carcinoma. The organization of the HPV-16 genome including adjustments in the E10RF (Matsukuraet al., 1986), the E50RF (Halbert & Galloway, 1988; Bubb et al., 1988)and in the URR E2 protein-bindingsite (Romanczuket al., 1990)is shown on top. Open boxes refer to ORFs. The positionof the firstATG in each ORF is marked with vertical lines. The nueleotide positions of 5' and 3' ends of each eDNA clone as well as splice donor and acceptor sites are indicated. V indicates spliced-out sequences. Zig-zaglines depict cellular sequences. (a) eDNA clones which map to the early regionof the viral genome. The oligo(dC)tail at the 5"end of each eDNA clone indicates the 5' end of the correspondingRNA. (b) eDNA clones of the URR, the early and the late regionof antisense orientation. The localizationof oligo(dC)tails indicates that the corresponding RNAs are complementaryto the coding strand of the viral genome. (c) eDNA clones of uncertain orientation.
was screened for HPV-16-positive clones by plaque hybridization using the entire cloned genome of HPV-16 as a hybridization probe. Fourteen clones were isolated and were mapped by restriction enzyme digestion and hybridization using subgenomic HPV-16 D N A fragments. Each c D N A clone was sequenced in both directions, using the dideoxynucleotide chain termination method of Sanger et al. (1977). In comparison to the HPV-16 prototype D N A sequence (Seedorf et al., 1985) numerous sequence alterations were found among the c D N A clones which are summarized in Table 1. Some sequence alterations were confined to individual c D N A clones and were not found in another clone spanning the same genomic region. This may be explained on the basis of two different HPV-16 populations within the same cancer or
may be attributed to cloning artefacts. N o D N A sequence mutations were found for the E6 and E 7 0 R F which is suggestive of selective pressure on the integrity of these genes. The locations of 5' and 3' ends and the splicing patterns of the c D N A s are shown in Fig. 1. For unknown reasons only one of the c D N A inserts contained an oligo(dA) sequence at its 3' end, although oligo(dT) had been used to prime reverse transcription. In contrast, all but three c D N A s had oligo(dC) at their 5' terminus. Since first-strand synthesis products had been tailed with dG, the oligo(dG) tail was used to determine the polarity of m R N A s from which the c D N A s were derived, c D N A clones 1, 3a, 9, 16 and 21 represent early viral m R N A s of sense orientation. Note the position of oligo(dC) tails at the 5' ends of c D N A clones in Fig. 1 (a) containing a spliced E6*I ORF and an unspliced E6
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Table 1. cDNA sequence mutations ORF E1 E5
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L1
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Mutation
cDNA clone
877 3813t 3866t 3903, 4155 4162 4163 4182t 4226t 4363 46731" 4736 4936 5184 5224 5246 5274 5276 5993 5996 6370 6371 6900* 6951, 7523t 7861,
del GCA A~G T-*A ins T ins T C-*T ins T A-.T T-*C A-~T G-~C G-,A G-~A G-*A A--,C G--*C ins C ins C C~T del T G~C C~G ins CAT del GAT A~C del A
3a 3a,9,16,21 3a,9,16,21 3a,9,16,21 3a 16 16 3a.16 6,7 6,7 4,6,7 4 5,6,7 4 5,6,7 5,6,7 4 4 5 5 14 14 5,11,18 5~11,18 5 5
* Nucleotide positions and the nature of each mutation (del, deletion; --,, change; ins, insertion) based on the HPV-16 prototype sequence (Seedorf et al., 1985) are listed. t Mutations detected in more than one cDNA clone which probably reflect natural sequence divergence among different HPV-16 isolates. , Mutations reflecting errors in the published HPV-176 DNA sequence. An additional thymidine at nt 3903 gave rise to a full-length E 5 0 R F as described by Halbert & Galloway (1988) and Bulb et al. (1988). A deletion of adenine at nt 7861 (Romanczuk et al., 1990) generated another E2-binding site ACC(N6)GGT (Spalhotz et al., 1987) in the URR whereas an insertion of CAT at nt 6900 and a deletion of GAT at nt 6951 led to a slight modification within the L1 ORF (Parton, 1990).
ORF, respectively. Their 5' ends seem to reflect the authentic 5' ends of the respective mRNAs because they map in the immediate vicinity of the major cap site at nucleotide (nt) 97 (Smotkin & Wettstein, 1986). For termination these transcripts probably utilized the early poly(A) signal at nt 4215. It is unlikely that the oligo(dA) tail of clone 1 represents an authentic early poly(A) site. These data are in general agreement with transcription data reported for other premalignant and malignant tumours or cell lines with the exception that the recently identified m R N A species E6*III (nt 226^3358), E1 ^El (nt 880^2709), El*^E2 (nt 1301^3358) and El*ALl (nt 1301^5637) (Dilts et al., 1990; Doorbar et al., 1990; Rohlfs et al., 1991; Sherman et a l . , 1992) were not detected. cDNA clones 19 and 45 represent another class of early region transcripts (Fig. 1 b). Both consist of viral
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and non-viral sequences and thus very likely represent virus-cell fusion transcripts. As deduced from the position of the oligo(dC) tails they are probably derived from antisense R N A [compare the positions of oligo(dC) tails of cDNA clones in (a) and (b) of Fig. 1]. This R N A species appears to be initiated in the E 1 0 R F at position 2364 and presumably terminates within the flanking cellular region. As there are no apparent consensus D N A sequences for splicing it is likely that nt 1482 actually reflects the break point within the viral genome at which integration into the host chromosome had taken place. Of the seven cDNA clones which map to the late region, four are clearly of antisense orientation (clones 4, 5, 6 and 18 in Fig. 1b). For the three remaining clones the orientation could not be determined with certainty (Fig. 1 c). The 5' ends of the putative antisense clones are heterogeneous. If they were to represent the authentic sites of transcriptional initiation, two of them would be located in the U R R (clones 5 and 18) and the other two in the 5" region of the L 1 0 R F (clones 4 and 6). However we cannot rule out the possibility that the 5' ends are artificial due to premature termination of first-strand cDNA synthesis which may occur as a result of secondary structures within the R N A template. Alternatively, activation of cryptic viral promoters due to conformational changes and/or cis-acting cellular elements as a consequence of viral integration are also plausible. The 3' ends of cDNA clones 4 and 6 are nearly identical (positions 4228 and 4224) and map to the 5' region of the L 2 0 R F . This particular region of the HPV16 genome contains poly(A)-rich stretches which probably permitted initiation of reverse transcription by oligo(dT), thus giving rise to cDNA clones with truncated 3' ends. This explanation may also apply to clone 7. Alternatively, although unlikely, the two viruscell hybrid cDNAs and the cDNAs with L1/L2 sequences could be derived from sin#e- or doublestranded genomic D N A fragments which might have contaminated the poly(A) + RNA. To examine whether synthesis of viral antisense R N A might be a common phenomenon in HPV-16-induced lesions, 11 premalignant lesions of the cervix (low grade and high grade cervical intraepithelial neoplasia) and eight additional squamous cervical cancers were analysed by R N A - R N A in situ hybridization for the presence of sense and antisense R N A using strandspecific probes which correspond to parts of the URR, the E6-E7 and the L1 region of the HPV-16 genome (for a detailed protocol, see Dfirst et al., 1991). Tissue sections were examined and photographed under a Zeiss Axiophot microscope equipped with a rotatable lightfield and darkfield condenser to reveal tissue histology and to enable good signal visualization respectively. The following observations were made. (i) E6-E7 m R N A could be
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Fig. 2. RNA-RNA in situ hybridization of an HPY-16-positive squamous cervical carcinoma. Low (a) and high (b) power magnifications of a haematoxylin-eosin-stained section are shown. Serial sections of the same tumour were hybridized with riboprobes derived from the URR (c,f) (nt 7455 to 7783),the E6-E70RF (d, g) (55 to 875) and the L10RF (e, h) (6153to 6788)of antisense orientation (c, d and e) and sense orientation (f, g and h) respectively.Hybridization probes of antisense orientation detect viral mRNA whereas hybridization probes of sense orientation detect viral antisense RNA. Darkfield optics were used for better signal visualization. Bar marker in (h) represents 50 ~tm for (b to h); that in (a) represents 100 p.m. detected in each biopsy (the term m R N A is used for HPV transcripts of sense orientation only) (see also Fig. 2 d). No m R N A s encoding structural (late) proteins were evident in high grade lesions and in the majority of cancers (see also Fig. 2e). (ii) Antisense R N A could be detected in each of three cancer biopsies with hybridization probes spanning, in part, the U R R , and the early and the late regions of the viral genome. Hybridization results of serial sections of one of the tumours are shown in Fig. 2 ( f , g and h). (iii) Viral antisense R N A signals were predominantly located in the nuclei of cells whereas in serial sections of the same tumour viral m R N A was detected in both cytoplasm and nucleus (Fig. 2 and data
not shown). (iv) Cells expressing viral antisense R N A invariably expressed viral m R N A . However in one of the biopsies there were also clusters of cancer cells which express m R N A only, indicative of clonal variability within a tumour (data not shown). A possible false interpretation of the in situ hybridization data due to partially denatured H P V D N A could be excluded since partially denatured D N A would give rise to hybridization signals with probes representing different parts of the genome both in sense and antisense orientation. This was not the case as demonstrated in Fig. 2. Moreover in serial sections of the same tumours RNase A digestion prior to hybridization prevented a positive hybridization signal. In the same experiment the integrity of the probe (residual RNase A could degrade the probe) was also monitored by treating serial sections with RNase A followed by heat denaturation prior to hybridization which permits the detection of viral D N A only (data not shown). The detection of HPV-16 antisense R N A s is in agreement with a recent report by Higgins et aL (1991). We have not detected viral antisense R N A in HPV-16-positive premalignant lesions which should predominantly harbour extrachromosomal viral D N A (Cullen et al., 1991; Diirst et al., 1985). Expression of viral antisense R N A therefore does not seem to be an intrinsic property of the episomal viral genome. Integration of viral D N A occurs frequently in cervical cancer (Dfirst et al., 1985) and is known to result in the production of virus-cell fusion m R N A s encoding viral genes and 3' cellular sequences (Schneider-G/idicke & Schwarz, 1986). Likewise, integration of viral D N A into a transcriptionally active region of the host chromosome could account for fusion transcripts which are initiated from a cellular promoter and which extend across the whole or part of the integrated viral genome thereby giving rise to viral sense and/or antisense transcripts. Indeed, such fusion transcripts were identified in a keratinocyte cell line immortalized by HPV-16 in vitro (Rohlfs et al., 1991). In this respect viral antisense RNA-containing tumours may turn out to be of value for the identification of cellular genes which have become inactivated or activated due to viral integration. It is not yet clear whether the synthesis of H P V antisense R N A in cervical cancers interferes with gene expression in analogy to other systems (for review see H61~ne & Toulm6, 1990; Takayama & Inouye, 1990). Under experimental conditions, inducible synthesis of R N A complementary to the HPV-18 E 6 - E 7 0 R F s in the HPV-18-positive cervical carcinoma cell line C4-I has been shown to correlate with a decrease in viral E7 protein and growth inhibition in vitro (von KnebelDoeberitz et aL, 1988). The same correlation, i.e. growth retardation concomitant with the expression of viral antisense RNA, was found when these cell lines were
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tested for tumorigenicity in nude mice (yon KnebelDoeberitz et al., 1992). This creates an apparent dilemma in trying to understand why antisense transcription, particulady of the E6 and E7 genes, is possible at all in cervical carcinomas. One likely explanation may be that in contrast to the naturally occurring antisense R N A in the cancers, the artificially expressed antisense R N A was correctly processed and transported to the cytoplasm. This may be a prerequisite for effective inhibition which may predominantly take place at the level of translation, rather than inhibition of R N A splicing and/or interference with m R N A transport to the cytoplasm. Clearly, despite high levels of viral antisense R N A in the nuclei of some cancers, viral m R N A is still transported to the cytoplasm, thereby ensuring the level of E6 and E7 expression necessary to maintain the transformed phenotype. Nevertheless, the possibility that viral antisense R N A might influence some biological properties of the carcinomas, such as tumour progression, requires further examination. We thank Dr Harald zur Hausen for encouragement and constant support. We are also grateful to Dr Achim Schneider for providing biopsy material. This work was supported in part by the Deutsche Forschungsgemeinschaft (grant D/i 162/1-2).
References BAKER, C. C., PHELPS, W. C., LINDGREN,V., BRAUN, M. J., GONDA, i . A. & HOWLEY, P M. (1987). Structural and transcriptional analysis of human papillomavirus type 16 in cervical carcinoma cell lines. Journal of Virology 61, 962-971. BARBO~A, M. S., EDMONDS,C., FISHER, C., SCHILLER,J. T., LOWY, D. R. & VOUSDEN,K. H. (1990). The region of the HPV 16 E7 oncoprotein homologous to adenovirus Ela and SV40 large T antigen contains separate domains for Rb binding and casein kinase II phosphorylation. EMBO Journal 9, 153-160. BARBOSA,M. S., VASS,W. C., LowY, D. R. & SCHILLER,J. T. (1991). In vitro biological activities of the E6 and E7 genes vary among human papillomaviruses of different oncogenic potential. Journal of Virology 65, 292-298. BEDELL, M. A., HUDSON,J. B., GOLUB,T. R., TURYK, M. E., HOSKEN, M., WILBANKS, G. D. & LAIMIN$, L. A. (1991). Amplification of human papillomavirus genomes in vitro is dependent on epithelial differentiation. Journal of Virology 65, 2254-2260. BUBB, V., MCCANCE,n. J. ~. SCHLEGEL,R. (1988). DNA sequences of the HPV 16 E 5 0 R F and the structural conservation of its encoded protein. Virology 163, 234-246. CORNELISSEN,M. T. E., SMITS,H. L., BRIt~T,M. A., VAN DEN TWEEL, J. G., STRUYK,A. P. H. B., VANDER NOORDAA,J. & TER SCHEGGET, J. (1990). Uniformity of the splicing pattern of the E6/E7 transcripts in human papillomavirus type 16-transformed human fibroblasts, human cervical premalignant lesions and carcinomas. Journal of General Virology 71, 1243-1246. CRUM, C. P., NuOVO, G., FRIEDMAN,D. & SILVERSTEIN,S. J. (1988). Accumulation of RNA homologous to the human papillomavirus type 16 open reading frames in genital precancers. Journal of Virology 62, 84-90. CRUM, C. P., BARBAR,S., SYMBULA,M., SNYDERS,K., SALEH,A. M. & ROCHE,J. K. (1990). Coexpression of the human papillomavirus type 16 E4 and L1 open reading frames in early cervical neoplasias. Virology 178, 238-246. CULLEN, A. P., REID, R., CAMPION, M. & LrRINCZ, A. T. (1991). Analysis of the physical state of different human papillomavirus
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ROHLPS, M., WINKENBACH,S., MEYER, S., RUPP, T. & Df2RST, M. (1991). Viral transcription in human keratinocyte cell lines immortalized by human papillomavirus type 16. Virology 183, 331-342. ROMANCZUK,H., THIERRY, F. & HOWLEY, P. i . (1990). Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P~o5 promoters. Journal of Virology 64, 2849-2859. SAMBROOK, J., FRITSCH, E. F. & MANIATIS, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory. SANGER,F., NICKLEN,S. & COULSON,A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A. 74, 5463-5467. SCHLEGEL, R., PHELPS, W. C., ZHANG, Y.-L. & BARBOSA,i . (1988). Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma. EMBO Journal 7, 3181-3187. SCHNEIDER-G.~d~ICKE,A. & SCHWARZ, E. (1986). Different human cervical carcinoma cell lines show similar transcription patterns of human papillomavirus type 18 early genes. EMBO Journal 5, 22852292. SCHNEIDER-MAUNOURY,S., PEHAU-ARNAUDET,G., BREITBURD,F. & ORTH, G. (1990). Expression of the human papillomavirns type 16 genome in SK-v cells, a line derived from a vulvar intraepithelial neoplasia. Journal of General Virology 71, 809-817. SCHWARZ, E., FREESE, U. K., GlSSMANN,L., MAYER, W., ROGGENBUCK, B., STREMLAU,A. & ZUR HAUSEN, H. (1985). Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature, London 314, 111-114. SEEDORF, K., KIL~MMER,G., Df3RST, i . , SLrHAI, S. & RSWEKAMP, W. G. (1985). Human papillomavirus type 16 DNA sequence. Virology 145, 181-185. SHERMAN,L., GOLAN,I., ALLOUL,N., DURST,M. & BARAM,A. (1992). Expression and splicing pattern of human papillomavirus type 16 mRNAs in preinvasive and invasive carcinomas of the cervix, in human keratinocytes immortalized by HPV 16, and in cell lines established from cervical cancers. InternationalJournal of Cancer 50, 356-364. SHIRASAWA, H., TOMITA, Y., KUBOTA, K., KASAI, T., SEKIYA, S., TAKAMIZAWA,H. & SIMIZU,B. (1988). Transcriptional differences of the human papillomavirus type 16 genome between precancerous lesions and invasive carcinomas. Journalof Virology 62, 1022-1027.
SMOTKn~, D. & WETTSTEIN,F. O. (1986). Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancerderived cell line and identification of the E7 protein. Proceedings of the National Academy of Sciences, U.S.A. 83, 4680-4684. SMOTKIN,D., PROKOPH,H. & WETTSTEIN,F. O. (1989). Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms. Journal of Virology 63, 1441-1 447. SPALHOLZ,B. A., LAMBERT,P. F., YEE, C. L. & HOWLEY,P. M. (1987). Bovine papillomavirus transcriptional regulation: localization of the E2-responsive elements in the long control region. Journalof Virology 61, 2128-2137. STOLER,M. H., RHODES,C. R., WHITBECK,A., CHOW,L. T. & BROKER, T. R. (1990). Gene expression of HPV 16 and 18 in cervical neoplasia. In Papillomaviruses, pp. 1-11. Edited by P. M. Howley & T. R. Broker. New York: Wiley-Liss. TAKAYAMA,K. M. & INOUYE,M. (1990). Antisense RNA. CRC Critical Reviews in Biochemistry and Molecular Biology 25, 155-184. YON KNEBEL-DOEBERITZ, i . , OLTERSDORF, T., SCHW/d?,Z, E. &. GISSMANN,L. (1988). Correlation of modified human papillomavirus early gene expression with altered cell growth in C4--1 cervical cancer cells. Cancer Research 48, 3780--3786. VON KNEBEL-DOEBERITZ,M., RITTMf3LLER,C., ZUR HAUSEN, H. & DORST, M. (1992). Inhibition of tumorigenicity of cervical cancer cells in nude mice by HPV E6-E7 antisense RNA. International Journal of Cancer (in press). WATANABE,S., KANDA,T. & YOSHIIKA,K. (1989). Human papillomavirus type 16 transformation of primary human embryonic fibroblasts requires expression of open reading frames E6 and ET. Journal of Virology 63, 965-969. WERNESS,B. A., LEVINE,A. J. & HOWLEY,P. M. (1990). Association of human papillomavirus type 16 and 18 E6 proteins with p53. Science 248, 76-79. WOODWORTH, C. D., DONINGER, J. & DIPAOLO, J. A. (1989). Immortalization of human foreskin keratinocytes by various human papillomavirus DNAs corresponds to their association with cervical carcinoma. Journal of Virology 63, 159-164. ZUR HAOSEN, H. (1989). Papillomaviruses as carcinomaviruses. Advances in Viral Oncology 8, 1-26.
(Received 6 January 1992; Accepted 2 March 1992)
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Sense and antisense transcripts of human papiHomavirus type 16 in cervical cancers Vera Vormwaid-Dogan, 1 Bertram Fischer, 2 Hubertus Bludau, 1 Ulrich Karl Freese, 1 Lutz Gissmann, 1 Dagmar Glitz, 1 Elisabeth Schwarz 1 and Matthias Diirst 1. Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-6900 Heidelberg and 2Department of Biochemistry, Boehringer/Mannheim, D-6800 Mannheim, Germany
Human papillomavirus type 16 (HPV-16) transcription was analysed in one squamous cervical carcinoma by cDNA cloning and DNA sequencing, and in eight additional squamous cervical carcinomas and 11 precancerous lesions by R N A - R N A in situ hybridization. The nucleotide sequences of the cDNA clones revealed structures of early HPV-16 mRNAs (E6*E7-E1 E4-E5) in agreement with data reported for other premalignant and malignant tumours, cDNA clones possibly representing viral RNA of antisense orientation were also detected. These RNAs included sequences of the upstream regulatory region, part of
the early and the late region of the genome. In three of eight squamous cervical carcinomas examined by in situ hybridization, signals specific for viral antisense RNA were also found. The antisense RNAs had a predominantly nuclear localization. Viral antisense RNA could not be detected in any of 11 HPV-16positive premalignant lesions. The expression of HPV antisense RNA is likely to be linked to viral integration into the host genome. The possible effects of viral antisense transcription with regard to tumour progression remain to be determined.
Human papillomavirus type 16 (HPV-16) together with a subset of other genital types are thought to play a key role in the genesis of cervical cancer. Evidence in support of this is largely of an experimental nature (for review see zur Hausen, 1989). The DNA of HPV types associated with anogenital carcinomas, particularly that of HPV-16 and HPV-18, but also that of some other cancer-linked HPV types, is able to immortalize normal human foreskin and cervical keratinocytes, and to extend the lifespan of fibroblasts (Diirst et al., 1987; Pecoraro et al., 1989; Pirisi et al., 1987; Schlegel et al., 1988; Woodworth et al., 1989). The viral genes E6 and E7 have been specifically identified as viral oncogenes responsible for an altered proliferation of the target cell (Barbosa et al., 1991; Hawley-Nelson et al., 1989; Mfinger et al., 1989a; Watanabe et at., 1989). Recent experiments suggest that an interaction of these viral proteins with cellular regulatory proteins could at least in part be responsible for these phenotypic alterations (Barbosa et al., 1990; Dyson et al., 1989; Gage et al., 1990; M/inger et al., 1989b; Werness et al., 1990). Maps of HPV-16-specific transcripts in cell lines derived from premalignant and malignant lesions have been produced but differences which might correlate with progression of cervical neoplasia were not apparent (Baker et al., 1987; Doorbar et al., 1990; Schneider-Maunoury et al., 1990; Schwarz et
al., 1985). Irrespective of the malignant potential of the cell line involved, the majority of transcripts encoding E6 and E7 were initiated from the viral promoter, P97, just downstream of the upstream regulatory region (URR). Three types of E6-E7 mRNAs were identified. Of these, two mRNA species (E6*I and E6*II) are spliced within the E6 open reading frame (ORF) and are presumed to encode the E7 protein which is abundantly expressed in HPV-16-positive cell lines (Smotkin & Wettstein, 1986; Smotkin et al., 1989). As HPV transcription is strongly dependent on epithelial cell differentiation (Bedell et al., 1991 ; Crum et al., 1988, 1990; Diirst et al., 1991 ; Stoler et al., 1990) some investigators have also analysed the viral transcriptional pattern in precursor lesions and cervical carcinomas (Cornelissen et al., 1990; Dilts et al., 1990; Johnson et al., 1990; Sherman et al., 1992; Shirasawa et al., 1988). However most of these studies have concentrated on the early region of the viral genome and were performed by reverse transcription and polymerase chain reaction. We have studied a cervical carcinoma which harboured both episomal and integrated HPV-16 DNA viral transcript structures by cDNA cloning and sequence analysis. For this purpose a cDNA library was constructed in 2 NM1149 using poly(A)÷ RNA according to standard procedures (Sambrook et al., 1989). The library
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Fig. 1. Schematic representation of the eDNA clones of an HPV-16-positive cervical carcinoma. The organization of the HPV-16 genome including adjustments in the E10RF (Matsukuraet al., 1986), the E50RF (Halbert & Galloway, 1988; Bubb et al., 1988)and in the URR E2 protein-bindingsite (Romanczuket al., 1990)is shown on top. Open boxes refer to ORFs. The positionof the firstATG in each ORF is marked with vertical lines. The nueleotide positions of 5' and 3' ends of each eDNA clone as well as splice donor and acceptor sites are indicated. V indicates spliced-out sequences. Zig-zaglines depict cellular sequences. (a) eDNA clones which map to the early regionof the viral genome. The oligo(dC)tail at the 5"end of each eDNA clone indicates the 5' end of the correspondingRNA. (b) eDNA clones of the URR, the early and the late regionof antisense orientation. The localizationof oligo(dC)tails indicates that the corresponding RNAs are complementaryto the coding strand of the viral genome. (c) eDNA clones of uncertain orientation.
was screened for HPV-16-positive clones by plaque hybridization using the entire cloned genome of HPV-16 as a hybridization probe. Fourteen clones were isolated and were mapped by restriction enzyme digestion and hybridization using subgenomic HPV-16 D N A fragments. Each c D N A clone was sequenced in both directions, using the dideoxynucleotide chain termination method of Sanger et al. (1977). In comparison to the HPV-16 prototype D N A sequence (Seedorf et al., 1985) numerous sequence alterations were found among the c D N A clones which are summarized in Table 1. Some sequence alterations were confined to individual c D N A clones and were not found in another clone spanning the same genomic region. This may be explained on the basis of two different HPV-16 populations within the same cancer or
may be attributed to cloning artefacts. N o D N A sequence mutations were found for the E6 and E 7 0 R F which is suggestive of selective pressure on the integrity of these genes. The locations of 5' and 3' ends and the splicing patterns of the c D N A s are shown in Fig. 1. For unknown reasons only one of the c D N A inserts contained an oligo(dA) sequence at its 3' end, although oligo(dT) had been used to prime reverse transcription. In contrast, all but three c D N A s had oligo(dC) at their 5' terminus. Since first-strand synthesis products had been tailed with dG, the oligo(dG) tail was used to determine the polarity of m R N A s from which the c D N A s were derived, c D N A clones 1, 3a, 9, 16 and 21 represent early viral m R N A s of sense orientation. Note the position of oligo(dC) tails at the 5' ends of c D N A clones in Fig. 1 (a) containing a spliced E6*I ORF and an unspliced E6
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Table 1. cDNA sequence mutations ORF E1 E5
L2
L1
URR
Nucleotide position*
Mutation
cDNA clone
877 3813t 3866t 3903, 4155 4162 4163 4182t 4226t 4363 46731" 4736 4936 5184 5224 5246 5274 5276 5993 5996 6370 6371 6900* 6951, 7523t 7861,
del GCA A~G T-*A ins T ins T C-*T ins T A-.T T-*C A-~T G-~C G-,A G-~A G-*A A--,C G--*C ins C ins C C~T del T G~C C~G ins CAT del GAT A~C del A
3a 3a,9,16,21 3a,9,16,21 3a,9,16,21 3a 16 16 3a.16 6,7 6,7 4,6,7 4 5,6,7 4 5,6,7 5,6,7 4 4 5 5 14 14 5,11,18 5~11,18 5 5
* Nucleotide positions and the nature of each mutation (del, deletion; --,, change; ins, insertion) based on the HPV-16 prototype sequence (Seedorf et al., 1985) are listed. t Mutations detected in more than one cDNA clone which probably reflect natural sequence divergence among different HPV-16 isolates. , Mutations reflecting errors in the published HPV-176 DNA sequence. An additional thymidine at nt 3903 gave rise to a full-length E 5 0 R F as described by Halbert & Galloway (1988) and Bulb et al. (1988). A deletion of adenine at nt 7861 (Romanczuk et al., 1990) generated another E2-binding site ACC(N6)GGT (Spalhotz et al., 1987) in the URR whereas an insertion of CAT at nt 6900 and a deletion of GAT at nt 6951 led to a slight modification within the L1 ORF (Parton, 1990).
ORF, respectively. Their 5' ends seem to reflect the authentic 5' ends of the respective mRNAs because they map in the immediate vicinity of the major cap site at nucleotide (nt) 97 (Smotkin & Wettstein, 1986). For termination these transcripts probably utilized the early poly(A) signal at nt 4215. It is unlikely that the oligo(dA) tail of clone 1 represents an authentic early poly(A) site. These data are in general agreement with transcription data reported for other premalignant and malignant tumours or cell lines with the exception that the recently identified m R N A species E6*III (nt 226^3358), E1 ^El (nt 880^2709), El*^E2 (nt 1301^3358) and El*ALl (nt 1301^5637) (Dilts et al., 1990; Doorbar et al., 1990; Rohlfs et al., 1991; Sherman et a l . , 1992) were not detected. cDNA clones 19 and 45 represent another class of early region transcripts (Fig. 1 b). Both consist of viral
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and non-viral sequences and thus very likely represent virus-cell fusion transcripts. As deduced from the position of the oligo(dC) tails they are probably derived from antisense R N A [compare the positions of oligo(dC) tails of cDNA clones in (a) and (b) of Fig. 1]. This R N A species appears to be initiated in the E 1 0 R F at position 2364 and presumably terminates within the flanking cellular region. As there are no apparent consensus D N A sequences for splicing it is likely that nt 1482 actually reflects the break point within the viral genome at which integration into the host chromosome had taken place. Of the seven cDNA clones which map to the late region, four are clearly of antisense orientation (clones 4, 5, 6 and 18 in Fig. 1b). For the three remaining clones the orientation could not be determined with certainty (Fig. 1 c). The 5' ends of the putative antisense clones are heterogeneous. If they were to represent the authentic sites of transcriptional initiation, two of them would be located in the U R R (clones 5 and 18) and the other two in the 5" region of the L 1 0 R F (clones 4 and 6). However we cannot rule out the possibility that the 5' ends are artificial due to premature termination of first-strand cDNA synthesis which may occur as a result of secondary structures within the R N A template. Alternatively, activation of cryptic viral promoters due to conformational changes and/or cis-acting cellular elements as a consequence of viral integration are also plausible. The 3' ends of cDNA clones 4 and 6 are nearly identical (positions 4228 and 4224) and map to the 5' region of the L 2 0 R F . This particular region of the HPV16 genome contains poly(A)-rich stretches which probably permitted initiation of reverse transcription by oligo(dT), thus giving rise to cDNA clones with truncated 3' ends. This explanation may also apply to clone 7. Alternatively, although unlikely, the two viruscell hybrid cDNAs and the cDNAs with L1/L2 sequences could be derived from sin#e- or doublestranded genomic D N A fragments which might have contaminated the poly(A) + RNA. To examine whether synthesis of viral antisense R N A might be a common phenomenon in HPV-16-induced lesions, 11 premalignant lesions of the cervix (low grade and high grade cervical intraepithelial neoplasia) and eight additional squamous cervical cancers were analysed by R N A - R N A in situ hybridization for the presence of sense and antisense R N A using strandspecific probes which correspond to parts of the URR, the E6-E7 and the L1 region of the HPV-16 genome (for a detailed protocol, see Dfirst et al., 1991). Tissue sections were examined and photographed under a Zeiss Axiophot microscope equipped with a rotatable lightfield and darkfield condenser to reveal tissue histology and to enable good signal visualization respectively. The following observations were made. (i) E6-E7 m R N A could be
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Fig. 2. RNA-RNA in situ hybridization of an HPY-16-positive squamous cervical carcinoma. Low (a) and high (b) power magnifications of a haematoxylin-eosin-stained section are shown. Serial sections of the same tumour were hybridized with riboprobes derived from the URR (c,f) (nt 7455 to 7783),the E6-E70RF (d, g) (55 to 875) and the L10RF (e, h) (6153to 6788)of antisense orientation (c, d and e) and sense orientation (f, g and h) respectively.Hybridization probes of antisense orientation detect viral mRNA whereas hybridization probes of sense orientation detect viral antisense RNA. Darkfield optics were used for better signal visualization. Bar marker in (h) represents 50 ~tm for (b to h); that in (a) represents 100 p.m. detected in each biopsy (the term m R N A is used for HPV transcripts of sense orientation only) (see also Fig. 2 d). No m R N A s encoding structural (late) proteins were evident in high grade lesions and in the majority of cancers (see also Fig. 2e). (ii) Antisense R N A could be detected in each of three cancer biopsies with hybridization probes spanning, in part, the U R R , and the early and the late regions of the viral genome. Hybridization results of serial sections of one of the tumours are shown in Fig. 2 ( f , g and h). (iii) Viral antisense R N A signals were predominantly located in the nuclei of cells whereas in serial sections of the same tumour viral m R N A was detected in both cytoplasm and nucleus (Fig. 2 and data
not shown). (iv) Cells expressing viral antisense R N A invariably expressed viral m R N A . However in one of the biopsies there were also clusters of cancer cells which express m R N A only, indicative of clonal variability within a tumour (data not shown). A possible false interpretation of the in situ hybridization data due to partially denatured H P V D N A could be excluded since partially denatured D N A would give rise to hybridization signals with probes representing different parts of the genome both in sense and antisense orientation. This was not the case as demonstrated in Fig. 2. Moreover in serial sections of the same tumours RNase A digestion prior to hybridization prevented a positive hybridization signal. In the same experiment the integrity of the probe (residual RNase A could degrade the probe) was also monitored by treating serial sections with RNase A followed by heat denaturation prior to hybridization which permits the detection of viral D N A only (data not shown). The detection of HPV-16 antisense R N A s is in agreement with a recent report by Higgins et aL (1991). We have not detected viral antisense R N A in HPV-16-positive premalignant lesions which should predominantly harbour extrachromosomal viral D N A (Cullen et al., 1991; Diirst et al., 1985). Expression of viral antisense R N A therefore does not seem to be an intrinsic property of the episomal viral genome. Integration of viral D N A occurs frequently in cervical cancer (Dfirst et al., 1985) and is known to result in the production of virus-cell fusion m R N A s encoding viral genes and 3' cellular sequences (Schneider-G/idicke & Schwarz, 1986). Likewise, integration of viral D N A into a transcriptionally active region of the host chromosome could account for fusion transcripts which are initiated from a cellular promoter and which extend across the whole or part of the integrated viral genome thereby giving rise to viral sense and/or antisense transcripts. Indeed, such fusion transcripts were identified in a keratinocyte cell line immortalized by HPV-16 in vitro (Rohlfs et al., 1991). In this respect viral antisense RNA-containing tumours may turn out to be of value for the identification of cellular genes which have become inactivated or activated due to viral integration. It is not yet clear whether the synthesis of H P V antisense R N A in cervical cancers interferes with gene expression in analogy to other systems (for review see H61~ne & Toulm6, 1990; Takayama & Inouye, 1990). Under experimental conditions, inducible synthesis of R N A complementary to the HPV-18 E 6 - E 7 0 R F s in the HPV-18-positive cervical carcinoma cell line C4-I has been shown to correlate with a decrease in viral E7 protein and growth inhibition in vitro (von KnebelDoeberitz et aL, 1988). The same correlation, i.e. growth retardation concomitant with the expression of viral antisense RNA, was found when these cell lines were
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tested for tumorigenicity in nude mice (yon KnebelDoeberitz et al., 1992). This creates an apparent dilemma in trying to understand why antisense transcription, particulady of the E6 and E7 genes, is possible at all in cervical carcinomas. One likely explanation may be that in contrast to the naturally occurring antisense R N A in the cancers, the artificially expressed antisense R N A was correctly processed and transported to the cytoplasm. This may be a prerequisite for effective inhibition which may predominantly take place at the level of translation, rather than inhibition of R N A splicing and/or interference with m R N A transport to the cytoplasm. Clearly, despite high levels of viral antisense R N A in the nuclei of some cancers, viral m R N A is still transported to the cytoplasm, thereby ensuring the level of E6 and E7 expression necessary to maintain the transformed phenotype. Nevertheless, the possibility that viral antisense R N A might influence some biological properties of the carcinomas, such as tumour progression, requires further examination. We thank Dr Harald zur Hausen for encouragement and constant support. We are also grateful to Dr Achim Schneider for providing biopsy material. This work was supported in part by the Deutsche Forschungsgemeinschaft (grant D/i 162/1-2).
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(Received 6 January 1992; Accepted 2 March 1992)
