MOLECULAR CARCINOGENESIS 6:88 99 (1992)

ARTICLES

Molecular Cloning, Analysis, and Chromosomal Localization of a Mouse Genomic Sequence Related to the Human Papillomavirus Type 18 E5 Region Tomas Kahn,’ Holger Friesl, Neal G. Copeland. Debra J. Gilbert, Nancy A. Jenkins, Lutz Gissmann, Judith Krarner, and Harald zur Hausen Deutsches Krebsforschungszentrum, Heidelberg, Germany (TK, HE LG, JK, HzH), and Mammalian Genetics Laborat04 ABL-Basic Research Program, NCi-Fredenck Cancer Research and Development Center, Frederick, Maryland (NGC, DIG, NAJ)

The E5 open reading frame (ORF) from bovine papillomavirus type 1 (BPV 1) as well as t h e E5 ORFs from human papillomaviruses (HPV) type 6 and type 16 have been reported to transform immortalized rodent cells. In an analysis o f murine and human tumors for t h e presence o f putative papillomavirus-related sequences, w e cloned amplified cellular sequences from t h e mouse cell line Eb t h a t cross-hybridized with t h e E5 ORF of HPV 18. A 2.1-kb fragment termed HC1 was sequenced. In normal murine cells, it was present as a single-copy genomic sequence located o n chromosome 8 . A region o f 21 3 nucleotides corresponded t o t h e E5 gene (HC1 E5), based o n t h e best alignments and on t h e presence of direct and inverted repeats bearing a central sequence motif. These structural elements are also present in t h e HPV 18 E5 ORE HCI E5 contained an ORF that was transcribed bidirectionally. The transcription in t h e E5 direction was enhanced i n RNA obtained from organs and tumors from carcinogen-treated animals and C127 cells. The polypeptide deduced from t h e sequence was related to E5 proteins from genital papillomaviruses, to t h e putative product o f t h e 4300 mouse gene, and to several viral and human growth factors. The data suggest t h a t there may b e several cellular counterparts t o t h e viral E5 proteins. o 1992 Wiley-Liss, Inc. Key words: DNA amplification, RNA overexpression, mapping, dimethylbenz[a]anthracene, skin tumors INTRODUCTION

Papillomaviruses (Pk) are a group of epitheliotropic DNA viruses that cause infections in mammals and birds. They are etiologically linked not only to benign epithelial tumors but also to certain epithelial malignancies of animals and humans [reviewed in 1-41, In animals, mainly the cottontail rabbit PV and the bovine PV (BPV) are known to produce lesions that under certain circumstances may undergo malignant conversion In the human PV (HPV) group, more than 60 different types have been isolated. From these, more than 20 have been found in anogenital lesions [5]. Consistent association of HPVs with human cancers has been found in patients with epidermodysplasia verruciformis, in whith warts containing HPV 5 or HPV 8 (and occasionally other types) may become malignant. This also applies to cervical, vulvar, penile, perlanai, and anal intraepithelial neoplasias, which are linked most prominrntly to HPV 16 and HPV 18 infections, as well as to a number of additional types. Intraepithrlial neoplasias are typical precursor lesions of cancer a t these sites. Studies on papillomavirus-specific gene functions led to the notion that the E6 and E7 open reading frames (ORFs) are important in the development and maintenance of the malignant phenotype in humans [6-8], whereas E5 and E6 are the transforming gene products in the BPV 1 system 191. However, a transforming capability has been reported for HPV 6 E5 and HPV 16 t i in immortalized 6 1992 WILEY-LISS, INC.

rodent cells [ 10,l 11 Since E5 mRNA and proteins are also present in premalignant lesions [12-151 and in HPV 16 -immortalized primary human keratinocytes [ 161, this could suggest a role of the E5 ORF in inducing cellular proliferation in humans as well [ 171. The E5 ORF is present in every genital HPV and in most of the other HPVs sequenced thus far. It is consistently located immediately upstream of the late g e m L2 and codes for only about 48-83 amino acids. The sequence i) not highly conserved a t the DNA or protein primarystructure levels between types. However, it always contains Cys-X-Cys dements, and the putative protein product has strongly hydrophobic domains in its amino-terminal two thirds, whereas the carboxyl-terminalthird contains a hydrophilic domain. ’Corresponding author: Deutsches Krebsforschungsrwtrum, Im Neuenheirner Feld 242, 6900 Heidelberg, Germany Abbreviations. ORF, open reading frame; DMBA, 7.12-dimethyl; benz[a]anthrarene; TPA, 12-0-tetrsdecanoylphorbol- 13 - ~ e t a t e PV. papillomavirus. BPV, bovine papillomavirus; HPV, human papillomavirus, EGF, epiderrn,il growth factor; PDGF, platelet-derived growth factor, rTGF-a, rat tumor growth factor-a, VGF, vaccinia virus growth factor; SFGF, Shope tibroma growth factor; MGF, myxoma virus qrowth factor, EDTA, ethylenediaminetetraaceticacid; TE, 10 m M Tri\and 1 rnM EDTA, pH 8.0, RFLF restridion fragment length polymorphism; DKFZ, Deutsches Krebsforschungsrentrum; UV, ultraviolet. KT. reverse transcriptase; PCR. polymerase chain reaction, SSC, standard saline citrate; BSA. Biological Sequence Analysis; HUSAR, Heidelberg Unix Sequence Analysis Resources; PIR, Protein Identification Resources; DHFR, dihydrofolate reductase.

MOUSE HOMOLOCUE OF HPVES REGlON

Since BPV 1 E5 products can transform rodent cells in culture efficiently, a functional analysis of E5 became feasible. It was found that the primary structure of BPV 1 E5 protein tolerates a variety of substitutions at many positions of the hydrophobic core without a complete loss of transforming activity, whereas some defined amino acids at the carboxyl-terminal domain are essential for efficient transformation [ I 8,191. Recent work showed that cotransfected human epidermal growth factor (EGF) cooperated with BPV E5 in the induction of stable transformation in mouse NIH 3T3 cells [20] and that the endogenous receptor for the platelet-derived growth factor (PDGF) is constitutively activated in C127 cells stably transformed by the E5 protein [21]. These experiments support the notion that the PV E5 proteins can activate cellular receptors that under physiologic conditions are activated by cellular growth factors. This activity resembles the effect of the gene products of other DNA viruses, like vaccinia virus growth factor (VGF), which is capable of binding and activating the EGF receptor [22]. It is noteworthy in this context that a polypeptide termed Q300 that is similar to PV E5 proteins was deduced from a novel transcription unit induced in simian virus 40-infected and -transformed mouse cells 1231. During a search of murine and human tumors for the presence of papillomavirus-related sequences, we found a mouse DNA fragment similar to the HPV 18 E5 gene. This paper describes the cloning and characterization of this fragment in terms of DNA amplification, sequence analysis, and relationships with HPV E5 genes, i.e., its transcription and chromosomal localization. The analysis was performed using cells from mouse cell lines, normal mice, and mice treated with carcinogens according to the tumor initiation-promotion model. MATERIALS AND METHODS Experimental Animals female NMRl mice were purchased from the lnstitut fur Versuchstierzucht, Hannover, Germany. Groups of 16 animals per experiment were randomly distributed into polycarbonate cages (four an ima Mcage). For u nequ ivocaI identification of individual animals, the mice were earmarked. They were kept under specific pathogen-free conditions on woodshavings with a diet of tap water and food (Altromin, Lage, Germany) ad libitum. At 6 wk of age, an area of approximately 3 x 4 cm of the dorsal surface of each mouse was shaved with electric clippers, and mice that were injured were replaced with uninjured animals. The mice were allowed to rest for 1 wk, and only those animals in the resting phase of the hair-growth cycle were used (other animals were replaced). Chemical Tumor-Induction Experiments 7,12-Dimethylbenz[a]anthracene (DMBA) and 12-0tetradecanoylphorbol-13-acetate (TPA) were synthesized by routine methods a t the Institute of Biochemistry. Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany. All chemicals were applied topically to the shaved region in 0.1 mL of acetone. For the induction of papillo-

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mas and carcinomas by multiple treatments with DMBA, the mice received 12.5 nmol or 50 nmol twice weekly for about 22 wk. Mice bearing presumably malignant tumors were killed 3-4 wk after that diagnosis. For the induction of papillomas by tumor initiation and promotion, the mice were treated with a single dose of 100 nmol of DMBA followed Iwk later by treatment with 10 nmol of TPA twice weekly. TPA application was stopped after 24 wk, and the animals were killed 1 wk later. The control groups were composed of mice that received acetone only for 6 wk, mice treated with a single application of 10 nmol of TPA at 13 w k of age, and mice treated with 10 nmol of TPA twice weekly for 6 wk. (A detailed description of the outcome of the different treatments from the point of view of skin carcinogenesis has been published [24,25].) Each experiment was conducted according to German law and U.S. National Institutes of Health guidelines. Radiation Tumor-Induction Experiments Ultraviolet (UV)-induced squamous cell carcinomas from hairless mouse Mu5 musculus strain HRA/Skh were kindly provided by Dr. G. Kelly(University of Sydney, Sydney, Australia). UV induction was performed as described by Reeve et al. [26]. Papillomavirus DNA DNAs of different types of human and animal PVs were kindly provided by Dr. E.-M. de Villiers of the Referenzzentrum fur Humanpathogene Papillomviren, DKFZ, Heidelberg. Cell Lines Eb lymphoblastotd mouse cells (kindly provided by Or. M . Schwarz, DKFZ, Heidelberg) were cultivated in RPMl 1640 medium. The cloned C 127 mouse mammary tumor cell line (furnished by Dr. R. Heilbronn, DKFZ, Heidelberg) was cultivated in Dulbecco's minimum essential medium at 37°C in a 5% COz atmosphere. The media were supplemented with 40 m M L-glutamine, 100 kg/mL streptomycin and 10% fetal calf serum (inactivated by heating for 30 min at 56°C). The C 127 cells were treated up to four times with 40 m M DMBA dissolved in acetone or with acetone alone as a control. After each 12-h treatment, the cells were supplied with fresh medium. After an additional 36 h, cells were subpassaged. This was repeated three times in the absence of carcinogen before DNA and RNA extraction or before reiteration of the treatment with DMBA to avoid acute cytotoxicity. Extraction of DNA and RNA From Cultured Cells Total high-molecular-weight DNA of treated and control cells was isolated by lysis of cells in 3% N-lauroxylsarcosine sodium salt, 0.07 M Tris, pH 8.0, 25 m M ethylenediaminetetraacetic acid (EDTA), and proteinase K (100 pg/mL heated at 55°C for 8 h, phenol and chloroform/isoamylalcohol (24: I), extraction, and dialysis against 10 m M Tris and 1 m M EDTA, pH 8.0 (TE). Cytoplasmic RNA was isolated by lysing cells in 0.6% NP-40, 0.15 M NaCI, 10 m M Tris-HCI, pH 7.4, and 1 m M EDTA followed by pelleting nuclei and

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KAHN ETAL.

mitochondria and repeatedly extracting the supernatant with phenol and chloroform/isoamylalcohol(24: 1) as previously described [71.

Reverse transcription-polymerasechain reaction (RT-PCR) were performed as previously described (31,321. Labeling of oligonucleotides at their 5' ends was performed according to the method of Maniatis et al. [33].

Extraction of DNA and RNA From Mouse Organs Mouse organs were removed and immediately frozen in small pieces in liquid nitrogen. DNA was obtained from some of the small tissue pieces by direct proteinase K digestion, dialysis against TE, RNase treatment, a second proteinase K treatment, repeated extractions with phenol and chloroform/isoamylalcohol(24: l), and dialysis against TE overnight. RNA was obtained using the guanidinium isothiocyanate/cesium chloride gradient ultracentrifugation protocol described by MacDonald et al. [271. Extraction o f Plasmid DNA for Sequencing Plasmid DNA was extracted and purified using Qiagen columns and protocols (Diagen, Dusseldorf, Germany). Cloning of DNA A genomic library was constructed from Eb cell DNA in the bacteriophage vector NM 1149 after cleavage with Hindlll. A genomic library was constructed from a UVinduced mouse skin squamous cell carcinoma in the bacteriophage vector NM1151 after cleavage with EcoRI. Positive recombinants were identified by the plaque hybridization technique 1281 using HPV 18 DNA as a radioactive probe. The complete procedure has been described elsewhere [29]. Subsequently, a 2.1-kb EcoRI-Hindlll subfragment from a relevant Eb library clone was subcloned into pUCl9 and named HCl. (Details are described in the Results section.) Analysis of Genomic DNA Ten micrograms of high-molecular-weight genomic DNA was digested with 50 U of restriction enzymes for 6-12 h under conditions specified by the commercial supplier, precipitated, resuspended in 30 p L of loading buffer, and electrophoresed on 0.8% agarose gels. The gels were blotted with 0.5 M NaOH and 1.5 M NaCl onto Genescreen membranes (Dupont, NEN Research Products, Boston, MA) after two 15-min periods in 0.25 N HCI and two 15-min periods in 0.5 M NaOH and l.5 M NaCI. The blots were crosslinked with UV and hybridized as described elsewhere [30]. Probeswere radiolabeled according to the random-priming method using a kit from Pharmacia (Freiburg, Germany). Analysis of RNA For gel fractionation and northern blot analyses, about 5 p g RNA per lane was denatured at 68°C for 15 min in H20, precipitated with EtOH, resuspended in a standard loading buffer, and immediately electrophoresed on agaroseMOPS gels, using MOPS buffer (20 m M 3-(N-morpholino)propanesulfonic acid, 5 m M sodium acetate, and 1 m M EDTA) as a running buffer. RNA was transferred from the gelsonto Genescreen membranes using 10 x SSC (1 x SSC = 0.1 5 M NaCI, 0.015 M sodium citrate).

DNA Sequencing The 2.1-kb HC 1 clone was completely sequenced by the dideoxy-chain termination method of Sanger et al. [34] on double-stranded DNA using the Sequenase and Pharmacia kits and a primer-walking strategy. The synthetic oligonucleotides used as primers are identified in Figure 2 below and were synthesized, as were the oligonucleotides used for the binding experiments, with an Applied Biosystems DNA synthesizer (Foster City, CA). Sequencing was performed in both directions, and oversequencing was always performed at least one additional time. DNA Sequence Analysis DNA sequence analysis was done with the aid of the Biological Sequence Analysis (BSA) program library developed at the DKFZ and the Heidelberg Unix Sequence Analysis Resources(HUSAR)program library, a software package with contributions from the University of Wisconsin Genetic Computer Group, BSA, Protein Identification Resources(PIR), UCSD (Doolittle), and EMBL (Vingrom, Zehetner). EMBL release 19.0, GenBank release 61 .O, NBRF-PIR release 22.0, and SwissProt 11.O were scrutinized for nucleotide or amino acid sequence homologies. lnterspecific Backcross Mapping lnterspecific backcross progeny were generated by mating (C57BU6J x M. spretus)Fl femalesand C57BU6J males as previously described 1351. A total of 205 N2 progeny were obtained, and a random subset of these N2 mice was used to map the Hc7 locus. DNA isolation, restrictionenzyme digestion, agarose gel electrophoresis, and Southern blot transfer and hybridization were performed essentially as described previously [36]. All blots were prepared with Zetabind nylon membranes(AMF-Cuno, Meriden, CT). The HC1 probe was labeled with [32PldCTP using a nicktranslation labeling kit (Boehringer Mannheim, Mannheim, Germany). Washing was done to a final stringency of 0.2 x standard saline citrate phosphate and 0.1YO sodium dodecyl sulfate at 65°C. A 3.7-kb fragment was detected in Hindlll-digested C57BU6J DNA, and a 5.5-kb fragment was detected in Hindlll-digested M. spretus DNA. The 5.5-kb M. spretus-specific fragment was followed in this analysis. Descriptions of the probes and restriction fragment length polyrnorphisms (RFLPs) for the loci linked to Hcl including plasminogen activator (Plat), fms-like gene (F/g),jun 8 proto-oncogene, and jun D proto-oncogene have been reported previously [37,38]. Recombination distances were calculated as described previously [39], using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.

MOUSE HOMOLOGUE OF HPV €5 REGION

RESULTS Molecular Cloning of a Murine Genomic DNA Fragment Similar t o t h e HPV 18 € 5 Gene and Amplified in Tumors Analysis of DNAs from cell lines and epithelial tumors of mouse and human origin for HPV-related sequences revealed that during Southern blot hybridization at T, = - 30°C. the mouse cell line Eb strongly hybridized with the complete HPV 18 DNA and with the 4.4-kb HPV 18 BamHIEcoRl fragment (nucleotides 2439-6930). This fragment contains part of the E2 and L1 ORFs and the complete E4, E5, and L2 ORFs. A mouse genomic lambda library was constructed using Eb DNA, and 7 58 clones that contained a 2.1-kb EcoRI-Hindlll fragment hybridizing with HPV 18 DNA were isolated. Five of these were subcloned into pUC19. Restriction analysis showed that they were identical. For further characterization, one clone was selected and designated as HCI. The HCI probe, under stringent hybridization conditions (T, = 18"C), detected a single copy of this sequence in various murine tissues. It obviously represents a conserved ancient mouse genomic sequence that is present in different mouse species and in M. musculus subspecies from all over the world (M. castaneus, M. musculus, M. molossinus, M. brevirostris, M. praetextus, M. m. shortridgei, M. m. saxicola, M . m. pahari, M. m. minutoides, M. m. booduga, M. m. cookii, M. m. cervicolor popaeus, M. m. caroli, M. m. spretus, M. m. hortolanus, and M. m. abbotti) (Figure 1A). HC 1 is not related to mouse PV since it does not react with Mastomys natalensis PV or with Micromys minutus PV DNA even under nonstringent hybridization conditions (T,,. = - 40°C) (data not shown). Normal murine DNA did not hybridize under stringent hybridization conditions with the HPV 18 fragments mentioned above (data not shown). The reason HC1 was detected using an HPV 18 probe was the high level of amplification of HCI in the Eb DNA (Figure 1 B). Cleaved DNAs from different mouse cell lines (NIH 3T3, primary mouse macrophages, and C127 cells) had modified bands hybridizing to the HCI probe. For instance, cloned C127 cells had a 5-kb band in addition to the 2. I-kb band expected after EcoRI-Hindlll cleavage of the genomic DNA. But in one of five tested UV-induced squamouscell skin carcinomas from hairless mice, high levels of amplification and rearrangements of the HCI sequence were observed using Southern blot hybridization and confirmed by molecular cloning (data not shown). In contrast, a high level of HC1 amplification could not be detected in four animals treated chronically with DMBA (initiation), in control animals, or in animals treated only once with DMBA and then repeatedly with TPA (initiation/promotion) (data not shown). ~

HC1 Sequence Analysis HCI Sequence Similarity to HPV 18 €5 The sequence of HCI is shown in Figure 2. (The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under the accession number X66285.) Because

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of HCl'sstructural elements, it could be divided into three regions: the 5' region (nucleotides 1-1 470). the E5-corresponding region (nucleotides 1471-1 684), and the 3' region (nucleotides 1685-21 31). The 5' end had 23 major purine-pyrimidine blocks 7-1 5 nucleotides long and six sequences seven nucleotides long that were repeated two times each. This part of the sequence shows no similarity to known sequences of the GenBank and EMBL sequence data banks. The region between nucleotides 1472 and 1684 (vertical black arrowheads in Figure 2) does not contain longer purine-pyrimidine blocks. It is rich in TG elements and contains two perfect direct repeats 37 bp long and two additional shorter versions of this repeated element (boxed in Figure 2). In these repeats, the motif TATGCAT is present five times. Allowing for minor variations, this sequence element can be seen as a part of a decanucleotide motif (G/A)T(G/A)T(A/G)KCATthat is present seven times in this region and resembles closely the Pit1-factor binding sequence [40] (Table 1). Using the Stemloop HUSAR program, a great number of inverted repeats with the theoretical capability of producing stem loops were found, as well as immediately juxtaposed inverted repeats. This 2 13-bp subfragment of HC 1 (HC 1 E5) (black arrowheads in Figure 2) is the part corresponding to the HPV 18 E5. This is shown at the DNA sequence level with the alignment depicted in Figure 3 . The primary direct repeats are marked to stress that the HPV 18 E5 also contains several direct repeats, as well as the above-mentioned consensus motif three times (Table 1). Inverted repeats were also found, albeit shorter than the ones in the HCI E5, but again with and without separation (the longest inverted repeat was 20 bp). Thus, the similarity of the alignment is not only indicated by the number of equal nucleotides, but also by the conservedstructural elements. These direct and inverted repeats are shared also with the Q300 ORF (longest direct repeat, 31 bp; longest inverted repeat, 29 bp). Moreover, using the 37-bp main repeat as a probe, not only was HPV 18 recognized, but also several other related HPV types, mainly types 6, 32, 34, 39, and 52 (data not shown). When the E5-corresponding region of HCI (HC1 E5) was compared with the sequences stored in the GenBank and EMBL libraries with the aid of the FASTN program, the best scores were obtained with sequences known to regulate the expression of tissue- or differentiation-specific genes, mainly the rat prolactin gene (69% identical nucleotides). Similar results were obtained with the HPV 18 E5 sequence itself. At the 3' end of the HCI sequence, an additional characteristic element rich in TTA and TTG elements is marked on Figure 2 (open vertical arrowheads). Comparing the complementary strand of HCI with the sequences stored in the DNA data banks, this element showed a very pronounced similarity to a coding sequence from the locus deformed, which belongs to the Antennapedia complex of Drosophila melanogaster This sequence also closely resembles the TAA-rich homeobox-protein binding domain described previously by Winslow et al. [41].

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KAHN ETAL.

Figure 1. HC1 in different mouse DNAs. (A) Clone HC1 represents a normal genomic sequence in mice. A Southern blot with BamHI-cleaved mouse liver genomic DNA (10 pgllane) was probed with HC1 at stringent conditions of hybridization (T, = - 18°C). Lane 1, Mus shortridgei (male); lanes 2 and 3, M. saxicola (male and female, respectively); lanes 4 and 5, M. pahori (male and female, respectively); lanes 6 and 7, M. minutoides (male and female, respectively); lane 8, M. booduga (male); lanes 9 and 10, M. cookii (male and female, respectively); lanes 11 and 12, M. cervicolor popaeus (male and female, respectively);

lanes 13 and 14, M. caro/i(male and female, respectively); lanes 15 and 16, M. spretus(male and female, respectively); lanes 17 and 18, M. hortolanus (male and female, respectively); lane 19, M. abbotti(ma1e). Molecular size markers (in kilobases) are indicated in lane A. (B) HC1 amplification in the Eb cell line. A Southern blot with EcoRI- and Hindlll-cleaved Eb DNA was probed with HC1 at stringent conditionsof hybridization (T, = - 18°C). Lane a, 10 pg of vector-free HCl DNA (equivalent to one genome copy); lane b, 10 pg of Eb DNA; lane c, 5 pg of Eb DNA.

HCl Sequence Homology With HPV 18 €5 at the Protein Level

era1 possible in-frame splice acceptor sites (positions 1409, 1428, and 1485; marked by arrows in Figure 4A). This ORF covers the complete E5-corresponding region, spanning nucleotides 1472-1691, and is the only one in the H C I sequence that codes as predicted by the method of McLachlan et al. [42]. In a comparison of the sequencederived protein of HC 1 with the HPV 18 E5 protein using the Bestfit program, 58.9% of similar amino acids were

The major ORFs of the HC1 sequence are located in the E5 corresponding region, in both the sense and antisense directions. The longest ORF on the sense strand (also sense strand for HPV 18 E5 transcription) extends from position 1383 to position 1737. It has no ATG, but we noted sev-

MOUSE HOMOLOCUE OF HPV €5 REGION ECOR1l

93

W C A G T GAGTGCAGTT ATAGAAAAAT CTTTATATCC TCTCACCTTA CATTATTCTT TCTATAATGT CCTTACCCAC AGACATTTAT ATAACTTCCC CTTAAGGTCA CTCACGTCAA TATCTTTTTA GAAATATACG ACACTCCAAT GTAATAAGAA AGCTATTACA G G A A T C G C I G TCTGTAAATA TATTCAACCG

101 CAGCTTCCGT AGGATTCCCG CATTCATCTC AGACGAACAC AATAOCTATA

ATCGTACCCA TTATAACGAC CATTCTTCTC AATTCTAAAG ACACACCGAA

-

GTCGAAGCCA TCCTAAGGGC GTAAGTAGAG TCTCCTTCTG TTATCGATAT TACCATCCOT AATATTGCTG GTAAGAAGAG TTAACATTTC TGTGTCGCTT

--

2 0 1 CTTGTGAAGA TGAAGCTGTG TCTTGATTGG CTGTTAAGTT GTGATCAATC AAACGTATTT ATCCTGTAAT TTTACACCCA AGCAAGAACC AGCCATCTCT

GAACACTTCT ACTTCGACAC AGAACTAACC GACAATTCAA CACTAGTTAC TTTGCATAAA TACGACATTA AAATGTGGGT TCGTTCTTGG TCCGTAGACA

50 I

CATAAATCAG CATGTGCATA CCGTCAAAAA TAAACTGTCT CACTTAGATG ACTTAACTGA ATTTTCTTTA TGTiAACAGG GCACAGCACA AGTTACACTT CTATTTAGTC GTACACGTAT GGCAGTTTTT ATTTCACAGA GTGAATCTAC TGAATTGACT TAAAAGAAAT ACAGTTGTCC CGTGTCGTGT TCAATGTGAA

4 0 1 AAAAATAGAA GGAAGCAAAA GAAAACCTAG ACATTGTTTT GGTTATTGGG TTAAAGAGAA TAACTGTATT ATAATGCACG GAACCTAAAG AGCATAGCAG

T T T T T A T C T T CCTTCGTTTT CTTTTGGATC TGTAACAAAA CCAATAACCC AATTTCTCTT ATTGACATAA TATTACGTGC CTTGGATTTC TCGTATCGTC

5 0 1 TCTTCTCAAA ATCACACCTT ACTTGTGGTG AGTTATCAGT GGGAGCCCAT GTTCTTCTTC

CCTTTTATCT

TAACCTCAGA AGAAGATGAT AAACTGAGAA

AGAAGAGTTT TAGTGTGGAA TGAACACCAC TCAATAGTCA CCCTCGGGTA CAAGAAGAAG GGAAAATAGA ATTGGAGTCT TCTTCTACTA TTTGACTCTT

601 CTACTTTCAG TATCAACAAC ATTTCCTGGA AAGGGCCCGA ATGTAAATAT TGTTGGCTTT AGAGGCCTTG TGTGGTTGAT GCTACCACAT GCTTCCTGCC

GATGAAAGTC ATAGTTGTTG TAAAGGACCT TTCCCGGGCT

TACATTTATA ACAACCGAAA TCTCCGGAAC ACACCAACTA CGATGGTGTA CGAAGGACGG

7 0 1 GGACAGCTCT TAGAATGTAA AACCAAGAGT TACCAAAGCA GCCTGGAGGC CAGCCTGTGA CCTATGGCTC AGTCTTAAGT CTTCTGATTT ATACGAATGT

CCTGTCGAGA ATCTTACATT TTGGTTCTCA ATGGTTTCGT CGGACCTCCG GTCGGACACT GGATACCGAG TCAGAATTCA CAAGACTAAA TATGCTTACA

1101 GCAAGTTGTG AGACTTAATA AAAGTAGTGC ACACAACTCT TTGATCTCCT GTGCACCACG CACTTTCATC TGCCTGGAAA AGTGATCTGG AAGGTCTCTC

CGTTCAACAC TCTGAATTAT TTTCATCACG TGTGTTGAGA AACTAGAGGA CACGTGGTGC GTGAAAGTAG ACGGACCTTT TCACTAGACC TTCCAGAGAG

901 AAGGTTTAAG AGCATGCTTA AAAACAGTGG CCA-TA

TCTCTGGGAA GCTGGGTGAG AAATGAATGC AACCCGTGGT CGTCTTCCTG CATGCTTGTG

TTCCAAATTC TCGTACGAAT TTTTGTCACC GGTCCCCGAT AGAGACCCTT CGACCCACTC TTTACTTACG TTGGGCACCA GCAGAAGGAC GTACGAACAC

1001 TGTAGGTTCT TGGTCTTGGG GGAGCCTGCT ACCTCTGTCC TTCTCAGATT TAGAAGGCAC TCAAAGAAAA TAGACTACAA CTAAAAGATC TCTTTCCAAC

ACATCCAAGA ACCAGAACCC CCTCGGACGA TGGAGACAGG AAGAGTCTAA ATCTTCCGTG AGTTTCTTTT ATCTCATGTT GATTTTCTAG AGAAAGGTTG

1 1 0 1 MCTCACAGA M A C T T A G C T GCTTACTTTT CAGATCCACA CACACTTATT ACCAGATCAT GTCTCATGAC TGCATGCCAA GCTGGCCTGC CTGCCAAGAT

TTGAGTGTCT TTTGAATCGA CGAATGAAAA GTCTAGGTGT GTGTGAATAA TGGTCTAGTA CAGAGTACTG ACGTACGGTT CGACCGGACG GACGGTTCTA

1201 M T T A C C T T G TTGTCCAAAG CGGTACTGAT TTGAAACCCT CTTTCCTTCA AGTCTAAAAC AGTTACTTGC CTTTACAGTG TTGGTATTTT TCTTGTCAGC

TTAATGGAAC AACAGGTTTC GCCATGACTA AACTTTGGGA GAAAGGAAGT TCAGATTTTG TCAATGAACG GAAATGTCAC AACCATAAAA AGAACAGTCG

1301 AGTCATATTC CAACAGCTGC CTTTTCAGTG ATATTTTATT GAATGGACAT ATTCTGGAAA TGCTATCTTC CATTAATGTT GAAGGAATAC ATATTGTATT

TCAGTATAAG GTTGTCGACG G M A A G T C A C T A T A A A A T A A CTTACCTGTA TAAGACCTTT ACGATAGAAG GTAATTACAA CTTCCTTATG T A T A A C A T A A

tpCR2

1401 CATAATAAGA GCAACAACAG GTTCGAGCTA TTGAGAGCCT CAAGCAGAGC ATGTACTCAT TCCATCATAA TAACAATCCA TGAGGTAGAC AGCATCTTTT

GTATTATTCT CGTTGTTGTC CAAGCTCGAT AACTCTCGGA GTTCGTCTCG TACATGAGTA AGGTAGTATT ATTCTTAGGT ACTCCATCTG TCGTAGAAAA

1501 CATGTGTGAG TGTGTGTGTA TGCATGTGTG T G I A T

GTACACACTC ACACACACAT ACG

1701 ATGAACTGTG AGCATGTGTG TATGGAATTA TGTGTATGAA GTTGACATCA GGTCTCTTCT ATCTCTCTCA ATCTCAGGTT TTTGAGACAA GATCTCTCAC TACTTGACAC TCGTACACAC ATACCTTAAT ACACATACTT CAACTGTAGT CCAGAGAAGA TAGAGAGAGT TAGAGTCCAA AAACTCTGTT CTAGAGAGTG

1801 CAAAACTGAG ATCATCAATT AGCTATATAG GCTGGTCAAT AAGCTCCAGA TATCTGCCTA TCATTGTGAC TGGTCTTACA GCAGTGGCCT GCCATGCATA

GTTTTGACTC TAGTAGTTAA TCGATATATC CGACCAGTTA TTCGAGGTCT ATAGACGGAT AGTAACACTG ACCAGAATGT CGTCACCGGA CGGTACGTAT

I901 GCTTTTTATA TAAGTGCCAA GGATCTGAAT T G G g C T C ATGTTTACAC AGAAATTTCT TTGCTGTCTC AGCCTGACTC CAGCTCCAGT AGATATTATT

-

CGAAAAATAT ATTCACGGTT CCTAGACTTA ACCCCAGGAG TACAAATGTG TCTTTAAAGA AACGACAGAG TCGGACTGAG GTCGAGCTCA TCTATAATAA

-

2001 GTTGTTGTTG TTGTTGTTAT T A T T A T T A T T ATTATTTCAA TTGTATAGGT AAAGAAAGTG ATAGTTAAGG AGTAGCCAGG GAGACATGAG TTGCTCCCTC

CAACAACAAC AACAACAATA ATAATAATAA TAATAAAGTT AACATATCCA TTTCTTTCAC TATCAATTCC TCATCGGTCC CTCTGTACTC AACGAGGGAG

2101 TACACCAAAT GCATTTCTCT C C T A G A 5 T

HlNDE

ATGTGGTTTA CGYAAAGAGA GGATCTTCGA A

A

Figure 2. (Legend appears on page 94.)

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KAHN ETAL. Table 1. HC1 E5 Conserved Sequence Motif*

Name

Position

Sequence

HC 1

1516 1560 1586 1602 1630 1637 1648

GTgTATGCAT GTgTATGCAT GTaTGTGCAT GTaTATGCAT GTaTGTGCAT aTgTATGCAT GTgTATGCAT

HPV 18

3967 4023 4089

GTgTATGCAT GcgTATGCAT GTaTATGtAT

HC 1 HPV 18 Pit I

consensus consensus consensus

GIA G A

T TIC

A

GIA GIA A R

A A

T T

T

T

A

T T T

G

C

G

C/T

N

C

A A A

T T T

*Position and sequence of the different versions of the HC1 and HPV 18 conserved motifs. Last three rows in table compare HC1 and HPV 18 consensus sequences with the Pit1 consensus sequence. Capital letters indicate the most conserved nucleotides within these sequences.

found over a length of 56 amino acids. (Figure 4A and Table 2). When this putative protein sequence was compared with the sequences in the PIR-NBR protein sequences library, the HPV 18 E5 protein was found to have the highest score. More important, additional characteristics of E5 proteins (Cys-X-Cys elements, the presence of a hydrophobic core, and a more hydrophilic carboxyl-terminal sequence) are conserved in the HC1 sequence. Although the carboxylterminal sequence is the part least similar to the HPV 18 E5 protein, it resembles the C-terminus of the BPV 1 E5 protein, bearing conserved amino acids shown to be necessary for the transformation activity of BPV 1 E5 (Figure 4Bf [19]. The similarity to the Q300-derived protein is even stronger than with HPV 18 E5 (Table 2 and Figure 4A). The opposite strand contains two possible ORFs. The longer one starts at position 1757, stops at position 1467, and bears an ATG at nucleotide 1647 spanning the whole HC1 E5 region. The shorter ORF, starting at position 1687, has an ATG at nucleotide 1656 and a stop codon at position 1459. Transcription of the HC1 E5-Corresponding Sequences The genital HPV E5 ORFs are protein-coding regions transcribed and most probably also translated in HPV-induced lesions and immortalized cells. The existence of an E5-corresponding ORF in the mouse genome prompted us to investigate whether the HCI E5 sequence is transcribed in vivo. Negative results were obtained with strand-specific oligonucleotides from the HC1 E5 region as a probe in northern blot hybridization of cytoplasmic RNAs from control and DMBA-treated C127 cells and RNAs from different Figure 2. Nucleotide sequence of HC1 (EMBL, GenBank, and DDBJ accession number X66285). Arrows indicate sense and sequence of the primers used for the primer-walking sequencing strategy and for the RT-PCR experiments (termed PCR 1, PCR 2, and PCR K). Vertical arrowheads mark the €5-corresponding region. Open vertical arrowheads indicate a region corresponding to the h 0 S O p h h melanogaster locus deformed. The main direct repeats are marked by boxes.

organs and papillomas obtained from animals treated once with DMBA and then repeatedlywith TPA (data not shown). In contrast, by testing RNAs from skin, skin carcinomas, and the livers of three mice treated chronically with DMBA, and by using a strand-specific oligonucleotide probe (see legend to Figure 5 for sequence), a strong signal of about 6 kb was observed in both the liver and carcinoma RNAs, but not in skin RNA from the same treated animals (Figure 511). An antisense probe (see legend to Figure 5) only detected signals of about 3 kb in liver RNA, very faint 3-kb signals in carcinoma RNA, and none at all in skin (Figure 51). Confirmation that comparable amounts of RNA were present on the northern blots was obtained by hybridization with p-actin DNA as probe (Figure 5111). To further test the bidirectional transcription of this region and to better understand the negative hybridization results obtained with RNAs from control and DMBA/TPA-treated animals, RT-PCR was performed (Figure 6). In these experiments, only a faint signal was detectable in the skin of one control animal after a long exposure and only when the opposite-strand oligonucleotide was used as primer. In the liver of treated and control animals, both strands were transcribed (the opposite strand at a higher level). The same occurred in a DMBAJTPA-induced papilloma and in untreated C 127 cells, but the proportions were inverted after chronic DMBA treatment of C127 cells. C hrornosoma I Locat ion

The mouse chromosomal location of the Hcl locus was determined by interspecific backcross analysis using progeny derived from matings of (C57BU6J x M. spretus)F, x C57BU6J mice. This interspecific backcross mapping panel has been typed for over 850 loci that are well distributed among all the autosomes as well as the X chromosome [35]. C57BU6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative RFLPs using the HC1 probe. A 5.5-kb M. spretus-specific Hindlll RFLP (see Materials and Methods) was used to follow the segregation of the Hcl locus

MOUSE HOMOLOGUE OF HPV €5 REGION

95

BEST ALIGNPlENT FOR (SEQ 1 ) COMPLETE (SEQ 2 ) HPV18 - CODING REGION E5A 10

20

40

30

50

60

1 GGTAGACAGCATCTTTTCA~GTGTGAGTGTGTGTGTATGCATGTGTGTG~ATTTGTGTGT N M R M Y Y W YMMYY M R M Y M Y M MYMMMMYYYY*MYRnMMMMKY* YKMY 2 TGTTATCA-CTTA'rTTT?TTAT-TTTGCTTGCT'rTTGTGTATGCATGTA'r~TG~GCTGCCATG-

-----

6 I A~TCATGTATGTGTGTGi'ATGCATGTGTGlGTGTATTTG~GTGTATGTGCAT~TGhGAGT 1( YY M Y YRM M M N YMYYYMMMKYYMMMY Y R M M Y Y X Y M Y K M Y Y Y M 5 9 -TCCCGCTTTTGCCATCTGTCTGTATGTGTGCGTATGCATGGGTATTGGTA~T~-~G~G~

121

A~A~GCATG~~TGTGTAT~G~G~G~G~~~TGCATGTATGCA-TGTG~A~G~A~

MMMW K M Y M YYM Y R Y M M Y Y YWY WYNM M M Y M K M Y Y M Y M M Y WX 1 1 6 AT_AT-----TGTG-GTAATAACGT-CCCCTGCCACAGCATTCACAGTATATGTAT-TTTG

1 80

TA~TTGTGTG'TTATETGTGTATGTTG

H HHM 168

HMY YRHMM

MRHMH

TTTTTTATTGCCCATGTTACTATTG

Figure 3. Alignment between the HC1 and HPV 18 € 5 sequences. *, identical nucleotides; R. purines; Y, pyrimidines.

The most prominent direct repeats of each sequence are marked with dots, dashes, or lines.

in backcross mice. The mapping results indicated that Hcl was located in the middle region of mouse chromosome 8 and linked to Plat, Flg, jun B, and jun D. Although 93 mice were analyzed for every marker and are shown in the segregation analysis (Figure 7). up to 182 mice were typed for some markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are as follows: centromere-Plat- 2/100 - flg - 261147 - Hcl - 8/168 - jun D - 11/182 - jun B. The recombination frequencies, expressed as genetic distances in centimorgans (cM) thestandarderror,arePlat-2.0? 1.4-Flg- 17.7 2 3 . 2 - H c l - 4 . 8 k 1 . 6 - i u n D - 6 . 0 k 1.8-junB.

ity of the E5s from BPV 1 [181, HPV 6 [ l o ] ,and also HPV 16 [ I I ] in vitro. The demonstration that BPV 1 E5 can activate exogenous human EGF [201 and endogenous mouse PDGF receptors 1211, as well as the presence of its mRNA and proteins in naturally occurring human HPV infections, also lend support to the assumption that the E5 protein has a role in the induction of epithelial cell proliferation. A possible therapeutic interference based on blocking the E5 protein activity has already been suggested [ 101. The E5 functions, their regulation, and even the identification of coding regions for E5 proteins and its mRNA, however, still leave many questions open. This is mainly due to the small size of the ORF, the polycistronic character of the E5 mRNAs, and the E5 region being silent or no longer present because of integrational events in most of the HPV-containing cell lines. In the course of attempts to isolate new types of PVs from various cell lines, we identified murine and human sequences that crosshybridize with the HPV 18 E5 region.

*

DISCUSSION Interest in the E5 region of HPk has been growing steadily ever since the demonstration of the transforming activA:

ALIGN

v HC1

I

18E5 1

v HC1

1

Q300

1

v

v

R N ' ~ Y C I H N K S N N R f F I . L ~ S S ~ C T f l S l I 1 l I H E V U S I F S C V S V C V C M C V Y L C V C S C M C V Y A C V C l C V Y V H V S V Y A C V C I ~ V C M C M Y A C V C M C V F V C M C V C C ~ W M P ~ C E H 1V 1~9~ M E l , C V I II l l l l l ~ l l l lI I I I I I I I I I I 1 I1 I I Il l II I I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M L S L l F L F C F C V C M Y V C C H V P L L P S V C M C A Y A ~ L V f V Y I W I T S ~ A T A F T V Y V F C E L L P M L L L H l H A l L S. L. Q. . . . . 7 3

v

v

RNTYCIHNKSNNRFELLNLSSRACTHSI~ITIH.EVDSIFSCVSVCVCMCVYLCVCSCMCVYACVCICVYVHVSVYACVCICVCMCMYACVCMCVFVCMCVCCHWMP~CEHVCMELCV 119 I I II 1 I I I I I IIIIIIIIIIIIIII I l l I I I I I I I I I I . . . . . . . . . . . . . . . MGKCHHAHLQFtlfYKFWWE(iETNLFYVCVCVCVCVCVCVCVCTLTCMCKSGGNLGCSSSGAIHCGVFVCVL1FEPGLTM . . . . . . . . . . . . . . . . . . . . . . . . . 77

B : MOTIF

BPVl20 HC1

SO

* * * * * v + F C C + L L L F L L L F F L V Y W D H F E C S C T G L P F I C V C M C M Y A C V C M C V F V C M C V

Figure 4. Alignment of the HC1-derived protein sequence and the HPV 18 E5.4300, and BPV 1 € 5 proteins. (A) Alignment of the HC1-derived protein and the HPV 18 E5(18E5)- and Q300-derived proteins using the Gap program. Vertical dashes indicate identical or similar amino acids. Triangles indicate potential splice-acceptor sites. (B) Comparison of the carboxyl-terminal

ends of the HC1-derived protein and the BPV 1 E5 polypeptide using the Motif program. The letters above the sequence indicate identical amino acids. The * and + symbols indicate similar amino acids. Bold letters indicate those residues shown to be essential for BPV 1 E5 transformation [19].

KAHN ETAL.

96

Figure 5. Northern blot analysis of HC1 E5 transcription. Mouse 1 was treated with 50 nmol of DMBA and mice 2 and 3 with 12.5 nmol of DMBA twice weekly for approximately 23 wk until skin carcinomas developed. Three to four weeks after diagnosis, the mice were killed, and RNA was extracted from different tissues. Five micrograms of RNA each from tissues of mice numbers 1, 2, and 3 were loaded as indicated. C, carcinoma; S, skin; L, liver. (I) Hybridization using a 37-bp single-strand oligonucleotide from the main direct repeat unit of HC1 as probe for antisense transcripts: 5’-tgtgtgtgtatgcatgtgtgtgtatttgtgtgtatgt3’. (11) Hybridization using a single strand oligonucleotidefrom the main direct repeat unit of HCl as probe for sense transcripts: 5’-acatacacacaaatacacacacatgcatacacacaca-3’. (Ill) Hybridization using p-actin DNA as probe. All hybridizations were performed on the same blot at high stringency 0, = 7”Cfor oligonucleotide hybridization;T, = - 18’Cfor p-actin hybridization).Markers in the right margin indicate the position of 285 (top) and 185 (bottom) RNA, respectively. ~

These findings prompted an analysis of these sequences, in the hope that they could constitute a model system for the study of the E5-like functions in a natural cellular environment and also provide information on the origin of viral E5 sequences. In this initial study, we concentrated on mouse E5-related sequences, thus permitting broader experimental approaches. The analysis of the mouse DNA not only revealed a surprisingly good fit in sequence alignment with HPV 18 E5 but also direct and inverted repeats including a consensus motif found in HPV 18 E5. Moreover, the cellular sequence encompassed an ORF corresponding to the E5 region, and

the putative protein derived from it bears some similarity to PV E5 proteins (Table 2). During the preparation of this paper, another sequence similar to the PV E5 oncoproteins, termed Q300, was reported [231. The Q300 sequence was isolated by an experimental approach completely different from ours, and the surrounding DNA sequences and the size of the RNA transcripts also are quite different, but the transcribed E5-like region bears striking similarities to the HC1 sequence: long direct and inverted repeats at the DNA level, including long GT dinucleotide repeats that are transcribed, and good agreement of the derived protein sequences. Even more important, both the HC1 and (2300 sequences are transcribed at high levels only in response to genotoxic stress: treatment with carcinogens in the first case and SV40 transformation in the second. Therefore, the data not only demonstrate a structural relationship, but also might indicate a functional relationship as well. The PV E5 products seem to trigger DNA replication and cell proliferation by activating growth-factor receptors.Other DNA viruses have gene products considered to be members of the family of growth factors, like EGF [43,44]. VGF binds and stimulates the activity of EGF receptors(221. Since we noted some sequence similarities between the putative HC1 product and different viral and cellular growth factors, we compared these polypeptides in a multiple alignment. Several amino acids known to be highly conserved between the Shope fibroma and the myxoma virus growth factors (SFGF and MGF, respectively) and between the rat tumor growth factor-a (rTGF-a) and human EGF are also conserved in the derived HC1 protein (Figure 8). To further characterize these relationships, we calculated a phylogenetic tree (data not shown) [451. Our results suggest a common ancestor for the HC1 and Q300 sequences; the HPV 16, 31, and 18 E5 gene products; and different viral and cellular growth factors. This theoretical result is in good agreement with our present knowledge of E5 activity and may be helpful for the design of future functional experiments, in particular, mutational analyses. The localization of HC1 on mouse chromosome 8 could be of interest in light of the report by Lindgren et al. [46], who studied transgenic mice carrying the BPV type 1 (BPV 1) genome. The presence of the BPV 1 genome elicited both benign fibromatoses and malignant fibrosarcomas in mice. Karyotypic analyses of both benign and malignant cells revealed consistent abnormalities of mouse chromosomes 8 and 14 in the fibrosarcomas that were not present in the preneoplastic fibromatoses. The abnormalities of chromosome 8 were generally trisomies or duplications involving region C 1 -C3. jun Bandjun D have both been mapped t o the C region of mouse chromosome 8 [47]. As we placed H c l 4.8 cM proximal to jun D (Figure 7), it is likely that Hcl resides in the C region as well. It is possible that H c l QunB or jun D) may be involved in tumor progressionassociated with papillomavirusgene expression. Finally, it is often possible to predict where a gene is on human chromosomes based on its position on mouse chromosomes.jun D has been localizedto human chromosome 19pl3 (Figure 7). Thus, the linkage between H c l and jun Dsuggests that Hcl resides on human 19p as well.

97

MOUSE HOMOLOCUE OF HPV ES REGION

Figure 6. Reverse transcription-polymerase chain reaction analysis of HCl E5 transcription. RNAs were extracted from tissues and papillomas (Pap) from treated and control animals and from cloned C127 mouse cells. Control animal (control mouse number 3) and cells without any treatment are marked with 0 Animal number 6 was treated with a single dose of 100 nmol of DMBA, followed 1 wk later by 10 nmol of TPA twice weekly for 24 wk. The animals were killed 1 wk later. C127 DMBA cells are C127 cells treated with 40 nmol of DMBA four times, with three passages between each treatment course without the addition of carcinogen, and three passages after the last treatment before extraction of cytoplasmatic RNA used in the assay. After DNase treatment, the RNA was divided into three aliquots, and

RT was performed using primer 1 (5’4gacaacatacacacat-3’; PCR 1 in Figure 2). primer 2 (5‘-acaatccatgaggtaga-3’; PCR 2 in Figure 2). or primer C (both primers 1 and 2 but without adding reverse transcriptase to the reaction). thus providing an internal negative control for each RNA. Lane + C contained 60 pg of clone HC1 DNA as a positive PCR control. PCR was performed immediately after RT and addition of the second primer; then the reaction products were separated on a 2.5% agarose gel, blotted, and hybridized with a 5’4abeled oligonucleotide (5‘-tgtatgttcatgtatgt-3’; PCR K in Figure 2). The lower right panel is a 48-h exposure of the lanes immediately above it (12-h exposure) to visualize the very faint skin signal.

Two additional findings require further analysis. One is the bidirectional transcription of the HCI region, and the other is the apparent relationship of HCI and PV E5 sequences with regulatory sequences. Bidirectional transcription has been demonstrated for the c-Ha-rasgene [481 and for the dihydrofolate reductase (DHFR) promoter in different organisms [49,50]. The latter example may be particularly pertinent in view of the DHFR gene‘s involvement in drug resistance due to overexpression after DNA amplification. The relationship with regulatory sequences, as determined by sequence analysis, suggests at the least an ancestor with regulatory functions; comparison of sequences stored in sequence data banks showed a remarkable similarity of the mouse E5-like sequence with upstream regulatory regions of genes known to be expressed in a tissue- or differentiation-specific manner or both. This may indicate a putative function of PV E5 sequences in the regulation of late protein expression. It is interesting to note that sequences that share homology with the E5 region upstream of the rat prolactin gene were shown to form Z DNA and inhibit gene transcription [Sl]. Our preliminary data revealed specific protein binding to these sequences, and functional assays with plasmids containing reporter genes cloned downstream of HPV 18 and HPV 16 E5 detected promoter activity (Geisen C, Kahn T, unpublished observations). The data show that the HCI region is transcribed at high levels under specific conditions. At this point, we cannot demonstrate whether or not these transcripts are translated into proteins. The same question remains unanswered for Q300. Cloning of cDNAs

and expression experimentsare underway as a first approach to answering this question. The evolutionary conservation of the HC1 sequence raises interesting questions concerning its origin in PV DNA. It has already been suggested that DNA tumor viruses may have evolved by acquiring from their host cells mutant growth suppressor genes, the products of which may interfere with the normal processes of cellular growth [52]. The HC 1 and Q300 E5-corresponding sequences are perhaps normal counterparts of an additional category of virally encoded proteins related to the EGF family and capable of deregulating normal epithelial cell proliferation. It will be interesting to analyze host cell DNA for additional genes sharing structural and functional aspem with specific genes of PVs. Such studies may yield further information on the role of PV in oncogenesis. ACKNOWLEDGMENTS

We thank D. Gallahan for providing blots with DNA from different mouse species and subspecies; E. Schwarz and D. Bartsch for helpful discussions; 5. Suhai, K . 4 . Glatting, and B. Drescher for help in the computer analysis; and H. Adldinger for critical reading of the manuscript. We thank M.B. Cybulski and D. A. Swing for excellent technical assistance. This research was supported, in part, by the National Cancer Institute under contract NO1-C0-74101 with ABL.

Received January 17, 1992; revised April 9, 1992; accepted April 15, 1992.

KAHN ETAL.

om om om om 0. om

0.01

cl.

O .

BO

. o

O .

O . .0

. a 1

1

6

O .

I 0

Bestfit

0.

3

0

4

2

1

8p12-q11.2 8pl2-p 1 1.2

1913

Figure 7. Position of the Hc7 locus on mouse chromosome 8. Hc7 was mapped t o mouse chromosome 8 by interspecific backcross analysis. The segregation patterns of Hc1 and flanking genes in 93 backcross animals that were typed in common for H c l is shown at the top o f the figure. For individual pairs o f loci, more than 93 animals were typed (see Materials and Methods). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BU6J x M. spretus)F, parent. The shaded boxes represent the presence of a C57BU6J allele, and white boxes represent the presence of a M. spretus allele. The number o f offspring inheriting each type o f chromosome is listed at the bottom o f each column. A partial chromosome 8 linkage map showing the location of Hc7 in relation t o linked genes is shown at the bottom of the figure. Recombination distances between loci in centimorgans are shown t o the left of the chromosome, and the positions o f all loci except Hc7 in human chromosomes are shown to the right. References for the map positions of most human loci can be obtained from Online Mendelian Inheritance in Man, a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD). Jund, jun D; Junb, jun B.

20

10

*

+++ + + rTGFa

(37) (35) (37) (44)

REFERENCES

19~13

hEGF

B P V l E5

1. Gissmann L. Papillomavirusesand their association with cancer inanimalsand in man. CancerSurv3:161-181, 1984. 2. Orth G. Epidermodysplasia verruciformis, a model for understanding the oncogenicity of human papillomavirus. Ciba Found Symp 120: 157-174,1986, 3. zur Hausen H. Papillomavirus as carcinomaviruses.In: Klein G (ed), Advances in Viral Oncology, Vol. 8. Raven Press Ltd., New York, 1989, pp. 1-26. 4. zur Hausen H. Human papillomaviruses in the pathogenesis of anogenital cancer. Virology 184:9-13, 1991. 5. devilhers EM. Heterogeneityof the human papillomavirusgroup. J Virol63:4898-4903, 1989. 6. CrookT, Morgenstern JP, Crawford C, Banks L. Continued expression of HPV 16 E7 protein is required for maintenance of the transformed phenotype of cells co-transformed by HPV 16 plus El-ras. EMBOJ8:513-519, 1989. 7. Schwarz E, Freese UK, Gissmann L, et al. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature314:111-114, 1985. 8. von Knebel Doeberitz M, Oltersdorf T, Schwarz E, Gissrnann L. Correlationof modified human papillomavirus early gene expression with altered growth properties in C4-I cervical carcinoma cells. Cancer Res 48:3780-3786, 1988. 9. Lambert P, Howley P The genetics of BPV 1. Annu Rev Genet 22:235-258, 1988. 10. Chen 5-L, Mounts P Transformingactivityof E5a protein of human papillomavirustype 6 in NIH 3T3 and C127 cells. J Virol 64:32263233, 1990. 11. Leptak C, Ramon y Cajal 5, Kulke R, et al. Tumorigenic transformation of murine keratinocytes by the €5 genes of bovine papillomavirus type 1 and human papillomavirus type 16. J Virol 65:7078-7083.1991. 12. Chen S-L, Mounts P Detection by antibody probes of human papillomavirus type 6 €5 proteins in respiratory papillomata. J Med Virol29:273-283, 1989. 13. Shirasawa H, Tomita Y, Kubota K, et al. Transcriptional differences of the HPV type 16 genome between precancerous lesions and invasive carcinomas. J Virol62: 1022-1027, 1988. 14. Stoler MH, Wolinsky S , Whitbeck A, Broker T, Chow LT. Differentiation-linked human papillomavirus types 6 and 11 transcription

6.0

HC1 SFGF MGF

HPV 18E5

Q300

*Calculated with the Bestfit program. Figures between brackets are length of the Bestfit alignment expressed in number of amino acids.

Hcl

Jwtb

HC 1

HCI 100 (70) 64.4 (59) 58.9 (56) 40.4 Q300 100 (77) 51.9 (59) 54.3 HPV18E5 100 (73) 59.4 100 B P V l E5

w 0 mu

8

i 4.8

Table 2. Percentage of Similarity (at Protein Level) Between HCl, HPV 18 E5, BPVl E5,and 4300

30

40

60

50

70

80

+ **+

TI-HEVDSIFSCVSVCV----CMCVYLCVYLCVCS----CMCVYACVCI----CVY-VHVSVYACVCICVCMCMYACVCMCV?VCMC----V MATRNLVASLLCIMYAVHA----MNDYLYIVKHVKVCNHDYENYCLNNGTC-FTIALD~SITP?CVCRINYE-GSRCUFINLV--TYMVPRDLVATLLCRMCIVQATMPSLDNYLYIIKRIKLCNDDYKNYCLNNGTC-FTVALNNVSLNP?CACHINYV-GSRCQFINLI--TIK WSHFNKCPDSHTQYCFH-GTCRFLVQEEKPA----CVCHSGYV-G~CEHADL----LA _________ NSD--SECPLSHDGYCLHDGVCMYIEALDKYA----CNCWGYI-GERCQYRDLKWWELR __-._______--------_ . ~ ~ ~ ~ _ _ _ _ _ _ _ _ _ _ _ _ ^ _ _ _ _ _ _ _ _ _ _

t

*

Figure 8. Comparison of the HCl sequence with members of t h e EGF-rTGF-a family. Multiple alignments between the HC1-derived protein and SFGF, MGF, and the secreted peptides of rTGF-a and human EGF (hEGF), as calculated by the Clustal similar amino acids. The program. *, identical amino acids;

+,

+

*+

* +

+ +

* *

*

+*

+

symbols used above the sequences refer only to HC1, SFGF, and MGF, whereas when used below the sequences, they apply t o all five sequences. Those residues known t o be highly conserved in the EGF-rTGF-afamilyare indicated by bold letters.

MOUSE HOMOLOCUE OF HPV €5 REGION

15

16. 17. 18.

in genital condylomata revealed by in situ hybridization with message-specificRNA probes. Virology 172:331-340, 1989. Wilbur DC, Bonfiglio 1, Stoler M. Continuity of human papillomavirus (HPV) type between neoplastic precursors and invasive cervical carcinoma: An in situ hybridization study. Am J Surg Pathol 12:182-186, 1988. Rohlfs M, Winkenbach 5, Meyer S, Rupp T, Durst M. Viral transcription in human keratinocyte cell lines immortalizedby human papillomavirus type 16. Virology 183:331-342, 1991 Vousden K. Human papillomavirus and cervical carcinoma. Cancer Cells 1 :43-50, 1989. Settleman J, Fazeli A, Malik J, Horwiz B, DiMaio D. Genetic evidence that acute morphologic transformation, induction of cellular DNA synthesis, and focus formation are mediated bya single activitv of the bovine DaDillomavirus E5 Drotein. Mol Cell Biol 9 5563-5572, 1989 Horwitz BH, Weinstadt DL, DiMaio D Transforminq activity of a 16-amino-acid segment of the bovine papillomavirus E5 protein linked to random sequences of hydrophobic amino acids. J Vlrol 63:4515-4519, 1989. Martin F: Vass W, Schiller 1, Lowy D, k l u T The bovine papillomavirus E5 transforming protein can stimulate the transforming activity of EGFand CSF-1 receptors. Cell 59.21-32, 1989. Petti L, Nilson LA, DiMaio D. Activation of the platelet-derived growth factor receptor by the bovine papillomavirus E5 transformingprotein. EMBOJ 10:845-855, 1991. King CS, Cooper JA, Moss 8, Twardzik DR. Vaccinia virus growth factor stimulates tyrosine protein kinase activity of A431 cell epidermal growth factor receptors. Mol Cell Biol 6:332-336, 1986. Wagner 5, Cullmann G, Knippers R. The Q300 gene: A novel transcription unit induced in simian virus 40-infected and -transformed mousecells. J Virol65:3259-3267, 1991. Fries1 5 , Schmidt R, Rippmann F, Steinbauer B, Hecker E. Cancerogenesis by 7.1 2-dimethylbenr[a]anthracene (DMBA): The influence of dose, dose frequency and tumor promotion. J Cancer Res Clin Oncol, in press. Edler L, Schmidt R, Weber E, Rippmann F, Hecker E. Biological assays for irritant, tumor-initiating and -promoting activities. J Cancer Res Clin Oncol 117:205-216, 1991. Reeve VE, Greenoak GE, Gallagher CH, Confield PJ, Wilkinson I. Effect of immunosuppressive agents and sunscreens on U.V carcinogenesis in the hairless mouse. Aust J Exp Biol Med Sci 63: 655-665, 1985. MacDonald RJ. Swift GH, Przybyk AE, Chirgwin JM. Isolation of RNA using guanidinium salts. Methods Enzymol 152:219227,1987. Benton W, Davis R. Screening lambda gt recombinant clones by hybridizationtosingle plaques in situ. Science 196: 180-182,1977. Gissmann L, Diehl V, Schulz-Coulon HJ, zur Hausen H. Molecular cloning and characterizationof human papillomavirusDNA derived from a laryngeal papilloma. J Virol44:393-400, 1982. Kahn T, Schwarz E, zur Hausen H. Molecular cloning and characterization of the DNA of a new human papillomavirus (HPV30) from a laryngeal carcinoma. l n t l Cancer 37:61-65, 1986 Herbarth F: Vosberg H-F! Enzymatic amplification of myosin heavychain mRNA sequences in vitro. DNA 7:297-306. 1988. Saiki RK, Gelfand D, Stoffel 5, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491, 1988. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1982. I

19

20. 21. 22. 23. 24.

25. 26.

27. 28. 29. 30. 31. 32. 33.

,

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