JOURNAL

OF

Vol. 30, No. 7

CLINICAL MICROBIOLOGY, July 1992, p. 1716-1721

0095-1137/92/071716-06$02.00/0 Copyright © 1992, American Society for Microbiology

General Primer Polymerase Chain Reaction in Combination with Sequence Analysis for Identification of Potentially Novel Human Papillomavirus Genotypes in Cervical Lesions ADRIAAN J. C. VAN DEN BRULE,1* PETER J. F. SNIJDERS,1 PETRA M. C. RAAPHORST,' HENRI F. J. SCHRIJNEMAKERS,1 HAJO DELIUS,2 LUTZ GISSMANN,2 CHRIS J. L. M. MEIJER,' AND JAN M. M. WALBOOMERS'

Department of Pathology, Section of Molecular Pathology, Free University Hospital, De Boelelaan 1117, 1081 HVAmsterdam, The Netherlands, 1 and Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Gernany2 Received 2 December 1991/Accepted 14 April 1992

We recently described the detection of potentially novel human papillomaviruses (HPV) genotypes (HPV smears (A. J. C. van den Brule, C. J. L. M. Meijer, V. Bakels, P. Kenemans, and J. M. M. Walboomers, J. Clin. Microbiol. 28:2739-2743, 1990) by using the general primer-mediated polymerase chain reaction method (GP-PCR). In this study, the HPV specificities of GP-PCR products were determined by sequence analyses. M13 bacteriophage clones of PCR products derived from cloned unsequenced HPV genotypes 13, 32, 35, 43, 44, 45, 51, and 56 were subjected to dideoxy sequencing. Analyses of the putative amino acid sequences of these HPV types in addition to published HPV sequence data revealed stretches of highly conserved amino acid residues present in all HPV types, resulting in an HPV amino acid consensus sequence. Subsequently, HPV X-specific PCR products found in premalignant cervical lesions (n = 3), carcinomas in situ (n 6), and invasive cancer (n = 6) were analyzed for their nucleotide sequences. Comparison of these sequences with published HPV nucleotide sequences and data obtained in this study revealed three HPV type 35, two HPV type 45, one HPV type 51, two HPV type 56, and six unique HPV X sequences, of which three types were present in four cases of carcinomas (in situ). The nucleotide sequences determined appeared to be unique after a data bank search. Furthermore, the sequences of all HPV X isolates matched the HPV amino acid consensus sequence, thus confirming HPV specificity. This study illustrates the power of GP-PCR in combination with sequence analysis to determine HPV specificity and genotyping of PCR products derived from sequenced as well as unsequenced HPVs, including novel, not yet identified HPV types. types X [HPV Xl) in cervical

detect a broad spectrum of HPVs, including unsequenced types (HPV types X [HPV X]), by using a single pair of primers. By the use of this approach, some unidentified HPV genotypes have been successfully detected in cervical scrapes (30, 31). To exclude the detection of coamplified cellular DNA, the HPV specificities of GP-PCR products were determined by expected size, additional hybridization (30, 31), and RsaI restriction mapping (29). In this study, nucleotide sequence analysis is used to confirm the specificity of HPV genotypes and for their identification. On the basis of the presence of both a conserved region and a polymorphic region, both the specificity and the genotype of an HPV could be determined. By using this approach, three carcinoma-associated HPV X types and three HPV X types present in cervical dysplasia, which could be novel, still unidentified HPV types, were detected.

At present, the heterogeneous family of human papillomaviruses (HPVs) consists of more than 60 different epitheliotropic viruses found in cutaneous and mucosal lesions (9). Only 11 of the 27 mucosotropic HPV types reported to date are thought to possess oncogenic potential. Several HPV genotypes, i.e., HPV types 16, 18, 33, 35, 56, and 58, have been isolated from carcinomas of the uterine cervix (1, 3, 10, 19, 20, 22). HPV types 31, 39, 45, 51, and 52 have been associated with cervical cancer (2, 18, 23, 24, 28). On the basis of their prevalence rates in cervical carcinomas and in vitro transforming capabilities (25), HPV genotypes have been grouped as high risk (HPV types 16 and 18), intermediate risk (HPV types 31, 33, 35, 39, 45, 51, 52, 56, and 58), and low risk (HPV types 6 and 11) for leading to the development of cervical cancer (12, 18, 19). The remainder of the 27 oral-anogenital HPV genotypes (9) have been found mainly in normal mucosa, benign lesions, and low-grade intraepithelial neoplasias, suggesting that they are low- or intermediate-risk types. With respect to the broad spectrum of the HPV family and the ongoing isolation of new types, it is interesting to study whether additional oncogenic HPV types are present in carcinomas of any origin. Squamous-cell carcinomas of the cervix uteri can be a valuable source of these types. The recently described general primer-mediated polymerase chain reaction (GP-PCR) (29, 31) has been proven to

*

MATERIALS AND METHODS HPV clones and clinical specimens. Cloned HPV types 6b, 11, 16, 18, and 30 were kindly provided by H. zur Hausen (Heidelberg, Germany), HPV type 31 was provided by A. Lorincz (Gaithersburg, Md.), HPV types 5, 33, and 39 were provided by G. Orth (Paris, France), HPV type 45 was provided by K. V. Shah (Baltimore, Md.), HPV type 51 was provided by G. Nuovo (New York, N.Y.), and HPV types la, 2a, 8, 13, and 32 were provided by E.-M. de Villiers (Heidelberg, Germany). Cloned HPV types 35, 43, 44, and

Corresponding author. 1716

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GP-PCR AND SEQUENCE ANALYSES TO DETECT HPV X

56 were derived from the American Type Culture Collection (Rockville, Md.). Cervical scrapes were used for cytological classification and for HPV detection studies (30). For the latter, spatulas were placed in 5 ml of phosphate-buffered saline and vigorously vortexed. In this way, Pap IV (carcinoma in situ)- and Pap V (invasive cancer)-assigned cell suspensions (n = 40) and scrapes with dysplastic cells (n = 3; selected group) were collected and stored at -40°C. Archival paraffin-embedded invasive cervical carcinomas (n = 65) were serially sectioned. The first and last sections were hematoxylin and eosin stained and histologically analyzed for the presence of neoplastic cells. HPV detection. HPV detection of cloned HPV types was performed by using PCR on purified DNA. Clinical specimens were pretreated before being subjected to PCR. HPV was directly detected in crude cervical cell suspensions by using PCR as previously described (30). Briefly, 10 ,ul of suspended cells was frozen at -40°C, thawed, boiled for 10 min, cooled on ice, and spun down before the PCR components were added. In addition, HPV was detected in a single paraffin-embedded section, which was deparaffinized by xylene and washed with 96% ethanol. After the tissue was centrifuged and air dried, the pellet was suspended in 50 ,ul of distilled water and frozen at -80°C for at least 30 min. After thawing, a 50-pul proteinase K mix (10 mM Tris HCI [pH 7.5], 1.5 mM MgCl2, 0.45% Tween 20, 60 ,ug of proteinase K per ml) (Boehringer, Mannheim, Germany) was added, and the mixture was incubated at 55°C for 1 h. Finally, samples were treated at 100°C for 10 min and centrifuged. Twenty microliters of the supernatant was used in the PCR mixture. Samples were subjected to GP-PCR followed by typespecific PCR (TS-PCR) in a total volume of 50 pul as previously described (30, 31; also see these references for primer sequences). Briefly, general primers GP5 and GP6 (GP5, 5'-TITlTGTTACTGTGGTAGATAC-3'; GP6, 5'-GA AAAATAAACTGTAAATCA-3') were used in the PCR, which permits the detection of the sequenced mucosotropic HPV types 6, 11, 16, 18, 31, and 33 but also that of unsequenced HPV types (HPV X) at the subpicogram level (29). After low-stringency Southern blot analyses with probes of HPV-specific PCR products, the GP-PCR-positive samples were subjected to TS-PCR. Primer sets specific for HPV types 6, 16, and 33 and HPV types 11, 18, and 31 (nucleotide localization of PCR product generated by TS primers: HPV type 6, nucleotides [nt] 4671 to 4950; HPV type 11, nt 6841 to 7200; HPV type 16, nt 6028 to 6179; HPV type 18, nt 6903 to 7119; HPV type 31, nt 3057 to 3570; HPV type 33, nt 2415 to 2870; for nucleotide sequences, see reference 30) were used to detect the sequenced HPV genotypes (30). TS-PCR products were confirmed for their specificities by using oligonucleotide hybridization (for sequences, see reference 30). Samples which were positive by GP-PCR and negative by TS-PCR were suspected of containing HPV X. M13 cloning and sequencing. Samples containing oncogenic HPV X types were subjected to GP-PCR in fourfold with general primers extended at the 5' end with an 8-nt tail including the BamHI restriction site (BGP5, 5'-acggatccTTIl GTTACTGTGGTAGATAC-3', and BGP6, 5'-acggatccGAA AAATAAACTGTAAATCA-3'). GP-PCR products were pooled. After the addition of 2 volumes of ethanol-2 M ammonium acetate at room temperature, the PCR products were centrifuged at 10,000 x g to get rid of the primers. The ends of the PCR products were filled in with 2 U of Klenow

1717

DNA polymerase (Pharmacia, Uppsala, Sweden) according procedures (21). After sodium-acetate-ethanol precipitation for 30 min at -80°C and centrifugation, the PCR products were digested by an excess of 40 U of BamHI restriction enzyme (Pharmacia) according to the manufacturer's instructions. Digested GP-PCR products were subsequently ligated into the BamHI cloning site of bacteriophage M13 mpl8. The ligation reaction was performed with the total amount of GP-PCR products, 50 ng of M13 DNA, and 2 U of T4 DNA ligase (Pharmacia) according to standard procedures (21) overnight at 16°C. Half of the ligation product was presented to CaCl2-competent cells of Escherichia coli JM101 (21). HPV-containing M13 clones were identified after a plaque lift to a DNA-binding nylon support (Biotrace; Gelman Sciences, Ann Arbor, Mich.). Filters were hybridized under conditions of low stringency (Tm 33°C) with a mixture GP-PCR products specific for HPV types 6, 11, 16, 18, 31, and 33 as a probe as previously described (30). Subsequent washings were done at low stringency, and autoradiography was performed with Kodak Royal X-Omat film and intensifying screens overnight at to standard

-800C.

Overnight 1-ml cultures of HPV-containing M13 clones pelleted, and 800 ,ul of supernatant was used for single-stranded DNA purification (21). The DNA was finally

were

[lA of distilled water. Five microliters of M13-HPV DNA was used for dideoxy sequencing by means of a T7 polymerase sequencing kit (Pharmacia) and [ac-32P]dCTP according to the manufacturer's instructions. Reaction products were analyzed after 8% polyacrylamide gel electrophoresis and autoradiography. dissolved in 10

RESULTS Sequence analyses of PCR products derived from cloned HPVs. GP-PCR permits the detection of a broad spectrum of HPV types by the amplification of approximately 150 bp of the Li region (29, 31). Additional hybridization under lowstringency conditions by using a cocktail probe of GP-PCR products specific for HPV types 6, 11, 16, 18, 31, and 33 confirms HPV specificity (31). Samples which were positive by GP-PCR and negative by TS-PCR (HPV types 6, 11, 16, 18, 31, and 33) were suspected of containing presently unsequenced HPV types designated HPV X in this study. So the group HPV X can consist of already identified types, such as HPV types 45 and 51, as well as still unidentified, novel HPV types. GP-PCR products derived from plasmid clones of unsequenced HPV types 13, 32, 35, 43, 44, 45, 51, and 56 were cloned into bacteriophage M13, and nucleotide sequences were determined (Fig. 1). Nucleotide sequences were compared with one another and with data bank information (Micro Genie release 6.0; Beckman, Berkeley, Calif.). HPV X sequences were not identical to those of sequenced HPV genotypes (HPV types 1, 2, 5, 6, 8, 11, 16, 18, 31, 33, 39, 47, 57, and 58; references are listed in the legend to Fig. 2), those of sequenced animal PV types (bovine PV [BPV] types 1, 2, and 4, cottontail rabbit PV [CRPV], deer

PV [DPV], European elk PV [EPV], and feline PV [FPV] type 1), or those of other viral and cellular genes known to date. In addition, the short stretches of nucleotide sequences from GP-PCR products flanked by both primers and the equivalent regions of HPVs with published sequence data were translated to the putative amino acid sequence, which was numbered according to the position of the Li start codon found in already sequenced HPV genotypes (6, 12).

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VAN DEN BRULE ET AL.

90 30 40 50 60 70 80 100 20 10 CAGACACATA TAAGGCCACA GAATATAAAC AGTACATGCG ACATGTAGAA GAATr HPV 13 TACACGCAGT ACrAACATGA CTGTGTGTGC AGCCACTACA TCATCIXTCT HPV 32 TACCCGTAGT ACTAACATGA CrGTGTGTGC TACTGTAACA ACrGAAGACA CATACAAGTC TACTAACTIT AAGGAATATC TACGCCATGC AGAGGAATA HPV 35 AACCCGTAGT ACAAATATGT CTGTGTGTTC TGCTGTGTCT TCrAGTGACA GTACATATAA AAATGACAAT TTrAAGGAAT ATTTAAGGCA TGGTGAAGAA TA HPV 43 CACTCGTAGT ACAAACTTGA CGTrATGTGC C=CFACTGAC CCrACTGTGC CCAGTACATA TGACAATGCA AAGTTTAAGG AATACTTGCG GCATGTGGAA GAATA HPV 44 TACCCGTAGT ACAAACATGA CAATATGTGC TGCCACTACA CAGTCCCCTC CGTCTACATA TACTAGTGAA CAATATAAGC AATACATGCG ACATGTTGAG GAGTr HPV 45 TACCCGCAGT ACTAATTrAA CATTATOTGC C=CTACACAA AATCCTGTGC CAAGTACATA TGACCCrACT AAGTTTAAGC AGTATAGTAG ACATGTGGAG GAATA HPV 51 TACCAGAAGT ACAAATTTAA CTATrAGCAC TGCCACTGCr GCGGTITCCC CAACATTTAC TCCAAGTAAC TITAAGCAAT ATATrAGGCA TGGGGAAGAG TA HPV 56 TACTAGAAGT ACTAACATGA CrATTAGTAC TGCTACAGAA CAGTrAAGTA AATATGATGC ACGAAAAATr AATCAGTACC TTAGACATGT GGAGGAATA HPV Xa CACTCGTAGC ACTAACATGA CTTTATGTGC TGAGGTTAAA AAGGAAAGCA CATATAAAAA TGAAAATTT AAGGAATACC TrCGTCATGG CGAGGAATT HPV Xc CACCCGTAGT ACTAACCTAA CATTGTGTGC TACAGCATCC ACGCAGGATA GCTTTAATAA TTCrGACTrr AGGGAGTATA TrAGACATGT GGAGGAATA HPV Xd CACCCGCAGT ACrAATITAA CCATTTGTAC TGCrACATCC CCCCCTGTAT CTGAATATAA AGCCACAAGC TTrAGGGAAT A1TnGCGCCA TACAGAGGAG TT HPV Xf TACACGTAGT ACAAACATGA CAATATGTGC TGCrACAACr CAGTCTCCAT CrACAACATA TAATAGTACA GAATATAAAC AATACATGCG ACATGTrGAG GAGTr HPV Xg CACTCGrAGC ACTAATTrAA CCITATGTGC TGCCACACAG TCCCCACAC CAACCCCATA TAATAACAGT AATTTCAAGG AATATTTGCG TCATGGGGAG GAGTr HPV Xh TACTAGAAGC ACTAATrTIT CrGTATGTGT AGGTACACAG GCrAGTAGCr CTACrACAAC GTATGCCAAC TCTAA1TTTA AAGAATATTT AAGACATGCA GAAGAGTA FIG. 1. Nucleotide sequences of GP-PCR fragments derived from cloned HPV types 13, 32, 35, 43, 44, 45, 51, and 56 and from potentially novel HPV X genotypes derived from clinical specimens. Primer sequences were excluded. Alignment of these amino acid sequences of cutaneous and mucosotropic HPV types (Fig. 2) revealed the presence of strongly conserved amino acid residues at both ends of the GP-PCR products, which allows the determination of an HPV consensus sequence. In contrast, the internal region, differing from 8 to 13 amino acid residues in length, was found to be polymorphic. Sequence analyses of carcinoma-associated HPV X. Carcinoma (in situ) specimens (n = 105) were analyzed by using GP-PCR followed by TS-PCR (data not shown). HPV types 16 and 18 were found in the majority of these clinical samples. In addition, HPV X genotypes were detected in 12 cases (Pap IV, n = 6; Pap V, n = 1; and paraffin-embedded cervical carcinomas, n = 5). Although this study aims to investigate carcinoma (in situ)-associated HPV X types, three HPV X-specific PCR products present in premalignant cervical lesions were also analyzed. HPV X-specific GPPCR products were verified by Southern blot analyses with a probe mixture of GP-PCR products of cloned HPV types 6, 11, 16, 18, 31, and 33 under stringent conditions (Tm) as previously described (31), resulting in the disappearance of hybridization signals. M13 clones of these HPV X-specific GP-PCR products were labelled by random priming (Pharmacia) and hybridized to the original amplified products derived from HPVpositive and HPV-negative clinical samples, which confirmed the specificities of the cloned products (data not shown). Subsequently, these M13 clones were sequenced. All separate M13 clones were analyzed for the presence of the HPV-specific consensus amino acid sequence (Fig. 3). These HPV-specific sequences were compared with the already known sequences of animal PVs (BPV types 1, 2, and 4, CRPV, DPV, EPV, and FPV type 1) and of HPV types (HPV types 1, 2, 5, 6, 8, 11, 16, 18, 31, 33, 39, 47, 57, and 58) in addition to the sequences of HPV types 13, 32, 35, 43, 44, 45, 51, and 56 obtained in this study. Seven carcinoma (in situ)-associated HPV X isolates were typed: HPV type 35 three times, HPV type 45 twice, HPV type 51 once, and HPV type 56 once. The remaining carcinoma-associated HPV genotypes (HPV Xa, Xc, and Xd) and dysplasiaassociated types (HPV Xf, Xg, and Xh) were unique in their sequences, and their sequences matched the HPV amino acid consensus sequence (Fig. 3). Table 1 shows the distribution of the different unique HPV genotypes, Xa, Xc, and Xd, in addition to HPV types 35, 45, 51, and 56 found in

5

10

15

20

25

30

35

A HPV

LSISMKN-N----ASTTYSNANFND[E] L RH

1

HPV 5 HPV 8 HPV 47

TRNTN

TRNTN TRNTN

FSISVYNQAGALKDVPDYNADQFRE

FSISVYTENGELKNITDYKSTQFRE FSISVYSQAGDIKDIQDYNADNFRE

T Ei

32

Y Q RH V EE Y L RH V EE Y Q RH V EE

37

* *

HPV HPV HPV HPV HPV HPV HPV HPV

HPV HPV HPV HPV

**

**

2a 6b 11

L RH M EE TRSTN VSLCA-TE----ASDTNYKATNFKE TRSTN MTLCAS-----VTTSSTYTNSDYKE Y M RH V EE TRSTN MTLCAS- VSKSATYTNSDYKE Y M RH V EE

32

TRSTN TRSTN TRSTN TRSTN TRSTN

33 56 57 58 16 31 35 51

HPV 13 HPV 18

MTVCA-T --- -VTTEDTYKSTNFKE Y L MTLC--TQ -- -VTSDSTYKNENFKE Y I MTIS--T---ATEQLSKYDARKINQ Y L VSLCA-T --- -VTTETNYKASNYKE Y L MTLC--TE ---VTKEGTYKNDNFKE Y V

TRSTN MSLCAAI----STSETTYKNTNFKE

L RH G EE TRSTN MSVCAAI---A-NSDTTFKSSNFKE Y L RH G EE TRSTN MSVCSA----VSSSDSTYKNDNFKE Y L RH G EE TRSTN LTISTATA--AVS-P-TFTPSNFKQ Y I RH G EE

33

TRSTN MTVCAATT---SSLSDTYKATEYKQ Y M RH V EE

34

HPV 39

TRSTN LTICASTQ---SPVPGQYHATKFKQ Y S RH V EE TRSTN FTLSTSIE---SSIPSTYDPSKFKE Y T RH V EE

HPV 43 HPV 44 HPV 45

TRSTN LTLCASTDPTV---PSTYDNAKFKE Y L RH V EE TRSTN MTICAATTQ---SPPSTYTSEQYKQ Y M RH V EE TRSTN LTLCASTQN--- PVPSTYDPTKFKQ Y S RH V EE * *

CONS.

32

RH A EE RH V EE RH V EE RH M EE RH V EE

TRSTN XTIC N S SLS V G

**

-

**

8-13 aa- YXXXXFKE Y X RH X EE F

YRQ F IND

FIG. 2. Putative amino acid sequences encoded by internal DNA segments (excluding the primers) of GP-PCR products or equivalent sequences of cutaneous HPV types (A) and mucosotropic HPV types (B), resulting in an HPV amino acid consensus sequence (CONS.). HPV types 2a and 57 are listed as mucosotropic types according to Hirsch-Behnam et al. (13). Boxes indicate conserved regions among mucosotropic HPV types, underlined residues indicate amino acid residues different from conserved amino acid residues of mucosotropic types, asterisks indicate amino acid residues strongly conserved among all PVs sequenced thus far, including animal PVs (the first amino acid residue is not marked, since it differs from those in animal PVs), and N is the number of amino acid residues. Dashes represent gaps in alignment. The published nucleotide sequence information used here was derived from references 4 (HPV type 18), 5 (HPV type 33), 7 (HPV type la), 8 (HPV type 11), 11 (HPV type 8), 12 (HPV type 31), 13 (HPV types 2 and 57), 16 (HPV type 58), 17 (HPV type 47), 26 (HPV type 6b), 34 (HPV type 39), and 35 (HPV type 5).

GP-PCR AND SEQUENCE ANALYSES TO DETECT HPV X

VOL. 30, 1992 5 CONS.

10

TRSTN XTIC N S SLS V G

15

20

25

30

35

YRQF

F

HPV-specific probes. However, additional nucleotide sequence analysis is the most reliable approach to rule out the detection of cellular or viral fragments cross-hybridizing with the HPV probe. Direct sequencing of PCR products failed in our hands, since this method needs large amounts of homogenous PCR products. Therefore, in this study, GP-

N

RIH X EE

- 8-13 aa- YXXXXFKE Y X

IND

_HGEE H G EE

HPV Xa HPV Xc

TRSTN MTLCA ----- EVKKESTYKNENFKE Y L [U TRSTN LTLCA-T---ASTQDS-FNNSDFRE Y I RI

HPV Xd

TRSTN LTICTATS--PPVSE--YKATSFRE Y L x

HPV Xf HPV Xg

TRSTN MTICAATTQ--SP-STTYNSTEYKQ Y M RI TRSTN LTLCAATQ --- SPTPTPYNNSNFKE Y L RI

HPV Xh

TRSTN FSVCVGTQA--SSSTTTYANSNFKE Y L RI H

ITEE

32

32

33

PCR products were cloned in M13 bacteriophage. At least three M13 clones of each HPV X-specific PCR product were sequenced in both directions to exclude reading errors of the

35

Taq DNA polymerase (15). Although most M13 clones were identical, variations were sometimes found at the nucleotide level at two to three different locations. This may indicate the presence of strain variants of a particular HPV genotype, as also recently shown by Ho et al. (14). Comparison of putative amino acid sequences of the GP-PCR products with published PV sequences resulted in a striking homology, as indicated in Fig. 2. Eighteen mucosotropic HPV genotypes and four cutaneous HPV types have been analyzed. The sequences of all HPV types match the amino acid consensus sequence as shown in Fig. 2. The mucosotropic HPVs contain the TRSTN amino acid sequence at the 5' part of the GP-PCR product. The sequenced cutaneous HPV types differ from this conserved region only at amino acid positions 3 (HPV types 1, 5, 8, and 47) and 5 (HPV type 1) (underlined in Fig. 2). HPV types 2 and 57 contain the mucosotropic-specific TRSTN sequence, which is in agreement with their recent designation as mucosotropic types (13). When the consensus is extended, it will be specific for all PVs, including animal PV types (data not shown). The amino acid residues marked with asterisks in Fig. 2 are strongly conserved among all PVs sequenced thus far, including the animal types BPV types 1, 2, and 4, CRPV, DPV, EPV, and FPV type 1. Of additional interest is the conserved pentamer RHXEE at the 3' end of the GP-PCR product, since this sequence is also reflected by a conservation at the nucleotide level. Glutamic acid (E) and histidine (H) are encoded by only two different triplets of nucleotides (GAG-GAA and CAT-CAC, respectively), which restrict the possible variation at the nucleotide level. Histidine has by now shown to be encoded only by triplet CAT (Fig. 1). These PV conserved regions were also found by Danos et al. (6), who previously compared Li-coded protein sequences of CRPV, HPV type la, HPV type 6b, and BPV type 1. HPV conserved amino acids can be of great value in confirming the HPV specificities of new nucleic acid sequences obtained by using PCR. Furthermore, these data confirm the conserved nature of the Li open reading frame (ORF) spanned by the general primers used in this PCR assay (29). Besides a consensus sequence to determine HPV specificity, a polymorphic region which allows HPV type differentiation also exists in the GP-PCR fragments. Comparison of the determined nucleotide sequence of a small part of the strongly conserved Li region of the carcinoma (in situ)-associated HPV X types with published HPV sequences and the sequences of cloned HPV X obtained in this study revealed HPV types 35, 45, 51, and 56 and three additional, still unidentified HPV genotypes (HPV Xa, Xc, and Xd). These potential oncogenic HPV X genotypes as well as all dysplasia-associated HPV X genotypes analyzed (HPV Xf through Xh) were unique in their sequences. Indeed, the sequences of all of these HPV types matched the amino acid consensus sequence, indicating HPV specificity (Fig. 3). In addition, a comparison was made

HVJEE A EE

FIG. 3. Comparison of putative amino acid se quences of HPV X-specific PCR products (excluding primer sequiences) with the HPV amino acid consensus sequence (CONS.). Boxes indicate conserved regions among mucosotropic HPV type-s. N is the number of amino acid residues. Dashes represent gaps in alignment.

cervical carcinomas (in situ), and the lengthIs of the generated GP amplification products. Homology (comparisons of these unique GP-PCR sequences by using the alignment function (Micro Genie; Beckman) with all o0 ther known PV sequences showed homology at the nucleotidle level of more than 55% with sequenced mucosotropic HP V types. Much lesser homology was observed with cellular and other viral sequences after data bank searches. The dettermined nucleotide sequences of coamplified GP-PCR prc)ducts of 80 to 120 bp in length that derived from human placenta DNA showed homology of less than 40% with H PV-derived sequences and did not match the HPV amino acid consensus sequence. DISCUSSION To date, the nucleotide sequences of only aa fraction of the 60 known HPV genotypes have been deteXrmined. Unsequenced HPVs (HPV X), including still-uniidentified types, are abundantly present in normal cells (2% 1HPV X; overall HPV prevalence rate of 3.5%) and dysplast ic lesions (20% HPV X; overall HPV prevalence of up t o 80%) of the cervical epithelium as shown in a large scree-ning cohort by using GP-PCR and TS-PCR (32). This study has focused on the presence of HPV X types in premalignanIt lesions and in cervical carcinomas (in situ) in an effort to icdentify all HPV genotypes with possible oncogenic potential. Cervical carcinoma (in situ)-associated unsequenced HP'V X types and some dysplasia-associated HPV X types deetected by GPPCR were further characterized. HPV speci ficities of these GP-PCR products are normally confirmed on the basis of their expected sizes and by Southern blot analyses with TABLE 1. Summary of sequenced HPV X genoltypesa associated with cervical carcinomas and carcinoma s in situ HPV genotype (no

Clinical sample (n)

Paraffin-embedded cervical carcinomas (5) Pap Vh (1) Pap IV" (6)

35

45

51

56

2

1

1

1

,.of times typed) Xa

Xc

Xd

1 1

1 1 " Lengths of sequences flanked by both general prime]rs: HPV type 56, 99 bp; HPV types 35 and 51, 102 bp; HPV type 45, 105 bp; HPV Xa and Xc, 99 1

1

1

1719

bp; HPV Xd, 102 bp. h Pap IV and Pap V were histologically confirmed as ca rcinomas in situ and invasive carcinomas, respectively.

between the determined sequences of HPV Xa, Xc, Xd, and Xf through Xh and HPV type 41 (13a) and preliminary HPV sequences which have been determined in a systematic

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VAN DEN BRULE ET AL.

sequencing effort at Heidelberg (HPV types 4, 7, 9, 10, 12, 14, 15, 17, 19, 25, 30, 51, 52, and 53 [8a]). The HPV Xa Li ORF sequence could be identified as that of HPV type 52, leaving HPV types Xc, Xd, Xf, Xg, and Xh still unidentified. Recently, the cervical carcinoma cell line ME180, which contained a novel HPV DNA (24a), was described. The sequence of this presumably new HPV type was different from all of the HPV sequences described in this paper, including the HPV X-derived sequences, but did match the HPV-specific amino acid consensus sequence. The sequences of these new HPV X types showed more than 55% homology to those of known sequenced mucosotropic HPVs. HPV Xa (HPV type 52) was highly homologous (71 to 75%) to HPV type 16 and HPV types of the 30s group. HPV Xc showed approximately 70% homology with HPV types 16, 32, and 45. These observed homologies of up to 75% are less than those found when the GP-PCR sequences of different, well-established, closely related HPV types, i.e., HPV types 6b and 11 (84%), HPV types 2 and 57 (82%), HPV types 18 and 45 (80%), and HPV types 33 and 58 (86%), are compared. This suggests that the HPV X-specific PCR products found have been derived from different HPV types and do not represent HPV strain variants. HPV typing based on the differences found in short stretches of 99 to 114 nt of these GP-PCR products seems therefore to be well possible. This is supported by the use of short sequences of the Li ORF (200 bp) in a phylogenetic study which revealed similar results using larger fragments of LI and other ORFs (33). In this study, it is shown that HPV Xc and Xd did not represent HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 58, which are known to be associated with cervical cancer. This means that at least two HPV X genotypes, detected by GP-PCR, may represent HPV types that until now have not been found in carcinomas, i.e., new oncogenic HPV types. Additional confirmation of the possible oncogenic potential of these HPV X types needs to be performed by DNA in situ hybridization studies which show their exclusive presence in carcinoma cells and not in the surrounding normal or dysplastic cells. Complete cloning of these putative new oncogenic HPV types may be facilitated by using the specific GP-PCR products as probes to screen the appropriate genomic library. In conclusion, GP-PCR is a very useful technique for collecting information concerning the great sequence variation within this virus group. This method, in combination with sequence analysis, is a rapid and efficient approach for HPV typing and the detection of novel HPV types in carcinomas of the cervix uteri and other neoplastic tissues suspected of HPV etiology as well. ACKNOWLEDGMENTS This work was supported by grants from the Prevention Fund (28-1502) and the Dutch Cancer Society "Koningin Wilhelmina Fonds" (IKA 89-16 and IKA-91-10), The Netherlands. REFERENCES 1. Beaudenon, S., D. Kremsdorf, 0. Croissant, S. Jablonska, S. Wain-Hobson, and G. Orth. 1986. A novel type of human papillomavirus associated with genital neoplasias. Nature (London) 335:246-249. 2. Beaudenon, S., D. Kremsdorf, S. Obalek, S. Jablonska, G. Pehau-Arnaudet, 0. Croissant, and G. Orth. 1987. Plurality of genital human papillomaviruses: characterization of two new types with distinct biological properties. Virology 161:374-384. 3. Boshart, M., L. Gissmann, H. Ikenberg, A. Kleinheinz, W. Scheurlen, and H. zur Hausen. 1984. A new type of papilloma-

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