Geographical dependence of sequence variation in ...

Journal of General Virology (1992), 73, 1829-1832. Printedin Great Britain

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Geographical dependence of sequence variation in the E7 gene of human papillomavirus type 16 Daniel Eschle, 1 Matthias Diirst, 2 J a n ter Meulen, 1 Jeff Luande, 2 H a n s C. Eberhardt, 2 Michael Pawlita I and Lutz Gissmann 1. IForschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 6900 Heidelberg, Germany and 2Tanzania Tumor Centre, Muhimbili University College of Health Sciences, P.O. Box 3592, Dar Es Salaam, Tanzania

We have determined nucleotide sequences of the E7 open reading frame (ORF) of human papillomavirus type 16 (HPV-16) isolates obtained from 32 genital tumours and two HPV-16-transformed human keratinocyte cell lines. In comparison to the prototype HPV-16 isolated from a German cervical cancer biopsy, no sequence variations were noticed in either the two cell lines or the 10 biopsies that were obtained from German patients. In contrast only three of 22 (13-6%) of Tanzanian isolates showed the prototype sequence. In 18 of these biopsies two alterations (T to C

and T to G) not affecting the amino acid sequence were found within the HPV-16 E 7 0 R F (nucleotide positions 789 and 795) but eight of these isolates contained an additional change (nucleotide position 647) coding for serine instead of asparagine (amino acid position 29). One tumour harbours HPV-16 D N A with a mutation (C to T) at nucleotide 790 changing the E7 amino acid sequence (arginine to cysteine) at position 76. Our findings suggest that clustering of E7 sequence variants may occur in different geographical regions of the world.

Human papillomavirus type 16 (HPV-16) is associated with premalignant and malignant tumours of the anogenital tract. The early proteins E6 and E7 seem to play a critical role in cell transformation as demonstrated in different in vitro systems (for review see zur Hausen, 1991). The HPV-16 genome has been identified in tumour biopsies from patients from different geographical regions (Diirst et al., 1983; Tsunokawa et al., 1986; Lorincz et al., 1987; ter Meulen et al., 1992). At this time, it is not clear whether HPV-16 isolates containing particular amino acid sequence variations are geographically clustered. In fact, little evidence for variation between the oncogenes of individual isolates is available (Icenogle et al., 1991). Recently, however, the clustering of sequence variations in the non-coding region (upstream regulatory region) of HPV-16 isolates obtained from four continents was reported (Chan et al., 1992). In this report an analysis of German and Tanzanian samples (including those already tested by Chan et al., 1992) with regard to possible changes within the E7 open reading frame (ORF) of the HPV-16 genome is described. Biopsies of 32 different genital tumours (two genital warts, 27 cervical cancers, two penile cancers and one

vaginal cancer), obtained from patients attending the Tanzania Tumor Center in Dar Es Salaam or from various hospitals in Germany, were analysed. By Southern blot hybridization they had been shown previously to contain HPV-16 DNA. Two human keratinocyte cell lines (HPK IB and H P K II; Diirst et al., 1987) transformed by the 'prototype' HPV-16 D N A (isolated from a German cancer biopsy designated WV 2916; Diirst et al., 1983) were also tested. D N A was extracted from all samples and the HPV-16 E7 sequences were determined after amplification of a 506 bp fragment by polymerase chain reaction (PCR). A Landgraf thermocycler (Vers Laborsysteme GmbH) was used to generate this fragment from 200 ng of cellular D N A with the following primers: V21, 5 ' A T A A T A T A A G G G G T C G G T G G 3' and MI8, 5" C A T T T T C G T T C T C G T C A T C T G 3", corresponding to nucleotide positions 480 to 499 and 985 to 965, respectively (numbering as in Seedorf et al., 1985). An initial denaturing step (95 °C for 5 min) was followed by 37 cycles at 95 °C for 1-5 min, 50 °C for 1 rain and 72 °C for 2 min. The last cycle was terminated by a prolonged extension reaction (72 °C for 4 min) followed by cooling to 4 °C. PCR-amplified D N A was electrophoresed in

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low melting point agarose and sequenced without further purification with Sequenase (USB). The protocol published by Kretz et al. (1989) and modified by A. Schmid & M. Billeter (personal communication) was used. Briefly, the agarose slice containing the PCR product was melted for 10 min at 70 °C and about 0.3 pmol of the DNA was denatured for 10min at 95 °C in a 15tx| reaction containing 5 x Sequenase buffer and 1.5 pmol of internal primer. The annealed DNA-primer complexes were incubated for 5 min at 37 °C with 5.5 ktl of labelling mix and 4.5 lxl aliquots were added to each of the four ddNTPs (2-5 Ixl). After incubation for 5 min at 37 °C 4 III of the formamide stop mix was added to each reaction. Electrophoresis was performed on a 5~o sequencing gel, after denaturation of the samples for 3 min at 80 °C. Depending on the individual reactions, overlapping sequences from both strands could be determined, representing between 240 and 294 nucleotides starting at the 5' end of the E 7 0 R F . HPV-16 DNA cloned in plasmids was sequenced with Sequenase according to the specifications of the manufacturer. In addition, HPV-16 DNA was cloned at the unique B a m H I site from bacteriophage 2 libraries which were constructed from cellular DNA from the biopsies designated WV 75, 1024 and 2965 as well as from DNA from the two keratinocyte cell lines. The different HPV16 DNA isolates were recloned into the pUC19 vector and their E7 nucleotide sequences were determined without prior amplification by PCR. Identical results were obtained from all the isolates when E7 sequences obtained from the PCR products and from the cloned HPV-16 genomes were compared. These data demonstrate that the viral genomes remained stable during the cloning procedures and that the error rate of the Taq polymerase was below detection level under the experimental conditions employed in this study. The fidelity of the reaction was confirmed when a second amplification and sequence reaction was performed in five cases and identical results were obtained in all instances (including sample A27 with the unique modification at nucleotide position 780; Table 1). Cross-contamination of samples was unlikely since the individual biopsies were collected over several years and the DNAs were extracted at different times. False positives due to contamination during the PCR were also unlikely as negative controls (human placental DNA and reaction mix with no DNA added) were included in all experiments and did not yield any amplified product. The results are summarized in Table 1. Between 240 and 294 nucleotides of each isolate (corresponding to amino acid positions between 1 and 80 and the fulllength E7 protein) were determined. All sequences were compared to the HPV-16 prototype DNA from biopsy WV 2916, described by Dfirst et al. (1983) and se-

Table 1. Nucleotide changes in various HPV-16 isolates Sample* H P K Ib:~ H P K I1:~ W V 75 W V 1024 W V 2965 W V 3096 W V 3097 W V 3270 W V 3471 W V 4042 W V 4043 W V 4044 A6 AI6 A21 A24 A27 A32 A36 A42 A46 A50 A54 A56 A61 A62 B20 B22 B23 B25 B29 B30 B33 B61

Tumour Nucleotides Changesat nucleotideposition typet sequenced 647 789 790 795 GeWa GeWa CxCa PeCa PeCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa VaCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa CxCa

562-855 562-855 562-855 562-855 562-855 562-855 562-855 562-855 562-855 562-826 562-855 562-826 562-801 562-801 562-848 562-848 562-837 562-848 562-848 562-840 562-840 562-840 562-840 562-840 562-840 562-840 562-850 562-848 562-848 562-848 562-848 562-848 562-848 562-848

. . . . . . . . . . . . A-~G§ A-*G§ A~G§ . A-*G§ A-*G§ . . A-*G§ A-*G§ A-*G§ -

. . . . . . . . . . . .

. . . . . . . . . . . . T~C T-*C T~C T~C . . T~C T-*C T--~C T~C T-~C T-*C T-*C . . . . T~C T~C T~C T-*C T~C T~C T-*C

. . . . . . . . . . . . C-*'I]I,¶ . . . -

T-*G T--,G T-*G T~G T-*G T-*G T-*G T-*G T--,G T-*G T-*G

T-*G T-*G T~G T-*G T-*G T-*G T-*G

* Biopsies d e s i g n a t e d W V are o f G e r m a n origin, s a m p l e s l a b e l l e d A or B w e r e o b t a i n e d f r o m T a n z a n i a (ter M e u l e n et al., 1992). S a m p l e s l a b e l l e d W V a n d A were also t e s t e d b y Chart et al. (1992) a n d the following d e s i g n a t i o n s were used in t h e i r p a p e r : W V 7 5 = G B 1 3 , WV 1024=GB14, WV 2965=GB1, WV 3096=GB3, W V 3097 = G B 4 , W V 3270 = G B 5 , W V 3471 = G B 6 , WV4042=GB10, WV4043=GBll, WV4044=GB12, A6=TB1, A16=TB3, A21=TB4, A24=TB5, A27=TB6, A32=TB7, A36=TB8, A42=TB9, A46=TBII, A50=TBI2, A54=TB14, A 5 6 = T B 1 5 , A61 = T B 1 6 , A 6 2 = T B 1 7 . t GeWa, genital wart; PeCa, penile carcinoma; CxCa, cervical carcinoma; VaCa, vaginal carcinoma. :~ H u m a n k e r a t i n o c y t e cell line t r a n s f o r m e d b y HPV-16. § C h a n g e from a s p a r a g i n e to s e r i n e at a m i n o a c i d p o s i t i o n 29 o f the E7 protein. IIC h a n g e from a r g i n i n e to cysteine at a m i n o acid p o s i t i o n 76 o f the E7 protein. ¶ Tested twice by PCR.

quenced by Seedorf et al. (1985). After several years in culture, the sequence of the HPV-16 E 7 0 R F s carried by the human keratinocyte cell lines HPK Ib and HPK II (Diirst et al., 1987) proved to be unchanged. In eight of the 32 biopsies (25~) nucleotide 647 showed a change from A to G, creating a codon for serine at amino acid position 29 (previously asparagine). The same eight

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biopsies and additional 10 samples (in total 56%) contained HPV-16 sequences with changes at positions 789 (T to C) and 795 (T to G). Neither change alters the predicted amino acid sequence but the change at position 795 creates a unique B s i W I restriction enzyme cleavage site (CGTACG) which is a useful marker for the screening of further isolates. An additional change was noted in biopsy A27, in which nucleotide 790 was changed from C to T, coding for cysteine instead of arginine at amino acid position 76. All changes were seen exclusively in biopsies of African origin. There was a strong geographical dependence of the E7 sequence variations observed. Whereas all 10 German samples showed an identical E7 sequence compared to the prototype HPV-16, only three of 22 (149/o) of the HPV-16 isolates from Tanzania exhibited prototype E7 sequences. Except for the alteration at position 790, all other changes have been previously reported for the E 7 0 R F of six of 24 HPV-16 isolates obtained from cervical cancer biopsies as well as from cervical swabs of asymptomatic women (Icenogle et al., 1991). These authors did not find additional modifications in the 3' part of the E 7 0 R F (nucleotides 802 to 855), a sequence which was not determined in some of our isolates. Our data confirm the earlier observation that in different HPV-16 isolates the E 7 0 R F is very stable (Icenogle et al., 1991). In the two HPV-16-transformed human keratinocyte cell lines HPK IB and HPK II analysed in this study the HPV-16 E 7 0 R F was shown to be unchanged when compared to the original HPV-16 isolate (Diirst et al., 1983; Seedorf et al., 1985) which was used for transformation (Diirst et al., 1987). Variations that occur within the E 7 0 R F in most instances do not affect the amino acid sequence. Alterations of the E7 (Leu to Phe at position 28; Asn to Ser at position 29; Pro to Leu at position 47; Arg to Cys at position 76) are not located at amino acid positions which so far have been identified as being important for the biological functions of E7 (Jones et al., 1990; Firzlaffet al., 1989; Storey et al., 1990). The fact that these variants were isolated from cervical cancer biopsies indicates that the different amino acid exchanges did not significantly alter the oncogenic properties of the protein. It remains to be tested, however, whether the individual HPV-16 isolates differ in their transforming activity in vitro or in their ability to bind the retinoblastoma gene product. Recently, HPV-16 E7-specific antibodies were detected in about half of cervical cancer patients with HPV-16positive tumours (M/filler et al., 1992). The HPV-16 E7 variations found by Icenogle et al. (1991) and this study are not located within the peptide (amino acids 10 to 19) that was used as the antigen for the ELISA (MiiUer et al., 1990). Hence, natural variability of HPV-16 is not a

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likely explanation for the lack of E7-specific antibodies in a proportion of cervical cancer patients. Biopsies designated WV and A (Table 1) analysed in this study were also examined by Chan et al. (1992). Examining the nucleotide sequence of part of the upstream regulatory region they also found specific European and African variants but with a higher frequency of nucleotide changes compared to the E7 ORF. This finding indicates that certain parts of the viral genome have evolved concomitantly, albeit to different extents, depending on the selection pressure acting upon protein-encoding and structural segments of the genome. We would like to thank Dr Harald zur Hausen for his support and stimulating discussions and Dr Hans Adldinger for his helpful comments. We are grateful to Anita Schmid and Martin Billeter (University of Ziirich, Switzerland) for communicating technical procedures. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Gi 128/2-1), the Roche Research Foundation and by the Science and Technology for Development Programme of the European Community (Contract TS2-0009-D).

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(Received 30 January 1992; Accepted 1 April 1992)