JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1991, p. 2656-2660 Vol. 29, No. 110095-1137/91/112656-05$02.00/0Copyright (O 1991, American Society for Microbiology

Characterization of Human Papillomavirus Type 66 from anInvasive Carcinoma of the Uterine Cervix

ANWAAR R. TAWHEED, SYLVIE BEAUDENON, MICHEL FAVRE, AND GÉRARD ORTH*Unité des Papillomavirus, Unité de l'Institut National de la Santé et de la Recherche

Médicale 190, Institut Pasteur, 75724 Paris Cedex 15, France

Received 9 May 1991/Accepted 14 August 1991

Human papillomavirus (HPV) DNA sequences coexisting with HPV16 and HPV45 were cloned from aninvasive cervical carcinoma. The cloned HPV was shown to be a novel type, named HPV66, and is related toHPV56 (an HPV detected in cervical cancer). After screening 160 anogenital biopsies, four specimens exhibitedhistological features of intraepithelial neoplasia and contained HPV66 sequences. Of these, three were found tobe associated with another HPV type.

More than 20 types of human papillomavirus (HPV) areassociated with anogenital infections, causing benign prolif-erations (condylomata acuminata) or premalignant lesions(intraepithelial neoplasia) (8). HPV types associated withintraepithelial neoplasia such as HPV16 and HPV18 havebeen detected in the majority of anogenital invasive carci-nomas (30). Several studies have shown the presence ofuncharacterized HPV types in 10 to 30% of HPV-positivegenital lesions (1, 17, 20, 29). These HPVs need to be

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characterized to clarify the role of the different HPV types ingenital carcinogenesis. Here, we report the molecular clon-ing and characterization of a novel type of HPV isolatedfrom an invasive cervical carcinoma.A biopsy specimen was taken from a 38-year-old patient

with a stage I invasive squamous-cell carcinoma of theuterine cervix. Total tumor DNA was extracted by theguanidinium isothiocyanate-cesium chloride method (21) andtested for the presence ofHPV DNA sequences by Southernblot analysis. The DNA was digested with both BglI andBglII restriction endonucleases, blotted onto nitrocellulose,and hybridized with a mixed probe consisting of 32P-labeledHPV16, -18, and -33 DNAs, under nonstringent conditions(Tm - 40°C). The total molecular size of the hybridizedfragments exceeded the size of the HPV genome (8 kb),suggesting the presence of more than one HPV (Fig. 1, lanea). Analysis of the tumor DNA by polymerase chain reactionwith primers specific for HPV6, -11, -16, -18, and -33 (20)revealed the presence of HPV16 (9) (data not shown). Theblot was dehybridized and subsequently rehybridized withHPV16 DNA probe under stringent conditions (T,,, - 20°C).The hybridization signal was restricted to a band larger than8 kb (Fig. 1, lane b). This suggested that the HPV16 genomewas integrated in the tumor DNA since HPV16 DNA does

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FIG. 1. Southern blot hybridization analysis of HPV DNA se-quences found in an invasive carcinoma of the uterine cervix. Thetotal cellular DNA extracted from the biopsy (6 ,ug; lanes a to d, f.and g) and the cloned HPV66 DNA (5 ng; lane e) were cleaved withendonucleases BglI and Bgtll (B1+B2), EcoRI (E), or with EcoRIand PvuII (E+Pv). The fragments were separated by electrophore-sis in 1% agarose gels, denatured in situ, and transferred tonitrocellulose filters. The filters were hybridized under nonstringent(NS) or stringent (S) conditions (Tm - 40°C and T,,, - 20°C,respectively), by using 32P-labeled DNA probes specific for HPV16,-18, -33, -45, and -66 DNAs, as indicated. The migrations of freemonomeric HPV DNA molecules, forms t, Il, III, are marked. Thesizes of the fragments are expressed in kilobases.

* Corresponding author.

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FIG. 2. Analysis of the extent of similarity among HPV DNAsby Southern blot hybridization. The purified cloned DNAs (5 ng) ofHPVs associated with cutaneous (HPV1 and HPV5) or genital(HPV6, -16, -18, -30, -33, -39, -42, -45, -51, -52, -53, -54, -55, -63, -56,and -66) lesions were electrophoresed in 1% agarose gels in thepresence of human placenta DNA (1 ,ug). Samples were blotted ontonitrocellulose membranes and hybridized under either stringent (Tm- 10°C) (A and C) or nonstringent (Tm - 40'C) (B) conditions, with32P-labeled DNA probes specific for HPV66 (A and B) and HPV56(C).

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FIG. 3. Physical map of HPV66 DNA. The sintaken as the origin. The HPV66 map was alignemap of HPV16 by DNA sequencing. The opecorresponding to the early (E) and late (L) genes a

bottom of the figure. The enzymes BglI, BgIII, kXbaI have no cleavage sites in HPV66 DNA.

not contain BglI and BglII restriction sitesments of 1.5, 1.7, and 4.8 kb (Fig. 1, lanespond to the BglI-plus-BglII pattern of HIThis was confirmed by hybridization vHPV45 DNA under stringent conditions (After hybridization of EcoRI-digested ttEcoRI site in HPV45), only bands correspî

coiled circular (form I), open circular forumu(form III) molecules were observed (Figappeared, therefore, that HPV45 was preset

form in this tumor.Two bands at the positions of forms I and

in the BglI-plus-BglII-digested tumor DN/

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presence of additional episomal HPV molecules (Fig. 1, lane=-~ a). This HPV was found to have a single EcoRI site (data not

£ Ezshown). We proceeded to isolate this HPV by constructing a

genomic library of the tumor DNA in bacteriophage lambdaL1 ,

zap Il (Stratagene, La Jolla, Calif.) (26) by using EcoRIendonuclease. The recombinant phages carrying the cloned

Isite was HPV DNA were identified by plaque hybridization by usinggle EcoRI

the was 32P-labeled DNA of HPV16, -18, and -33 as a mixed probe,n reading frames under nonstringent conditions (Tm - 40'C). The HPV DNAare indicated at the was subsequently subcloned into plasmid pBR322. AfterlindII, SmaI, and cleavage with a mixture of EcoRI and PvuII endonucleases,

the cloned HPV DNA and the tumor cellular DNA yieldedfragments of identical sizes (with a sum of 8 kb), by using thecloned HPV DNA as a probe (Fig. 1, lanes e and f). Thisindicated that the complete genome of the HPV had been

s (25). The frag- cloned. Hybridization of the BglI-plus-BglII-digested tumora) might corre- DNA with the 32P-labeled cloned DNA confirmed the pres-

>V45 DNA (19). ence of this HPV as free episomal DNA molecules (Fig. 1,vith 32P-labeled lane g).(Fig. 1, lane c). The degree of similarity between the newly cloned HPVimor DNA (no DNA and the DNA of HPV types 1 to 56 (8) was examinedending to super- by Southern blot hybridization under different conditions ofn 11), and linear stringency. At high stringency (60% formamide; Tm - 10°C),,. 1, lane d). It the 32P-labeled, newly cloned HPV DNA showed a strongnt in an episomal cross-hybridization with the DNA of HPV56, an HPV type

associated with cervical cancer (16), and a weak cross-

Il were detected hybridization with the DNAs of HPV30 (14) and HPV53 (12)&, indicating the (Fig. 2A). Similar results were observed when HPV56 DNA

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FIG. 4. (A) Nucleotide sequences of the 3' end of long control region and the E6 and E7 open reading frames of HPV66. The predictedE6 and E7 genes begin at nucleotides 437 and 907 and terminate at nucleotides 901 and 1221, respectively. The putative HpaI site used to alignthe papillomavirus sequences is double underlined (7). The TATA and CAAT sequences are boxed. The ACCN6GGT binding motifs of theE2 gene products are indicated by arrows. The putative consensus sequences of splice donor and acceptor sites are underlined. (B) Alignmentof the deduced amino acid sequences of E6 and E7 proteins from HPV16, -56, and -66. Residues that are identical between the aligned regionsare shaded. The putative zinc finger motifs (Cys-X-X-Cys) (6) are underlined. The minimal regions needed for binding HPV16 E7 to p105-RB(18) and the corresponding regions in HPV56 and HPV66 are boxed.

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FIG. 5. Detection of HPV66 DNA in tumor specimen by in situ hybridization. A small biopsy was fixed with Carnoy's fluid and embeddedin paraffin. Sections (5 pum thick) were used for histology and in situ hybridization. (A) Histological features. An invasive squamous-cellcarcinomae merges from a low-grade intraepithelial neoplasia. Hematoxylin-and-eosin staining was used. Magnification, x 124. (B) In situhybridization. HPV66 DNA molecules are detected in the nuclei of differentiating cells of the intraepithelial neoplasia after hybridization witha tritiated HPV66 DNA probe by using conditions previously described (11). Magnification, x 140.

was used as a probe (Fig. 2C). However, when the hybrid-ization was carried out under conditions of lower stringency(20% formamide; Tm - 40°C), the newly cloned HPV DNAshowed a strong cross-hybridization with the DNAs of thegenital HPV types and a weak cross-hybridization with theDNAs of cutaneous HPV types, as illustrated for HPV1 andHPV5 (Fig. 2B). No cross-hybridization was shown understringent conditions between the cloned HPV and HPVtypes 57 to 65 (8a). The extent of similarity between thenewly cloned HPV and HPV56 was estimated as 40% (meanvalue of four independent experiments) by using liquid-phase hybridization at saturation followed by Si endonucle-ase digestion (15). By using different restriction enzymes,

the physical map of HPV66 was constructed (Fig. 3), and itwas clearly different from that of other known types. Therewere no conserved restriction sites found between HPV66and the published HPV56 physical maps. According to theconvention for the classification ofHPV isolates (5), an HPVis considered as a new type if it exhibits less than 50%cross-hybridization with other HPV types. Therefore, thecloned HPV was designated as a new type, named HPV66.The E6 and E7 genes of HPV16 were found to have

transforming and immortalizing activities (13), and theirproducts are able to form complexes with the cellular p53

protein (28) and retinoblastoma tumor suppressor gene prod-uct (p105-RB) (10, 18), respectively. Alignment of the phys-ical map of HPV66 with the genetic map of HPV16 wasdeduced from electron microscopy analysis of the heterodu-plex molecules (2) (data not shown). HPV66 E6 and E7genes were located in an EcoRI-SacI fragment (Fig. 3). Thisfragment was subcloned in M13 phage, and the completeDNA sequences of these two open reading frames, as well asthe 3' end (436 nucleotides) of the long control region, weredetermined by using the dideoxy chain termination method(Fig. 4A) (22). In the long control region, several conservedsequence elements, known to be involved in the regulationofHPV expression, were identified, including the TATA andCAAT boxes and two consensus-binding motifs of the viralE2 transregulating proteins (6). In the E6 region, potentialsplice donor and acceptor sites were found, signifying theexistence of an intron, the splicing of which generates a

truncated E6 protein. This has been recognized as a commonfeature of HPV types associated with genital carcinoma (24,27).Comparison of the nucleotide sequences of E6 and E7 of

HPV66 and HPV16 (25) revealed 56 and 54% similarity,respectively. Alignment of their deduced proteins showedthe presence of 50 and 54% identical amino acids in E6 and

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E7, respectively (Fig. 4B). A region was found in the HPV66E7 protein sharing homology with the p105-RB binding motifof HPV16 (18). Nine of the 12 amino acids known to beessential for binding to p105-RB were identical (Fig. 4B)(18). A similar comparison between HPV66 and HPV56 (16)in the E6 and E7 regions showed that the two HPVs shared84.5 and 93% nucleotides and 81.8 and 88.6% identical aminoacids (Fig. 4B), respectively. Thus, HPV66 and HPV56 aremuch more closely related than anticipated from liquid-phase hybridization studies. Similar results were previouslyfound for other HPV types, such as HPV6 and HPV11 (4) orHPV32 and HPV42 (3). It is hoped, therefore, that theaccumulation of nucleotide sequences of HPVs will providenew bases for their classification.

In this article we have shown that HPV66 was detectedalong with HPV16 and HPV45 in a biopsy taken from acervical cancer. To address the role of HPV66 in thedevelopment of the tumor, in situ hybridization experimentswere performed on tumor sections by using tritiated HPV16,HPV45, or HPV66 DNA probes. The specimen showed alow-grade intraepithelial neoplasia which continues as apoorly differentiated invasive carcinoma (Fig. 5A). Highlevels of HPV66 DNA vegetative replication were detectedin nuclei of terminally differentiating cells of the intraepithe-lial neoplasia (Fig. 5B). No hybridizationr signals were foundin deeper layers or in the carcinoma. This was expectedsince these cells are most likely to contain viral episomes inlow number (less than 50) which are undetectable by in situhybridization (23). The inability to detect HPV16 and HPV45DNA might be due to their presence in another region of thetumor specimen. The in situ detection of HPV66 DNA in alesion with histological evidence for malignant transforma-tion suggests an oncogenic potential for this virus. This wasfurther supported by identifying HPV66 sequences in DNAisolated from precancerous lesions, by using Southern blothybridization. Upon screening 160 anogenital biopsies withprobes specific for 20 HPV types, HPV66 was found in threecervical lesions diagnosed as high-grade cervical intraepithe-lial neoplasia and one penile intraepithelial neoplasia. Inthree of these four specimens, HPV66 was found associatedwith another HPV type (HPV16, HPV51, or HPV58).

In conclusion, HPV56 and HPV66 can be classified asmembers of an HPV group associated mainly with genitalneoplasia.

Nucleotide sequence accession number. The nucleotide se-quence accession number M75123 was assigned to HPV66.

We thank G. Riou for providing the tumor DNA preparation forthe cloning of HPV 66 and the biopsy specimen for in situ hybrid-ization experiments. We thank E.-M. De Villiers for her help in thecharacterization of HPV66, L. Gissmann and H. zur Hausen fortheir kind gifts of cloned HPV DNAs (HPV6, -11, -16, -18, -41, -48),T. Kahn for HPV30 DNA, D. Gallahan for HPV53 DNA, and A. T.Lorincz for HPV56 DNA. We thank P. Flamant for in situ hybrid-ization experiments, G. Pehau-Arnaudet for heteroduplex analysis,P. Cassonnet for skillful technical assistance, and C. Bergeron andO. Croissant for helpful discussions. We would also like to thank E.Gormley for critically reading the manuscript.One of us (A.T.) is a recipient of a fellowship from the Ligue

Nationale Francaise Contre le Cancer.

REFERENCES1. Barrasso, R., J. de Brux, O. Croissant, and G. Orth. 1987. High

prevalence of papillomavirus-associated penile intra-epithelialneoplasia in partners of women with cervical intra-epithelialneoplasia. N. Engl. J. Med. 317:916-923.

2. Beaudenon, S., D. Kremsdorf, O. Croissant, S. Jablonska, S.

Wain-Hobson, and G. Orth. 1986. A novel type of humanpapillomavirus associated with genital neoplasias. Nature(London) 321:246-249.

3. Beaudenon, S., D. Kremsdorf, S. Obalek, S. Jablonska, G.Pehau-Arnaudet, O. Croissant, and G. Orth. 1987. Plurality ofgenital human papillomavirus: characterization of two newtypes with distinct biological properties. Virology 161:374-384.

4. Broker, T. R., and L. T. Chow. 1986. Human papillomavirusesof the genital mucosa: electron microscopic analysis of DNAheteroduplexes formed with HPV types 6, 11, and 18. CancerCells (Cold Spring Harbor) 4:589-594.

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8. de Villiers, E.-M. 1989. Heterogeneity of the human papilloma-virus group. J. Virol. 63:4898-4903.

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11. Favre, M., D. Kremsdorf, S. Jablonska, S. Obalek, G. Pehau-Arnaudet, O. Croissant, and G. Orth. 1990. Two new humanpapillomavirus types (HPV54 and 55) characterized from genitaltumours illustrate the plurality of genital HPVs. Int. J. Cancer45:40-46.

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14. Kahn, T., E. Schwarz, and H. zur Hausen. 1986. Molecularcloning and characterization of the DNA of a new humanpapillomavirus (HPV30) from a laryngeal carcinoma. Int. J.Cancer 37:61-65.

15. Kremsdorf, D., S. Jablonska, M. Favre, and G. Orth. 1982.Biochemical characterization of two types of human papilloma-viruses associated with epidermodysplasia verruciformis. J.Virol. 43:436-447.

16. Lorincz, A. T., A. P. Quinn, M. D. Goldsborough, P. McAllister,and G. F. Temple. 1989. Human papillomavirus type 56: a newvirus detected in cervical cancers. J. Gen. Virol. 70:3099-3104.

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18. Münger, K., B. A. Werness, N. Dyson, W. C. Phetps, E. Harlow,and P. Howley. 1989. Complex formation of human papilloma-virus E7 proteins with the retinoblastoma tumor suppressorgene product. EMBO J. 1:4099-4105.

19. Naghashfar, Z. S., N. B. Rosenshein, A. T. Lorincz, J. Buscema,and K. V. Shah. 1987. Characterization of human papillomavi-rus type 45, a new type 18-related virus of the genital tract. J.Gen. Virol. 68:3073-3079.

20. Riou, G., M. Favre, D. Jeannel, J. Bourhis, V. Le Doussal, andG. Orth. 1990. Association between poor prognosis in early-stage invasive cervical carcinomas and non-detection of HPVDNA. Lancet 335:1171-1174.

21. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

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