Biologic properties and nucleotide sequence analysis of human papillomavirus type 51.

0022-538X/91/084216-10$02.00/0

Copyright ©1991, AmericanSociety forMicrobiology

Biologic

Properties

and

Nucleotide

Sequence Analysis

of Human

Papillomavirus

Type 51

OCTAVIAN LUNGU,1 CHRISTOPHERP. CRUM,2 AND SAUL

SILVERSTEIN'*

DepartmentofMicrobiology and Cancer Research Center, Columbia University, 701 West 168th Street,New York, New York 10032,1 and Department of Pathology, Brighamand WomensHospital, Boston, Massachusetts021152

Received 25 March 1991/Accepted16May 1991

Humanpapillomaviruses (HPVs) may be grouped accordingto the site from whichtheyareisolated andthe disease with which they are associated. We recently identified and cloned HPV type 51 (HPV-51) from a low-grade precancerous lesion (G. Nuovo, E. DeVilliers, R. Levine, S. Silverstein, and C. Crum. J. Virol. 62:1452-1455, 1988). Molecular epidemiologic analysis of cervical lesions, including condylomataand low- and high-grade precancers, revealed that HPV-51 was presentin about5% of the samplesweexamined. We have now determined the completenucleotide sequence of this virusandcomparedit withother sequencedHPVs. Ouranalysis revealsthat the7,808-bp genomeiscomposed ofeight open reading frameswhich are encoded on the same strand and that this virusismost closely relatedto HPV-31. Sequence comparisons placethis virus in the group of high-risk viruses (those with an increased risk ofprogressing to malignancy) along with HPV-16, -18, -31, and -33. Morphologic transformation experiments demonstrated that HPV-51 had transformation potential and that transformed cellscontainedRNAshomologoustoE6 and E7.

Human papillomaviruses (HPVs) are small icosahedral viruses that contain a double-stranded DNA genome of approximately 8 kbp. The genomes of more than 60 indepen-dent HPVs have been isolated as molecular clones. A new isolate is designated a novel strain if it is.50%homologous toanyotherknown HPV as determined by liquid hybridiza-tion analysis (13). The best criteria for the classificahybridiza-tion of novel isolates are direct DNA sequence analysis and com-parison of the amino acid coding potential of novel isolates with known oncogenic and benign strains. Members of this virusfamily are the causative agents for a variety of benign lesions that result from abnormal epithelial cell differentia-tion, and HPVs are frequently subgrouped according to their association with cutaneous or mucosal sites. Some of these viruses are associated with genital neoplasias which range in severity from mild dysplasia to frank carcinoma (7, 24, 27, 38, 70, 71). The ability of specific virus early gene products tointeractwithhost proteins such asp53and RB105 (25, 43,

54, 66)maybe the molecular basis that differentiates viruses with theability to cause malignant disease from those which causebenign papillomas.

We previously used Southern blot hybridization, in a molecularepidemiologic study, to correlate HPVs with their

segregation patterns according to the pathology of the le-sions atgenital sites (18). In this study several HPV DNAs with novel restriction endonuclease digestion profiles were detected. One of these DNAs, originally identified in a condyloma as an -8 kbpHindIIIfragment, was cloned and used to screen a panel of virus DNAs. Molecular hybridiza-tionanalysis revealed that this DNA genome represented a novelisolate which was designated HPV type 51 (HPV-51)

(47) The prevalence of HPV-51 in a small sampling of

biopsiesderived from patients with abnormal Papanicolaou smears wasexamined, and it was shown to be present in 7 of 127 samples derived from patients with condylomata, cervi-cal intraepithelial neoplasia, or carcinoma. A more recent prevalence study using polymerase chain reaction (PCR)

* Correspondingauthor.

analysis suggestedthatthis viruswaspresentinapercentage

(-3%) of thepopulation similarto thatof other virus types (e.g., HPV-6, -16, -18, -31, and -33) which were more frequently associatedwithgenitallesions (4).

We havedetermined thecomplete nucleotide sequence of HPV-51 and compared the arrangement and homologies of its open reading frames (ORFs) withthose of othergenital

isolates. Transfection of HPV-51 DNA into either the BALB 3T3 or the CREF rodent fibroblast cell line results in morphological transformation of these cells. The HPV-51-transformed lines express RNAs that have the potentialto encode the E6 andE7 oncoproteins.

MATERIALS ANDMETHODS

Cloning and sequence analysis. The HPV-51 genome as described in Nuovo etal (47) was excised fromvectorDNA

by digestion with HindIII, and the 7.6-kb virus band was purifiedfroma0.5% agarosegel by solubilizingthegel and

binding the DNA to glass powder (64). The DNA was cleaved withPstI, and the threePstI and the two HindIll-PstI fragments were subcloned into M13mpl8 and -19. Nucleotide sequence was determined by the dideoxy chain terminator method (53) both manually and by using an Applied Biosystems automated DNA sequencer. Where necessarythelengthoftheinsertwas pared with exonucle-ase III,and thefamilyof nested deletions that resultedwas sequenced. In several instances oligonucleotides that flanked regions for which no sequence information was availablewereprepared and used todetermine the

interven-ing sequence. Sequences were compiled and analyzed by usinga commercially available sequence analysis package,

DNASIS,from LKB.

The 232-bpHindIII fragmentwhich encodes the E6 ORF was cloned after amplification of DNA isolated from two

biopsies containing HPV-51 by using primers that flanked the HindIII site. Isolated DNA was heat denatured in the presence of thefollowing primers: 5'AAGTATAGAAGAA

CACCATGT, which is complementary to the anticoding

strand at positions 80 to 99, and 5'CTTTGACATCTATGA 4216

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FIG. 1. Sequencingstrategyand ORFsof HPV-51. TheM13 clones thatwereusedtodetermine thesequenceof the HPV-51genome are

shown beneathalinear representation of thegenome. Arrowswithoutnamesrefertooligonucleotides thatwereusedasprimersto"walk" through regions of thesequenceforwhichnocloneswereavailable. Fragments FltoF6representPstI-PstIorPstI-HindIII subclones that were used to generate the various subclones in M13. The letters above the line referto restriction endonuclease sites for the following

enzymes:H, HindIII; Spe, SpeI; P, Pstl; Rv, EcoRv; M,MluI; Pv, PvuII; B, BamHI; and Hc, HincII. The circlesrepresentthe potentialor

verified E2 binding sites, and the squares representpotential polyadenylation sites. The lowerpartof the figure depicts the location and boundaries ofthe variousORFs of HPV-51. The distribution of termination codons (long vertical lines) and initiation codons (short vertical lines) is shown.

CACCT, which is complementary to the coding strand at

nucleotides419 to 400.ThisDNA wassubjected to 35 cycles

of denaturing, annealing,and extensionat95, 55, and72°C,

respectively, with Taq polymerase in aPerkin-Elmer-Cetus

Thermocycler. The resulting product was gel purified and clonedinto the filled-inBamHIsiteofpIBI31. The sequence was determined from double-stranded plasmid DNA by

usingtheprimerssynthesizedforamplificationand T7 DNA

polymerase.

Morphologic Transformation. Continuous rat embryo

fi-broblasts (CREF) (26) wereobtained fromPaul Fisher, and BALB 3T3 cells were obtained from Mitchell Goldfarb. These cells weremaintainedatsubconfluenceinDulbecco's

modificationof Eagle medium containing 5% fetal calf serum andplatedat7 x 105cells per 5-cmdish priortotransfection.

Eachdishofcellswastransfected with 3.3 ,ugof linearized

HPV-51 or -18 DNA and 6.7 ,ug of salmon sperm carrier DNA or with only 10 ,ug of salmon sperm DNA, as

previ-ously described(67). Twodaysposttransfectioneachdish of cellswassplit 1:3, and the cells were fed fresh medium every third day. After 21 days, foci of transformed cells were

photographed, cloned, and counted.

PCRamplificationand RNAanalysis. DNAsfrombiopsies, plasmids,ortransformed cellsweretestedfor HPVDNAby PCR amplification of the Li region with the MY09 and MY11 primers described by Manos et al. (37). These DNAs were examined for the presence of HPV DNA

ho-mologousto the E6ORFbyusing primerscomplementaryto the coding strand (RT primer) 5'GGGAATTCCTTCA CAGTCCATCGCCGTTG and anticoding strand (5' E6 and E7 primer) 5'GGGGGATCCAACACCATGTTCGAA GACAAG, respectively. These oligonucleotides hybridize to HPV-51 DNA at nucleotides 887 to 866 and 91 to 111. RNA synthesized in transformed cells was analyzed by primer extending 25 jig of RNA with the RT primer and subsequently isolatingthecDNAproductandsubjectingitto 35 cycles of amplification with the RT and 5' E6 and E7 primers. The product was extracted with phenol and chlo-roform-isoamyl alcohol and precipitated withethanol. One-fifth of the purified product wasthen electrophoresed on a 3% GTG agarose gel that consisted of 3:1 NuSieve to SeaPlaqueinTris-borate buffer. The resolvedproductswere isolatedfrom the gel as previously described (64) and ana-lyzed for the presence of sequenceshomologoustoE6orE7 by slot blot hybridizationwith32P-labeledprobes that were specific forone orthe other of these genes.

Nucleotidesequence accessionnumber.TheHPVsequence in this paper was assigned GenBank accession number M62877.

RESULTS ANDDISCUSSION

Nucleotide sequence of the HPV-51 genome. To further characterize the HPV-51 genome, a detailed restriction

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AACAATTATC TTGTAAAAAC TAGGGTGTAA TCGAAGACAA GAGGGAAAGA CCACGAACGC

GGAATTATGT AGAGCAGATG TATATAATGT

TTTTATTCAA AAATTAGAGA GTATAGACGT GGTGTCATAG ATGTCAAAGA CCACTTGGGC

ATGCGCTAAT TGCTGGCAAC GTACACGACA

TTTAACACCA CAGACTGAAA TTGACTTGCA

AGACGGGCTG GACAGGCTAC GTGTTACAGA

GCGTTGTACA GCAGATGTTA ATGGGCGAAC GGGGCGGGGT GTAATGGGTG GTTTTTTGTT GATCTGATTT AATAAACTTT ATAGATAGTG

AAACAAAGAG GCTGTGCATC AGTTAAAACG AGTCAGGCAA ACGAGTCACA AGTTAAAAGG AGGTAGATGG GCAACATGGC GGTTCACAGA ACTAAACAGT ATATGTGAAG TATTAAAAAG

CGGGTGTTTA AAAGTGATAA AACATGTTGT CATTTTGCAT GTACTACCAT ATACAATGTT

TGCTAAGTGT TTAAGTACAT TAGTAAATAT

ACAGGCATAT CAAACATTAG CAATACATAT

TATCACAAAT GGTGCAATGG GCATTTGACC

AGCGTTTTTA AAGAGTAATT GCCAAGCAAA

GCCTGGATAA GGTATAGATG TGATAGAGCA

TTCAAATGTT TAAACAGTTT TTAAAAGGAA

AATGAAGTTT ATGCAAGGGT CCATTATTTC

GCTACGTATG GGTGTTGGAC ATATATTGAT

TAGTATGTCC ACCATTACTA ATAACGTCAA

TACATTTCCA TTTGATAACA ATGGGAATGC

GAGGAAGAGG ACAAAGAAAA TGGAGACCCT

GATCAAATTA ACTATTGGAC ATTGTTACGA

CAACAACAGT ATCAAAACAA AAGGCCTGTC

GCGGGAGACA TGTTATGAAC TATGGTGTGT

GCAATGGACT ATACAAGCTG GAAATTTATA

TAAATTCAAA AAAAGAATAT TATGTACAGT

CCGAAAAGGG TTATGACCGA AAACGGTGCA TATAAAAGTG CAGTGGTAAA AGTATAGAAG AACACCATGT TGCATGAATT ATGTGAAGCT TTGAACGTTT CTATGCACAA TATACAGGTA GTGTGTGTGT ATTGTAAAAA AGCATTTACT GAAATTAAGA TTGTATATAG GGATAATAAT CCATATGCAG TATGCAAACA ATGTTTACTG

TATAGCAGGT CTGTGTATGG TACTACATTA GAGGCAATTA CTAAAAAAAG CTTATATGAT TTATCGATAA CTGAAGAAAA GCAAAAATTG GTGGACGAAA AAAAAAGGTT CCATGAAATA GCGGGACGTT GGACGGGGCA ACGTAACGAA ACCCAAGTGT AATAAAGCCA TGCGTGGTAA TGTACCACAA TTAAAAGATG TAGTATTGCA ATGCTACGAG CAATTTGACA GCTCAGAGGA GGAGGATGAA GTAGATAATA TGCGTGACCA GCTACCAGAA ATTGAAGCTC CGTGTTGCAG GTGTTCAAGT GTAGTACAAC TGGCAGTGGA AAGCAGTGGA GACACCCTTC TAAGCCTGGT TTGCCCGTGT TGTGCGAACA ACTAGCAACG GCGATGGACT GTGAAGGTAC AGAGGATGAG GAAGCAATAG TAGAAAAAAA AACAGGAGAT AATGTTTCGG ATGATGAGGA TGAAAATGCA GATGATACAG

AAACTAGTAT TTGCAGTCAG GCGGAACAGG AGACAGCACG GGCGTTGTTT CAGGCCCAAG AATTACAGGC AAAGTTTCTA GTCAGCCCGC GAAGCAGCCC ATTAGGAGAC ATTACAAATC AAAACAACAC ACACAGCCAT AGATTACTGG ACAGTTATCC GGACAGCGGA TATGGCAATA CACAAGTGGA AACTGTGGAA GCAACGTTGC ACAGTGTGTG TAGTAGCGGG GGGGGCAGTG TTATGGATGT GGAAACAACA GAAAGCTGTG CAAATGTAGA CAGTAATGCA AAAGCAACGT TAATGGCAAA ATTTAAAGAG TTGTATGGTA TTAGTTATAA TGAGTTGGTA ATAGATTGGG TTTGTGCATT GTTTGGCGTT TCCCCAATGG TAGCAGAAAA TTTAAAAACA CTAATTAAGC TATCATGTGA TTGGGGCACC ATTGTATTAA TGCTAATTAG GTTTTCATGT GCAAAAAACA GAACAACAAT

CCCACAATCA CAAATGTTTA TAGAACCACC AAAATTACGT AGTACACCTG TGGCATTATA TTTTTATAGA GGAGAGACAC CTGAATGGAT TACACGACAA ACGCAACTAC AACATAGTTT TGAGGATAGT ACCTTTGAAT ATGAAGTATT AGATGATAGT GAAATAGCAT TTCATTATGC ACAATTAGCA GATATAGATA GTAATGCTGC

ATATGTAAAA GATTGTGGGA CCATGGCACG GCATTACAAA CGAGCACAAA GAAAATCATT ATCTATGTCA

AAGGATGGAG GCAACTGGAG AGAAATTGCT AAATTTTTAA GATATCAAGG TGTAAACTTT ATGTCCTTTA

CACCAAAACA CAATTGCATA GTCATATATG GCCCACCAAA CACAGGCAAG TCATTATTTG CAATGAGCCT

ATATGTAAAC TCTGGTAGTC ATTTTTGGTT ACAGCCACTA GAGGATGCTA AAATAGCATT GTTAGATGAT CAGTATTTAA GAAACTTTTT AGATGGTAAT CCATGTAGTA TAGATAGAAA ACATAGGAGT TTAATACAAT

ACATAAATCC ACAAGAGGAT GCAAACCTAA TGTATTTACA TACAAGGGTA ACAGTATTAA AGTTTTTAAA

TGTGTATACA TTGAATGATG AAAATTGGAA AAATTTTTTT TCCACCACAT GGTCCAGATT AGATTTGGAG

ATGCCACCGT TTAAATGTGT GCCAGGAGAA AATACTAGAC TGTTATGAAC TGGACAGTGA TAAATTAGTA

TATGAAGCTG CTATGTTTTA TGCAGCACGG GAAAGAAACT TACGAACAAT CAATCACCAG GTAGTACCAG

AAGCAATTGA AATGCACATG GCCTTACAAT CGCTTAACAA ATCAGACTAT AACATGGAAC CATGGACAAT

GGCTCCCAAG CAATGTTTCA AAAAGGGGGG CATAACTGTA ACAGTTATAT TTGATGGAAA TAAGGACAAT

TATATATATG ATAATGATAA GTGGGTAAAG ACAAATGGAA ATGTGGACTA TACGGGTATA TATTACACTG

TTAAAGATGA AGCCAAAATA TATGGGGCAC AACAGTGGGA GGTCTATATG TATGGTACTG TAATAACATG

3301 TCCTGAATAT GTATCTAGTA CCTGCAGCGA CGCGTTATCC ACTACTACAA CTGTTGAACA ACTATCAAAC ACCCCAACGA CCAATCCCCT TACCACCTGC

3401 GTGGGCGCCA AAGAAGCCCA GACACAACAG CGAAAACGAC AGCGACTTAC TGAGCCCGAC TCCTCCACAA TCTCCCCACT GTCCGTGGAC AATACAAACA

3501 ACCAAATACA CTGTGGAAGT GGAAGCACTA ACACTGGAGG GCACCAAAGT GCAACTCAGA CTGCGTTTAT AGTGCATTTA AAAGGTGATA CAAATTGTTT

3601 AAAATGTTTT AGATACAGAT TTACAAAACA CAAAGGGTTA TATAAAAACG TATCCTCAAC CTGGCATTGG ACCAGTAATA CTAAAACAGG CATTGTTACC

3701 ATTGTGTTTG ACAGTGCACA TCAACGGGAA ACATTTATAA AAACCATTAA AGTACCCCCA AGTGTAACAC TGTCATTGGG AATTATGACA CTGTAACTAG

3801 TGTAATATAT GTATTGTACA TATATACTGT CACAAGCCAA TATGTGCTGC TAATTGTATA GACATATTGT AACCATTGCA GTGTTTATTA TTTTGCTATT

3901 TGTGCTTTGC TTGTGTGTGT GTCTTGTGTT GTGTTGTTTG TTGCCGCTAC TGCTGTCCCA ATACGTGTTT GCAGCTGCCT TATTATTAAT TTTATGTTTT

4001 TGGTTTGTTG TTGCAACATC CCAATTAACT ACATTTTTTG TATATTTGAT TTTTTTTTAC TTACCTTGTT TACTTTTACA TCTATATACA TTTTTACTTT

4101 TGCAATAAAC TTGTTATATT TTTGTGATTA AATATGGTGG CTACACGTGC ACGGCGTCGG AAGCGAGCAT CTGTAACACA ATTATATTCT ACATGCAAAG

4201 CTGCTGGTAC ATGTCCTCCT GATGTTGTGA ATAAGGTTGA AGGTACTACA TTGGCCGATA AAATATTACA GTGGAGTGGG TTGGGTATAT TTTTGGGTGG

4301 CCTAGGTATT GGTACTGGGT CTGGATCTGG GGGGCGTACT GGATATATCC CTTTAGGTGG TGGGGGTCGC CCAGGCGTGG TGGATATTGC TCCTGCAAGG

4401 CCACCTATTA TAATTGACCT ATGGCACCAT ACTGAACCTT CTATAGTAAA TTTGGTTGAG GACTCTAGTA TTATTCAGTC TGGGTCTCCT ATACCTACCT

4501 TTACTGGTAC CGATGGCTTT GAAATTACTT CATCTTCCAC AACAACCCCT GCTGTGTTGG ACATCACCCC ATCTGCTGGT ACTGTACATG TTTCTAGTAC

4601 TAACATTGAA AATCCTTTAT ATATTGAACC TCCATCCATT GAGGCTCCAC AATCTGGAGA AGTGTCAGAT ATATATTTAC TAGTACACTA CTCTGGTACT

4701 CATGGGTATG AAGAAATACC TATGGAAGTG TTTGCATCCA ATGTCAGTAC TGGTACTGAA CCTATTAGCA GCACACCTAC TCCAGGGGTT AGTCGCATAG

4801 CTGCTCCCCG CTTGTATAGT AAGTCCTACA CACAGGTTAA AGTTACAAAT CCTGATTTTA TTAGTAAGCC ATCCACATTT GTTACATTTA ATAATCCTGC 4901 TTTTGAGCCT ATTGACACAT CCATAACTTT TGAGGAACCT GATGCTGTTG CACCTGATCC TGATTTTCTG GATATTATTA CACTGCACCG CCCTGCCCTT

5001 ACATCTCGTA GAGGCACAGT ACGCTTTAGT AGGTTAGGTC AAAAGGCCAC CATGCGCACT CGTAGTGGCA AACAAATTGG TGCTCGTGTA CATTATTATC

5101 ATGATATTAG TAGAATTGCA CCAGCTGATG AACTTGAAAT GCAGCCTTTA CTTTCACCTT CTAATAATTA TAGTTATGAC ATTTATGCTG ATTTAGATGA

5201 AGCTGAAACA GGTTTTATAC AGCCCACACA CACCACACCT ATGTCACACT CCTCTTTGTC TAGGCAGTTG CCCTCCTTAT CTTCATCTAT GTCTTCATCT

5301 TATGCAAATG TTACTATTCC ATTTTCAACT ACATATTCTG TTCCTATTCA TACAGGGCCT GATGTGGTAT TGCCCACATC TCCTACAGTA TGGCCTTATG

5401 TTCCCCACAC TTCCATTGAC ACCAAGCATT CTATTGTTAT ACTAGGTGGG GATTACTATT TGTGGCCCTA TACACATTTA CTACGCAAAC GCCGTAAACG

5501 TATACCCTAT TTTTTTACAG ATGGCATTGT GGCGCACTAA TGACAGCAAG GTGTATTTGC CACCTGCACC TGTGTCTCGA ATTGTGAATA CAGAAGAATA

5601 TATCACACGC ACCGGCATAT ATTACTATGC AGGCAGTTCC AGACTAATAA CATTAGGACA TCCCTATTTT CCAATACCTA AAACCTCAAC GCGTGCTGCT

5701 ATTCCTAAAG TATCTGCATT TCAATACAGG GTATTTAGGG TACAGTTACC AGATCCTAAC AAGTTTGGAC TCCCGGATCC AAATTTATAT AATCCAGACA

5801 CAGATAGGTT GGTGTGGGGT TGTGTGGGCG TTGAGGTGGG CAGAGGACAG CCCCTTGGTG TTGGCCTTAG TGGTCATCCC TTATTTAATA AATATGATGA

5901 CACAGAAAAT TCACGCATAG CAAATGGCAA TGCACAACAA GATGTTAGAG ATAACACATC TGTTGACAAC AAACAGACTC AGTTATGTAT AATAGGCTGT

6001 GCTCCACCTA TTGGGGAACA CTGGGGTATT GGCACTACAT GCAAAAACAC ACCTGTACCT CCAGGAGACT GCCCCCCCCT GGAACTTGTA TCCTCTGTCA

6101 TTCAGGATGG CGATATGATT GATACAGGGT TTGGAGCTAT GGATTTCGCT GCCCTACAGG CCACCAAATC AGACGTCCCT TTGGATATTT CACAGTCTGT

6201 TTGTAAATAT CCTGATTATT TAAAAATGTC TGCAGACACA TATGGTAATT CCATGTTTTT TCATTTACGC AGGGAGCAAA TCTTTGCTAG GCACTATTAT

6301 AATAAACTTG TAGGTGTTGG GGAAGACATT CCTAACGATT ATTATATTAA GGGTAGTGGT AATGGCCGTG ACCCTATAGA AAGTTATATA TACTCTGCTA

6401 CTCCCAGTGG GTCTATGATA ACATCTGATT CTCAAATTTT TAATAAGCCT TATTGGCTCC ACCGTGCGCA GGGTCACAAT AATGGCATTT GCTGGAACAA

6501 TCAGCTTTTT ATTACCTGTG TTGATACTAC CAGAAGTACA AATTTAACTA TTAGCACTGC CACTGCTGCG GTTTCCCCAA CATTTACTCC AAGTAACTTT

6601 AAGCAATATA TTAGGCATGG GGAAGAGTAT GAATTGCAAT TTATTTTTCA ATTATGTAAA ATTACTTTAA CTACAGAGGT AATGGCTTAT TTACACACAA

6701 TGGATCCTAC CATTCTTGAA CAGTGGAATT TTGGATTAAC ATTACCTCCG TCTGCTAGTT TGGAGGATGC ATATAGGTTT GTTAGAAATG CAGCTACTAG

6801 CTGTCAAAAG GACACCCCTC CACAGGCTAA GCCAGATCCT TTGGCCAAAT ATAAATTTTG GGATGTTGAT TTAAAGGAAC GATTTTCTTT AGATTTAGAC

6901 CAATTTGCAT TGGGTCGCAA GTTTTTGTTG CAGGTTGGCG TACAACGCAA GCCCAGACCA GGCCTTAAAC GCCCGGCCTC ATCGGCATCC TCTTCCTCTT

7001 CCTCTTCAGC CAAACGTAAA CGTGTTAAAA AGTAATGTAT GTTAGTTTTT GTATGCTTGT GCACACTGTT GTATGCCTGT ATGTATATGT TTGTGTATGT

7101 ACTGTATGTG TTTTTGTGTG TGTGTGTGTT GTTGTTCCTG TATGTATGAG TTATGTATGT TTATTATTAA TAAACTATGT GGTGTGTGTG TGTGTGTTTT

7201 TGCATGACTG CATTTGTATG ACATGTACGG GTGTATGTGG GTATTACATT ATCCCCGTAG GTCAAGGGTG GTGTTTCGGT GGCGTCCCTA TTGCCCTACC

7301 CATTTTTTGC AGCACAACAG TTTATATTTG TGCTATTTAG TTATACTTTG TAGCTTCCAT TTTGTTACAG CTGCAGCCAT TTTGAGTGCA ACCGATTTCG

7401 GTTCGTGTAC TTTTAGTATA TTTGCCAAGT TTTAAACCAC AACTGCCAGT TGTTTTTGGC ATAAACCATC ATTTTTTTAT GACATAGTGC ATACATCCGC

7501 CCGCCCACGC CTTGTACTTG GCGCGCCTTA CCGGCGCTAG TCATACAACC TATTAGTCAT TTGTACTTTA ACAATTGTTG GCACACTGTT TTCCGCCCTA

7601 TAATAATTTA ACTGCTTATA GGCATGTATT TTTTGGCATA TTTTATCTTA CTAATTGCAT AGTTGGCAGG TCAAATACTA TGTTTTTAGT GCCAAGTTTC

7701 TATCCTACTT ATAAACCATC TTACTCATAT GCAGGTGTGC TACACAAATG TGTTACCTAA CCGATTTGTG TTCTGCCTAT GCTTGCAACA TTTTTTCTTA

7801 TAACATTT

FIG. 2. Complete 7,808-bpsequence of the HPV-51genome. Theposition of nucleotide 1wasdetermined by alignment with HPV-16

DNA.

1 101 201 301 401 501 601 701 801 901

1001

1101 1201 1301 1401

1501 1601 1701

1801 1901 2001 2101

2201

2301 2401

2501

2601 2701 2801 2901 3001 3101

3201

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TABLE 1. Homologies of HPV-51 and other HPV ORFsa

% Homology

HORFP1

HPV-6 HPV-11 HPV-16 HPV-18 HPV-31 HPV-33 NA AA NA AA NA AA NA AA NA AA NA AA

E6 59 57 57 56 70 72 58 61 67 73 69 77 E7 58 63 55 63 60 67 62 62 58 64 54 67

El 66 67 63 67 66 65 63 67 67 74 67 73 E2 61 62 62 62 60 66 62 61 61 65 51 64 E4 58 54 57 54 58 62 65 60 50 60 58 58 E5 58 55 59 56 59 63 62 65 65 66 57 61 L2 58 70 59 70 63 68 66 77 63 70 54 67 Li 66 82 66 80 66 78 66 84 68 83 50 83

aSequenceswerealigned andcompared byusing theNeedleman-Wunsch algorithm(46) asimplementedby the DNASIS analysis package running on an MS-DOS-basedcomputer. NA, nucleic acid homology; AA, predicted amino acid homology withinthe ORF. Complete ORFs were used for comparison of thenucleicacids, andORFsbeginning with the firstin-frameATG codon were used for the amino acids, except for the E4 of HPV-16, -31, and -33 and the E5 ofHPV-16 and -51, which have noin-frame ATG; in these instances the complete ORFswereused.Onlythe E4AORFof HPV-6 and the3' ElORF ofHPV-16 were usedforcomparison.

endonuclease map of the original Hindlll fragment was prepared and each of the PstI-PstI and PstI-HindIII frag-ments was subcloned in both orientations in M13 vectors. These subclones were subjected to sequence analysis as described in Materials and Methods. Where the ability to read the sequence was exceeded by the length of the clone, thatpieceof DNA was subcloned or pared with exonuclease III, orspecificoligonucleotideprimers were synthesized and used to "walk" through the sequence. The sequencing strategy and clones analyzed are displayed in Fig. 1; more than95% of the sequence was determined in both

orienta-tions.The sequencesderivedfromtheoriginalHindlIl clone were assembled and aligned with the genomes of other HPVs. Thisanalysis revealedthatthe originalclone lacked the potential to encode anE6 protein. To determine if the

missingsequenceresulted fromisolation ofadefectivevirus or ifa small HindIII fragment had gone undetected in our original analysis, primers which flanked the single HindlIl

site weresynthesizedand usedtoamplifyDNAfromseveral HPV-51-containing lesions, including DNAfrom which the

original clonewasisolated. Each sample testedwas shown tocontain DNA that servedas atemplatefor thesynthesis of a 340-bp DNA which released a 232-bp fragment after digestion withHindlIl. The entire PCR-amplified fragment

wascloned and sequenced. Insertionof this informationinto the original compiled sequence resulted in

generation

ofa viralgenomeof7,808 bp.

Thecomplete nucleotidesequenceoftheHPV-51genome

isshown inFig. 2.Thefirst nucleotide was

assigned

onthe

basis ofhomologywith HPV-16 DNA. The G+C contentof

thisDNA is 39%. Alignment ofthe nucleotide sequence of HPV-51 with that of the other HPVs for which

complete

sequence information was available revealed that HPV-51 most closelyresembled HPV-31 (Table 1).

ORFs.The

pattern

of ORFswasdetermined and shownto be conserved with othersequencedHPVs. Allof the

ORFs,

with theexceptionof thatencodingtheE5homolog, contain an initiating methionine (Tables 1 and 2). A schematic

representation of the

major

ORFs is shown in

Fig.

1. Note that all of the major ORFs are derived as a result of translation of mRNAs that would be transcribed from

only

one strand of the DNA.

Comparisons

of the amino acids

TABLE 2. ORFs ofHPV-51

Nucleotideposition No. of No. of

ORF bss amino

StartORF First ATG Stopcodon bases acids'

E6 85 97 550 465 151

E7 479 560 863 666 101

El 733 874 2776 2,043 634

E2 2690 2720 3794 1,104 358

E4 3294 3309 3570 276 87

E5 3854 4106 255 84

L2 4125 4134 5538 1,413 468

Ll 5443 5521 7033 1,590 504

a Number of aminoacid which wouldconstitute thepredictedproteinsif translation initiatesatthefirst ATG ofeachORF,exceptinthe caseofE5,

which does nothave an initiating ATG and therefore begins at the first in-frame amino acid.

encoded

by

these ORFs allowed us to localize them with respecttotheirhomologsinother HPVs. The firstnucleotide

of each of the

major

ORFs along with the

positions

of the first ATG and stop codon is shown in Table 2. The number

of amino acids

composing

eachof the

presumptive

ORFs is also

presented

in this table.

The E6and E7 ORFsare

adjacent

but indifferent

reading

frames, and the E6 stop codon is

separated

from the first ATG in E7

by

10 nucleotides. E6from the

high-risk

HPVs

associateswith

p53

and targets itforthe

ubiquitin-dependent

degradation pathway (54, 66).

The E6 ORF in HPV-51is151 amino acids and contains tworepeats ofa

Cys-X2-Cys-X29-Cys-X2-Cys

motifthatare

separated by

36aminoacids.The sequenceand arrangement of this

motif,

whichis involved in

coordinating

Zn2+

(2, 29),

is conserved in all

sequenced

HPVs(14). Cole and Danos

(14)

proposed

that the

periodic-ity

of these repeats and the presence of other

highly

con-servedamino acidsat

regularly spaced

intervals suggest that E6 hasarisenas aresultofa

duplication

event.Inadditionto the motifs

they

describewe note

that,

amongthe

high-risk

types

(HPV-16,

-18, -31,

-33 and

-51),

there are additional

highly

conserved blocks of amino acids at invariant dis-tancesfrom the

metal-binding

domains

(Table

3).

The role of theseamino acids is

unknown; however,

theirconservation

in the E6ORF may

potentiate

their

oncogenicity

by

targeting

p53

for

degradation. Furthermore,

HPV-51,

like HPVs

as-sociated with

anogenital

cancer and in contrast to those

associated with

benign

condylomata,

possesses

potential

splice

donors at

positions

177 and 219 and a consensus

acceptorat402 within E6

(57, 60,

61).

If the donorat177is used and iftranslationinitiates atthe same AUG codon as

for

E6,

then the

resulting product

would bea43-amino-acid E6*-like

protein

(58).

Use ofthe donorat219would

require

internal initiation to maintain the

reading

frame and there-fore would not conserve the amino terminus of the E6*

protein.

The E7

proteins

from

high-risk

viruses associate with

p105-RB

with

high

affinity,

whereas those from low-risk

groups do not

(43).

This

protein family

shares

significant

amino acid

homologies,

which include the conserved

Cys-X2-Cys-X29-Cys-X2-Cys

motif

(14)

and a

string

of seven

amino acids

(DLXC'EQ),

which in HPV-18areinvolved in

binding pRB-105.

Mutagenic

analysis

of this

region

of the HPV-16 E7 ORFdemonstrated that the amino acids which compose this

string

are

important

in

binding

pRB-105

(43).

Analysis

of the E7ORF fromHPV-51reveals thatit encodes a 101-amino-acid

protein

with both the

Cys

motifand the

binding

domain for the

product

of the retinoblastomagene.

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TABLE 3. Organization of E6 ORFs from high-risk HPVs

Sequencea

CVYCKLELCRADVYNVAFTEIKIVYRDNNEYAVCKQCLLEYaLIRFYRRY CVECKKPLQRSEYYDFAFADLTVVYREGNPFGICKLCLRELaKISEYRH_H CVYCKGQLTETEVLDFAFTDLTIVYRDDTPHGVCTKCLREY5KVSEFRWY CVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDEYSRIRELRHY CVYCKQQLLRREvYDFAFRDLCIVYRDGNPYAVCDKCLKEY5KISEYRHY SRaVYGTTLEAITKKSLYDLSIRCHRCQ.RPLGPEEKQKLVAEKKRFHQIA NY8VYGNTLEQTVKKPLNEILIRCIICQRPLCPQEKKRHVDLNKRFHNIS RYaVYGTTLEKLTNKGICDLLIRCITCORPLCPEEKQRHLDKKKRFHNIG SDSVYGDILEKLTNTGLYNLLIRCLRCQRPLNPAEKLBHLNEKRREHNIA CYSLYGTTLEQQYNKPLCDLLIRCINCQBPLCPEEKQRHLDKKQRFHNIR

GRYTGQCANCW GRWAGRCAACW GRWTGRCIACW aHYRGQCHSCC aRWTGRCMSCC

aThe amino acidsequences of the E6 ORFs from these high risk HPVswerealignedasdescribed in Cole and Danos(14). Highlyconserved amino acids

previously noted by these authorsareinboldface. Those conserved amino acids whichweredetected inthismoreextensiveanalysis, whichincludedHPV-31 and-51,areunderlined.

The El ORF is thelargest foundinpapillomaviruses, and it encodes a protein which has been shown to dock to the virus-specified E2 DNA-binding protein when it interacts withsequencesintheupstreamregulatory region (URR)(6, 40). Phylogenetic analysis of this ORF in human viruses suggeststhat itmaybecomposedoftwodomains(14). The ElORF inHPV-51 hasthe potentialtoencode aprotein of

634 amino acids. There is considerable conservation of

sequence homology at boththe nucleotide and aminoacid levels betweenthe HPV-51 El ORF and othermembers of the HPVfamily (Table 1).

E2isadimericDNA-binding protein which recognizes the

sequence ACCN6GGT (1, 41) and has both activator and repressorfunctionsontheURR from bovine papillomavirus

(BPV). These functionscan beattributedtothepresenceof

two functional domains which are separated by a spacer

whose length is associated with the virus subgroup from which it is derived. Thus, viruses associated with epider-modysplasia verruciformis contain longer and more

hydro-philic spacers than those associated with infections of the

genital tract (36). The N- and C-terminal segments encode the transactivation and repressor-dimerization domains,

re-spectively. Repressor may be independently derived by

transcription fromaninternalpromoterincells infected with BPV (62, 68) or by alternative splicing as detected in transcriptional analyses of cells and biopsy samples contain-ingHPV-6, -11, or-16 (11, 22,52). Inthe genital

papilloma-viruses E2 bindsto twosites thatareproximal to transcrip-tional elements of the E6 promoter and represses

transcription (5, 51,63).The E2ORF of HPV-51canencode a 358-amino-acid protein that has the characteristic

three-domain structure ofother papillomaviruses (28). The 185-amino-acid N-terminaldomain is rich inarginine and lysine residuesandterminatesataconservedV-185.The

70-amino-acidC-terminaldomainwhich beginsatG-289is preceded by

a 104-amino-acid spacer. The relatively short size of the

middle segment and thepaucity of arginic and lysinic resi-dues distinguish this virus from those associated with epi-dermodysplasia verruciformis (36).

InHPVs the mRNA encodingthe E4 ORFis mostlikely derived by splicing fromadonor in Eltoanacceptorin E4 (11, 12,45), because the bulkof ElRNA, asdetected byin

situhybridization analysis,is found in the nucleus incervical intraepithelial neoplasms (19). The spliced mRNA can

en-codea functional E4gene product, and

immunohistochem-ical analysisofwartscontainingHPV-1 andearly neoplasias containing HPV-16revealsthatE4 is expressed principally

as a cytoplasmic protein in cells that stain positive for Li (16,20, 21).TheE4ORF in HPV-51 iscompletely contained within E2, asit is in other HPVs. Justupstream of the E4 ORF, atposition 3270, is apotential splice acceptorwhich could be usedto generate this species from any ofseveral potential donorsiteswithintheEl ORF. Demonstration of this spliced species will require the analysis of mRNAs expressedin lesionscontainingHPV-51orcell lines

express-ingthe virusgenome.

The E5 ORFfrom BPV has been studied in greatdetail. This 44-amino-acidproteinispresentintransformedcellsas a membrane-associated homodimer (55). The protein

con-tains ahydrophobiccore anda hydrophilic

carboxyl-termi-nal tail which defines a single functional domain that is

responsible for its ability to induce focus formation and stimulate cellular DNA synthesis (33, 59). Recently, this proteinwasshowntoactivate theendogenous P-type

recep-torfor theplatelet-derived growth factorintransformed cells (48).Althoughnoclearfunction has beenascribedtotheE5 ORFs in HPVs, the ESa ORF of HPV-6c was shown to transform 3T3 cells (10). The ES ORF in HPV-51 lacks an

initiator methionine and can encode a protein of 84 amino

acids witha predicted molecular weight of 9,764. As inthe otherHPVES ORFs there is aconservedCys-X-Cysin the

amino-terminal third of the protein, and there are three hydrophobicdomainswhich havebeen suggestedto consti-tuteamembrane spanning region (8).

The L2 and Li ORFswhich encode the minor and major capsid proteins, respectively, are the products of lategene

expression andare highly conserved betweenHPVs(Table 1). They are typically only expressed in well-differentiated

epithelium in the apical portion ofa lesion (17, 44). This

sequence conservation has formed the basis for the

devel-opment ofa sensitive PCR assay which uses primers that

specifically amplifythe Li ORFfor thedetection andtyping of HPV genomes(37). Amplification of viral DNAorDNA

from lesions containing HPV-51 with the MY09and MY11

HPV

HPV-51 HPV-33 HPV-31 HPV-18 HPV-16

HPV-51 HPV-33 HPV-31 HPV-18 HPV-16

HPV-51 HPV-33 HPV-31 HPV-18 HPV-16

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* N ,~\5 ~ ~ ~ G)\

a

~ ~ ~ ~ ~ 77

FIG. 3. Amplification of HPV DNAs. DNA was isolated from

biopsies containingHPVsequences, and the virus typewas deter-minedbytherestriction endonucleasedigestionpattern of the DNA after Southern blothybridization. (A) Theproducts of DNA from these samples were amplified with the MY09 and MY11 primers

described in Manosetal. (37). (B)Thetwopotential hybridization

sites within the Li region of HPV-51 DNAfor the MY09 primer; annealing at these sites results in generation of the two PCR

productsseeninpanelA.

primersdescribed by Manos et al. (37)generates two

prod-ucts (Fig. 3A): one of 452 bp is similar in size to what is

amplified in othergenital HPVs, andanother that is 272bp

which results from mispriming of the MY09 primer at

position 6718 to 6737 (Fig. 3B). The shorter amplification product can be eliminated if the annealing temperature is raised to 550C. The Li ORF in HPV-51 can encode a

504-amino-acid proteinwhich is approximately 80%

homol-ogous atthe amino acid level toLlsfrom other HPVs. The

L2 ORF consists of468 amino acids and contains the motif

T-T-P-A-V-L at position 148 along with clusters of basic residuesatthe amino andcarboxy termini(56);thecarboxy

terminus overlaps the amino terminus of Li by 6 amino acids. TheT-T-P-A-V-Lmotif ishighlyconservedamongthe mucosal HPVs,and its presencefurthercements the classi-fication of this virus among these members of the HPV

family (32).

Regulatory motifs in the HPV-51 genome. The URR of HPVsis operationallydefined as the sequencebetween the

C

FIG. 4. Transformation of CREF cells. Freshly plated CREF cells were transfected with HPV-51 (a), salmon sperm (b), or

HPV-18 DNA(c)as described in Materials and Methods. The cells

weresplit 1:3 after 24h, andthe mediumwaschangedevery third

day. Foci were photographed after 21 days by using a Nikon invertedmicroscopeand T-MAX 400film (Kodak).

laststopcodon of Li and theinitiator ATG in E6.

Computer-based searches ofHPV URRs and DNA-binding studies of

the URRfromHPV-16 have revealed that thegenitalHPVs

contain a glucocorticoid response element (GRE) and a keratinocyte-dependentenhancer(15). Inaddition,thereare

otheroverlapping cis-actingsequenceswhichrespondtoand

interact with general, tissue-specific, and virus-specified trans-actingproteins.Theseinclude sites forinteraction with

APi, NFI, TFIID, Spi, and the E2 activator-repressor

protein. The URR of HPV-51 is 869 bp and contains three

copies of the sequence ACCN6GGT, to which E2 is known

A

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TABLE 4. Location ofsequencemotifsin the HPV-51genome

Sequencemotif' Consensussequence Sequencein HPV-51 Locationb

Keratinocyte-dependent enhancer TTTGGCTT TTTcGgTT 7396-7403(+)

TTTGGCaT 7455-7462(+)

TTTatCTT 7402-7409(+)

TTTGgcTT 7765-7772(+)

TTTcGgTT 29-37(-)

E2binding site ACCN6GGT ACCGATTTCGGT 7391-7402(+)

ACCGAAAAGGGT 30-41(+)

46-57(+)

ACCTTTTACTGG 4497-4508(+)

NFI TTGGCA TTGGCA 7423-7428(-)

7456-7461(+) 7578-7583(+) 7633-7638 (+) 7563-7568(+) 7690-7695 (-)

Spl GGGCGG GGGCGG 7497-7502 (-)

7501-7506(-) 7593-7598(-)

TFIID ATATA ATATA 60 64(+)c

AP1 TGAcTCA TtAGTCA 53-59(+)

Oct-1 ATTTGCAT AaTGCAT 7653-7660(+)

GRE GGTACANNNTGTTCTCd GcTtcATTTtTTgT 7356-7372(+)

TGTACATTGTGTCATe TGTACATTaTtATCAT 5090-5105(+)

TCTACATTTTATACTf aCTACATTTTtTg_T 4030-4044(+)

AGAACATACTGTCCCf AcAttATACTGTCCC 3818-3833(+)

Poly(A)addition AATAAA AATAAA 4103-4109(+)

7169-7173 (+)

GT repeat GTGTGTGTGT GTGTGTGTGT 181-190(+)

3913-3922(+) 7116-7125 (+) 7186-7195(+)

Sequencemotifsfor interaction withknowntranscription factorswere asdescribedinMitchellandTjian (39). b+, motifs that areinthe senseorientation; -, motifs in theantisense orientation.

There areadditional copies of thissequencethroughoutthegenome; only thesitein URRisshown here.

dThe GRE consensususedwasfrom Chanet al. (9).

eHPV-16 GRE.

fHPV-39 GRE (65).

to bind (1, 41). These sequences arelocated at nucleotides 7391, 30, and 46 within URR, and a fourth copy of this sequence motif is found at position 4497 within L2. A mobility shift gel electrophoresis assay with end-labeled

fragmentsfrom the HPV-51 URR (that contained the canon-ical E2binding site sequence) was used to demonstrate that recombinantE2from BPV would bind and alter the

electro-phoretic mobility of thosefragments which contained these sites (data not shown).

Nucleotide sequenceanalysis of the HPV-51 URR reveals the presence of the many tightly clustered and overlapping

regulatory elements; there is a homolog of the GRE, and therearesevencopies ofthe NFIbindingmotif. Inaddition,

there are single sites for AP1, Oct-1, Spl, and TFIID. The orientation and location of these sites is summarized in Table 4. Inaddition to the single GRE in URR there are four other

homologsof this sequence, three of which are clustered near the E5ORF and one which is in L2.

Poly(A) signals for mRNA termination are bipartite, con-sisting of a signal AATAAA followed at a distance of 20 to 30 nucleotides by a GT-rich sequence (50). Sequences homol-ogous to this termination signal are found in all HPVs. In HPV-51 there aresix copies of the AATAAA motif, and two ofthese, located at 4104 and 7169, are downstream of the

earlyandlate genes, respectively, and contain both compo-nents of the polyadenylation signal.

Finally, we notethe occurrence ofadecameric sequence

that is composed of alternating G T residues which is repeated fourtimes inthe virus genome.

Transforming activity of HPV-51DNA.Cloned DNAsfrom those HPVs which are associated with lesions that have a high risk for malignant progression have the capacity to transform rodent cells in culture (69) and to immortalize humankeratinocytes (23, 35, 49). Immortalization of kerati-nocytes results from cooperation between the E6 and E7 geneproducts (31). Sequence analysisrevealed that HPV-51 was more closely related to the oncogenic HPVs than to thoseassociated withbenignlesions. Toextend this associ-ation further, we next asked if DNA from HPV-51 would transform a continuous line of rat embryo fibroblasts. Be-cause the original clone of virus DNA was isolated as a HindIIIfragmentandlackedtheE6ORF,the intactgenome was reconstructed after cleavage with Hindlll and self-ligationtogenerateconcatamers. ThisDNA wasdigestedat theunique HincIIsite in theoriginalclone(47),downstream ofE7, and cloned intoaHindlIl- vector at auniqueHincIl site. The missing 232-bp Hindlll fragment (which encodes most ofE6)was then insertedatthe uniqueHindIII site in the virus genome.This DNAwasreleasedfrom the resulting

plasmid,and10,ug(each) of HPV-51 and HPV-18 DNAwas compared for their abilities to transform CREF cells. HPV-51 DNA induced transformed cells with a frequency

similar to that of HPV-18 DNA (30 CFU/,ug versus 20 CFU/,ug). Moreover, control transfections that included

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97 549

1 E6

599 862

P97 E7 1

r01

0 622

527

404

309

F2

F3 242

217!

201 r

is

,

160 147

F4

I

4F5

F6

..

122 110

90

76

67

PCR Productsof DNA 5'E6,E7

5'E6 3'E6

I

1000bp

Size RT

- 796bp

F2

F4 Predicted PCR Products of cDNA

796bp

554bp 540bp 191bp

... 90bp 80bp Hybndization Probes

_

Probe

1

--- --- Probe 2

F7 F8

F5

F6

FS

Probe1

[image:8.612.52.550.73.378.2]

Probe2

FIG. 5. Analysis of RNA and DNA inacellline transformed with HPV-51DNA. (A)The productsofPCRamplificationof DNA derived

from purified plasmid (lanes 2 and 4)ortransformedcells(lanes3and 5) with primers that would amplify E6 and E7 (lanes 4 and 5)orE6

(lanes 2 and 3) along with the PCRamplification products of thecDNAs synthesizedwith theRTprimeronRNAisolatedfrom the same

transformed cell line(lane 6)wereanalyzed by electrophoresis in 3% GTGagarosegels. Lane 1 containsanMspIdigestof pBR322 DNAas

asizemarker. (B) Aschematic of the E6 and E7 regions of the HPV-51genomeis shown along with the positions of the primers usedfor amplification of E6 (5' E6 [nt 79to99] and 3' E6 [nt 399to419])and E6 and E7 DNA (5' E6 and E7 [nt 91to111] and RT[nt 866to887]) andforsynthesis of cDNA (RT [nt 866to887]) and its amplification. Thetheoretical spliced products with their potential donor andacceptor

sitesareindicatedalong with the predicted size of the amplification products. The probes usedtoanalyze the products of amplification of the cDNAs(panel A, lane 6), i.e., Fl, 780 bp; F2, 580 bp; F3, 220 bp; F4, 162 bp; F5, 135 bp; F6, 120 bp; F7, 88 bp; and F8, 80 bp, for thepresence

of E6(Probe1)orE7(Probe 2)sequences arealso shown. (C) Results ofhybridization of each of the amplified products of the cDNA reaction

to32P-labeledprobes specific for E6 (Probe 1) and E7 (Probe 2).

HPV-6, HPV-11, orplacental DNA gave rise to no

trans-formed colonies, whereas transfection with similaramounts

ofsrc DNAresulted in >500 CFU/ptg. Comparison of the morphologictransformation frequency of thesesameDNAs on BALB 3T3 cells resulted in too few transformants with either HPV-18 or HPV-51 DNAs to quantitate. A typical morphologically transformed CREF colonyisshown in Fig. 4. Several transformants were isolated and recloned twice.

The cloned colonies werethen expandedand examinedfor thepresenceofsequences homologoustoHPV-51 DNA by PCR amplification of the E6, E7, and Li regions of this virus. The transformed cells contained amplification prod-ucts ofthe appropriate size (Fig. 5A and B). Analysis of these products by digestion with restriction endonucleases demonstrated that they were derived from HPV-51 DNA

(data notshown).

Thestateof virus DNAinasingle PCR-positive clonewas

examined by Southern blot hybridization. Hybridization of transformed-cell DNA cleaved with Hincll (which should not cleave integrated virus DNA because the site is de-stroyedafterrelease of the insert from thevector) revealed

the presence ofa single 14-kb species; after digestion with

EcoRV(asingle cutter), twobandswere detected(datanot shown). This resultsuggeststhatonlyasinglecopyofvirus

DNAwasintegratedinto thegenomeof thetransformed cell. Because products of both E6 and E7 are required for

completemorphologictransformation(3, 30,31, 34, 42),we

next examined the RNAs present in a line oftransformed

cells that were homologous to virus sequences from these regionsof the HPV-51 chromosome. RNA isolated from the single-copy transformant was reverse transcribed with a

primer (RTprimer)that isdownstream of the E7 termination codon (Fig. SB). The resultingcDNAs werethen amplified with primers that flank these ORFs and examined by gel electrophoresis. Theelectrophoretic mobilities of the prod-ucts are shown in Fig. 5A. The slowest migrating band resulted from contamination of the RNA with virusDNA,as

judged by its appearance in the absence of any reverse

transcriptionand the fact thatitwasnotpresentif RNAwas

preselected for poly(A)-containing molecules (data not shown). Bands corresponding to products that might be derivedas aresult of utilization ofthevarioussplicedonors

A

1

2

3 4 5 6

B

R1 R2

C

N %

Ft

4E_

t,

P1

F2

_gopm

P3 340bp

F4

-_ F5

F6

F7

P8

ProbeI

Probe 2

"X

I.lb"I

oI"

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and acceptors in this region ofthe genome (Fig. 5B) were

excised from the gel and examined for the presence of

sequences homologous to either E6 or E7 by molecular hybridization by using probes that were specific for each ORF. Theresults ofthisanalysisare shown inFig. SC. The three slowest migrating bands contained amplimers that hybridized with both the E6 and E7probes, while the other products appeared to contain sequences thatwere

predom-inantly derived from E7. Because the E6 probe only

con-tained 30 bp of homology with the region upstream of the splicedonor atposition 177, itsability tohybridizewith E6

sequences is somewhat limited. We conclude from these

data that thereare splicedRNAs present in this transformed cell thatcan encode both the E6 and E7 proteins. Further characterization ofthe products of these reactions will be requiredtodetermine theutilization of donors andacceptors and theexactcoding potentialof each of the RNAs identified in thispreliminary analysis.

Insummary, wehave determined the nucleotidesequence

ofHPV-51. Analysisof thegenome sequence,inconjunction withassayof its transformation potential, placesthis isolate

amongthehigh-risk groupof HPVs.

ACKNOWLEDGMENTS

Studies in the authors' laboratories were supported by grants

CA23767(S.S.) and CA47676 (C.P.C.)from the National Institutes

of Health andSIG13 (S.S.) fromthe American CancerSociety. We

gratefully acknowledge the Perkin-Elmer Foundation forthe

dona-tion ofathermalcycler.

Wethank Rey Sia forperforming the E2binding assays, Elliott

Androphy foracloneexpressing BPVE2, and Li Hui forsomeof

theclones used indetermining thesequenceof HPV-51.

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