Nucleic Acids Research, Vol. 18, No. 13 3919
k.) 1990 Oxford University Press
Epidermodysplasia verruciformis associated human papillomaviruses present a subgenus-specific organization of the regulatory genome region Armin Ensser and Herbert Pfister Institut fur Klinische und Molekulare Virologie, Universitat Erlangen-Nurnberg, LoschgestraBe 7, 8520 Erlangen, FRG Received March 6, 1990; Revised and Accepted May 30, 1990
EMBL accession nos X52059-X52062 (incl.)
ABSTRACT The regulatory regions of the human papillomaviruses (HPVs) 9, 17, 20, and 36 were mapped, sequenced, and aligned. They revealed an arrangement of putative protein binding sites specific for the epidermodysplasia verruciformis associated HPVs. Three subgroups could be differentiated.
important cis-acting regulatory elements of papillomaviruses (6) and is therefore called long control region (LCR). All ev-LCRs showed good homology in the regions of the putative binding sites of the viral, transcription-activating E2 protein, and particularly in two motifs with 33 and 29 bp, respectively, called M33 and M29 (5).
INTRODUCTION
MATERIALS AND METHODS
At least 18 human papillomavirus (HPV) types seem to be specifically associated with epidermodysplasia verruciformis (ev), a lifelong skin disease characterized by the development of multiple flat warts and macular skin lesions (1,2). In skin cancers, which occur in 30% of the patients, HPVs 5 and 8 are most prevalent; HPV 14, 17 and 20 have been found once each. The nucleotide sequences of HPVs 5, 8, 19, and 25 (3-5) revealed the typical genome organisation of papillomaviruses except for a surprisingly short noncoding region between open reading frames (ORF) Ll and E6. This genome segment contains
The cloning of DNAs of HPV 9, 17a, 20, and 36 was described previously (7-10). Standard protocols were used for all restriction enzyme digestions, preparations of plasmid and phage DNAs, molecular cloning, radioactive labelling and blot hybridisation (11). 35S-Dideoxy-plasmid-sequencing of both strands with commercially available kits (pUC-Sequencing-kit, Boehringer Mannheim and T7-Sequencing-kit, Pharmacia) followed the instructions of the manufacturers. For sequence analysis, the UWGCG-program package (version 5.3) was used (12).
HPV 9 HPV 17 HPV 20 HPV 36
B
E H
H
H
B
I
B
E
E
_I
BH H H E
E L
S
E
eiI
E
B
S
B
E B
B HP
B BE
11
/ sequenced region _ subcloned region LCR (E6- LI)
4--
Fig.l. Physical maps of restriction enzymes cleavage sites in the genomes of HPVs 9, 17, 20, and 36, and localisation of the sequenced regions and the LCRs. B = Bam HI; E = Eco RI; H = Hind III; P = Pvu II; S = Sac I.
3920 Nucleic Acids Research, Vol. 18, No. 13
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Hpv2O gaattcaaat atattagagg agtggcagtt aggatttgtt cctgcaccag ataatcctat ccacGAtAca TAcaGATATA TTaaTTCtgc Hpv36 gaattcttct ttattggaag attggcagtt aggttttgta cctactccag ataaccctat tcaaGAcAcc TAtcGATATA TTgaTTCatt HPV9 GAtAtc TAcaGATATA TTgcTTCaaa Cons GA-A-- TA--GATATA TT--TTC--101 Hpv2O TGtCCtGAca Hpv36 TGtCCtGAta Hpv9 TGcCCaGAtg Hpvl 7 TG-CC-GA-Cons
201 Hpv2O AATATtCTcT Hpv36 AATActCTtT Hpv9 AATATcCTtT Hpvl 7 AgTATcCccT AATAT-CT-T Cons
aaaatcctCC aAaaGAAAgA GAaGATCCtT acaaggatct aaacTTtTGG aAtGTtGAcC TatCaGAAAG aaacccccCC tAagGAAAaA GAgGATCCcT acaaggggtt aaagTTcTGG gAtGTgGAtC TtACtGAAcG ctgttgagCC tAcaGAAAaA GAaGATCCcT ttgccaaata ctcaTTtTGG &AaGTgGAtC TaACtGAAAG TaaAcC TtACatttAG --------CC -A--GAAA-A GA-GATCC-T ---------- ----TT-TGG -A-GT-GA-C T-AC-GAAAG
AGGactcAAA AGGtaGAAAg AGGtaGAAAA AGGacGAAAA
AGG--GAAAA
Ifn-RE TTctTaTTTC TTTcTaTTTC TTTcTtTTTC TTTaTtTTTC TTT-T-TTTC
401 APM Hpv2O Hpv36 Hpv9 Hpv17 Cons
ATTGTC--TA GATTT-GATC
P 0 300 AAGCaGGTTT aCAacaggcg ACcgtAAaCg GTACaAaaac tgtaTCTtca AggttaTCta ctAggGGcgt AAGCcGGcTT aCAgcagacg ACcgtAAgCg GTACaAaatc agtgTCTtat cgAgggTtca ccAgaGGaac AAGCtGGTTT gCAa ...... ACacgAAaac GlcCtAttaa aacaTCTgtt AaAacaTCt. .Aaaaatgc AgtCaGGTTT gCAg ...... gCaaggccCa GaACtAttcg gaccTCTgta AaAgtgcCc. . .AagGGtat AAGC-GGTTT -CA------- AC---AA-C- GTAC----- ---- C--- A-A---TC-- - -A- -GG- - p 1 GtTTTCGGT. GtTTTtGGT. GaTaTCGGTt GcTTTCGGTc G-TTTCGGT-
CK-OCT ..aCAATAAA g.TcAAcTTT ..aCAgattt a.TaAAcTTT tc.CAATAAA atTtAAgTTa tctCAATAAA .. cAAaTaa ---CAATAAA --T-AA-TT-
N 33 400 TACACggTat TcaagGAtg tTTaTTTAct C. TG TACACagTat TcaagGAAtg tTTgTTTAct C......TG TcCAattTgg TatgtGAAgc aTTtTTTAac CatcttcgTG acCAaaaTgg TatgtGAAgt aTTtTTTtac CatgttcgTG ...
T-CA--- T-- T ---- GAA-- -TT-TTTA-- C------- TG
PEA-2
500
CRE P 2 NFI NFl ACTAA .. cta agatAcCaAC CGCACCCGac acAT.aaAgg tgAgTtgTGt gccaaatgag gtgagttgtg agccagaaga gatcacaWcc ACTAAgtata actctaCcAa gggaAcCgAC_CGCACCCGGT acAgtcaAcg ATAcT..gct gccaatatag a...................
ACTAAaccgt acaagtCaAc acagAgCgAC CGCACCCGGT ttATctgAtt ATAaT.gTGc ACTAAa.cgt cttcgtCaAc gc.cAgaaAC CGCACCCGGT taATcagAtt ATAaT..TGc ACTAA ----- ------ C-A- ----A-C-AC CGCACCCGGT - -AT --- A- - ATA-A- -TG-
501 NF1 NF1 NF1 Hpv2O aagtcaggct tgagccaaat cagataca.c tgcgtgccag agttggctcA Hpv36 ..ctctgttt agtgccagaa catat.catc ttggaagcag atc ...... g Hpv9 . ..........A..........A Hpvl 7 .A..........A ---------- ---------- ---------- ---------- ---------A Cons .........
..........
....................
.........
...................
601
NFl NFI gg CTGCCAA... CGcttTTGG Ctc ...- ttCt ..gAtTtaat CTa.CAA... TCGctgTTGG CAa... tCgc ..AAtTtca. CTGCCAAggt TCG... TTGG .... CAg aCCg Hpvl 7 gaAAcTgg.. aGCCAAggt T . TTGG CAgaacaCCa - -AA-T- - - - CTGCCAA- - - TCG --- -TTGG CA ----- CCCons
Hpv2O Hpv36 Hpv9
AGCcACaaaa AGC -AC--- --
200 ATTGTCctTA GAaTTgGATC gTTGTCacTA GATTTgGATC ATTaTCgtTg GATcTtGATC AaTGaCtccA GATTTaGATC
I fn-RE
301 CK-OKT STOP cAAaCGaAAa CGcaaaatt aaactcGACC Hpv36 cAAgCGcAAg CGaaaacagt aatat.GACC Hpv9 tAAgaGaAgg CGa... acc . taACC Hpvl 7 tAAaCGcAAa CGt . tca . tGACC Cons -AA-CG-AA- CG-------- ------GACC Hpv2O
AGCtACtaga AGCcACtcgc
..A
NFI TtTTGGCacA TtTTGGCtgA TccTGGC. -t TcTTGGaacA T-TTGGC- -A
NF1 701 Hpv2O .cgtacaTTa Hpv36 actggaaTTt Hpv9 .... tatTT. Hpvl 7 .... actTTc -------TTCons
P36
agcttcatcg aCcGtgttcg cCtGgtgcaa cCtGgtgcga
.. * --
* -
.
...-
--
-
-
*- .
.-
.. --.-..... ............
...... ......
.......... ..........
...... ......
..........
NFI 600 TcccAAC ... ACgtTCGgaa cAggA ..... GgaAatGta .
TTgtAAC ... ACgcTCGgat tA.gAgacat TTtgAACaat ACtaTCGtgg aAtcAccgca TTttAtCact gCttTtGtgg aAgcAccgca -C-G------ TT--AAC--- AC--TCG--- -A--A----P 3
tGCcAagGaa gGCaAccGcc gGCgcccGcc -GC-A--G-P 4 700
NF1
GCagaa.GAC CGtTAACGGT AAGttt .... tTaTTtGtAt CGggcGCGGT GatagctGAC CGgTAACGGT AAGttattta aTtTatGtAc CaggtGCGGT tCaaaacGAC CGaTAACGGT AAG ....... TcTTGGCA. CGtagGtGGT GC . AC CGaTAACGGT AG. .attat aTcTTgGaAc CGtagGCGGT GC ----- GAC CG-TAACGGT AMG ------- - T-TT-G-A- CG - -- GCGGT .
NF1 CAAT N 29 P 5 800 ctcATttggt aGtTG.TTGT tGccAaCtAC cATCaa...g CATA.gCATg tttttgcctg taacgtTaTC g..gcACaGt gaTteaTAta cacAataaTa a.tTG.TTGT tGccAACtAC cATtgc.... tATAttCAag tttttgcctg tatcgtTtTC gTaTcAtgtg a.ataaTAta ..gATcgtTg gGaTGaTTGT gGttAACaAC aATCta .... CATAcaCAT . TtTC aTaTgACcGc ctTcgtTA.. tgatTggtTt gGcTGaTaGT aGttACaAC aATCttctct CATAaatAT .aTgTgACcGc ctTcgtT... ---AT ---T- -G-TG-TTGT -G--AAC-AC -ATC------ CATA--CAT- ---------- ------T-TC -T-T-AC-G- --T---TA-......
801 TATA CK-OKT/rev Hpv2O tatatataTA TATAtAtATA tATAtATATA Tatatatata tataaataga tacataTag. Hpv36 ctgtAtagTA TAaAtAaATA aATAaATATA Tatatatat. gcttcaaagg ttggGtTttt Hpv9 .ataAgctTA TATAgAcATA aATAtATAaA .................... GgTgcc Hpvl 7 AcctTA MAT.gAtcTA cATAcA.ATA T .Gagagc - -- -A- - - TA TATA-A-ATA -ATA-ATA ------------ ---------- --- G-T --Cons ....
....
START 900 acagAtaTca tAGAGCtaat gcagagagtg caggctcatg
taaTAaTT........... .aag atgTAtTTaa cAGAGCagat t................... ..........
tctTAcTTa. --- TA-TT- -
901
AGAGCat.. *
-AGAGC- - - START
.
...*.-.--.-.-
1000
Hpv2O gctacacctc cttcttcaga agacagcgct gatgaaggac catccaatat tggagagaca aaacctttta ttcTaGAgcc aCCAttgCCT gcAACAaTct Hpv36 gcagagcaag cctcc... ga acagcag............ tacagaaaaa gaa ....... .aatat aAaga aCagctgCCT ttAACtaTtA .....
Hpv9 Hpvl7
..........
..........
............. .......... ....... ..... . . . . . . . . . .
..........
..........
............. .......... ....... ..... .. ..
Cons
----------
Hpv2O Hpv36 H pv9 Hpvl 7
1001 gtGgcCTaGC aGGgcCTgtC aGGaaCTaGC gGGagCT tGC -GG- -CT-GC
Cons
.....
gaAccttTTa GaaATaCCgc aGAatCaTTa GgcATTCCgT aGAcaCtcTt GtgATTCCtT tGAtaCcTTg tgtATTCCaT -GA--C-TT-
Fig.2. Alignment of the newly determined
.
. . . . .
a gAA CA g T aA T..g .a.. T.g...G g gCCAaaaCC T c aAA CA gTgA T-GA-CCA- - -CCT - -AACA-T-A
. . . . . . . . . . GA ca g g CCA aa a C C T ... . . .
. . .. ....... .. . . . .. Ata ---------- ---------- ---------- ----
G--ATTCC-T
..
..
TAGatGATTg TTTgATACCT TGTAAcTTcT TtGTaGAcTg TcTaATACCT TGTAAcTTTT TAaTaGATTt gTTgATACCT TGTAAaTTTT TAGTgGATat TTTatTACCT TGcAgaTTTT TAGT-GATT- TTT-ATACCT TGTAA-TTTT
.
GcggTAatTT GtggcAaaTT GcaaTAgaTT GtaaTAggTT
.
1100 TcTtaCacAT TTAGAAgTtt gtgagTTTGA
TTTAgaTTAT TTAGAAgctt
..........
TTTAtCTTAT TTtGAgcTac ttaatTTTGA TTTAgCTTAc aTAGAAtTgg tggcgTTTGA G --- TA--TT TTTA-CTTAT TTAGAA-T-- -----TTTGA
sequences of HPVs 20 and 36 and HPVs 9 and 17. Putative functionally important elements and protein binding sites indicated in bold letters or underlined. Ifn-RE=Interferon responsive element. CK-OKT=Cytokeratin-octamer (19). PO-5,P36=E2-protein binding sites. STOP=Translation stop codon of ORF LI. CRE=cAMP responsive element. PEA-2=Polyomavirus enhancer element. v-myb= Binding site for v-myb protein. NFl =Nuclear Factor 1 binding site. M33,M29=conserved motifs of 33 and 29bp respectively. CAAT= putative CAAT-box. TATA=stretch of A and T nucleotides. START=Translation start codon of ORF E6.
are
Nucleic Acids Research, Vol. 18, No. 13 3921
PO Pt M33 AP1 P2 HPV 5
*O
HPV 8
O 0
Nfl J0 0 0 ]0 00
HPV 36
* 0
]0 0
HPV 19 HPV 20 HPV 25
*u
P3 0
P3 P4 NFl CAAT M29 P5 TATA 00 000 C]m 0000 00 0-] 0 00 000 a C]
NFl
IIoo-oo_000 0.0*0o00000 0o a00 o ?
+
o
o -o *C]*.00
C
-00 0.-
C
00 000
=
HPV 9 HPV 17
Fig.3. Schematic distribution pattern of conserved, putative regulatory elements in the LCR sequences of ev-associated HPV 5 (3), HPV 8 (4), HPVs 19 and 25 (5), and HPVs 9, 17, 20, 36 (this paper). PO-5, P36 = E2 protein binding sites: 0 = ACCGN4CGGT; (3 =ACCGN4CGTT; 0 = ACCN6GGT; a= degenerated palindrome. * =AP1 binding site. a = NFl binding site. af = M33, M29 conserved motifs of 33 and 29bp, respectively. *= CAAT box. TATA = TATAstretch: +++ = -SObp; + + = -20bp; + = TATA-like sequence in HPV 17. ? in HPV 25: DNA-sequence not available.
RESULTS AND DISCUSSION The LCRs in the genomes of HPVs 9, 17, 20 and 36 were localized by Southern blot analysis using the homologous HPV 8 DNA as probe (Fig. 1). The cross-hybridising fragments were subcloned into the Bluescribe vector (Vector Cloning Systems) and sequenced. The lengths of the LCRs from the translation stop codon of LI to the start codon of E6 were 513 bp in HPV 20, 480 bp in HPV 36, 387 bp in HPV 9, and 379 bp in HPV 17. The aligned nucleotide sequences and the locations of conserved elements and putative recognition sites for DNA binding proteins (13) are shown in Fig.2. A comparison with published LCR sequences of ev-associated HPVs confirmed the following, highly conserved landmarks (Fig.3): 1. Four palindromic E2 binding sites ACCGN4CGGT or slightly degenerated versions thereof (P1-4). Palindrome P3 is the most conserved among them and includes a putative binding site for the v-myb protein (14). 2. One binding site for the transcription factor jun/API (13). 3. A cluster of direct or inverted repeats TGCCAA and related sequences, which were shown to bind the transcription factor NF1 (15). 4. A 'CAATmotif', which is generally conserved among papillomaviruses (5). HPVs 5, 8, 19, 20, 25, and 36 (group A) share the previously described conserved blocks with 33 and 29bp, respectively, an additional palindrome PO within the 3'-part of ORF LI, and a striking stretch of about 50 A and T nucleotides. HPVs 5, 8, and 36, on the one hand, and HPVs 19, 20, and 25, on the other hand, form two slightly different subgroups Al and A2. In A2-viruses, palindromes P2 and P4 are incomplete, and 12bp are deleted between API and P2; the AT rich stretch consists of monotonously alternating A and T, in contrast to subgroup Al viruses, which show two to four TAAA blocks followed by a shorter AT run. This AT stretch is without precedent among papillomaviruses, but similar motifs were identified within the autonomously replicating sequences (ARS) and promoter regions of yeast. Promoter activity was there shown to improve with increasing length of this element (16,17). Against the background
of a rather uniform organization of group A virus LCRs, an apparently recent duplication can be noted in the case of HPV 20, which has a 21bp direct repeat with 3 mismatches between pos. 451 and 492 (Fig.2). The same duplication was observed in a second HPV 20 clone from another patient, which renders it unlikely to represent a cloning artefact. The sequence ACCGN4CGTT, which was recently shown to bind E2 of bovine papillomavirus 1 (18), occurs once in HPV 36 (P36). In HPV 9 and 17 (group,B) no M29 and only a slimed down M33 could be detected. The AT stretch is shortened in HPV 9 to about 20bp and interrupted by a few other nucleotides. No classical TATA-box was found in HPV 17 in this region, but TATA-like sequences TTAAATGA and TACATACAATAT are located at pos. -36 and -22 relative to the putative E6-protein translation start codon. The group B virus sequences thus indicate that the characteristic TATA stretch and the M29 motif discussed before are not consistent features of ev-associated HPVs, and can therefore not account for their host-specificity. Despite good general homologies with group A viruses in the 3'-part of ORF LI, group B viruses display no P0 in this region, but they contain an ACCGN4CGTT following the CAAT-box (P5). No obvious correlation exists between the LCR classification into subgroups Al, A2, and B and the assumed oncogenic potential of ev-HPVs. This analysis provides another example, however, that LCRs of papillomaviruses, which belong to the generally less conserved genome segments, show a subgenousspecific organization pattern in viruses with similar tropism and pathogenic properties as observed for viruses associated with anogenital lesions.
ACKNOWLEDGEMENTS We are indebted to Dr. G. Orth who provided cloned DNAs of HPVs 9, 17a, and 36. We gratefully acknowledge helpful discussions with Dr. P.G. Fuchs. This work was supported by the Deutsche Forschungsgemeinschaft (PF 123/3 -2).
3922 Nucleic Acids Research, Vol. 18, No. 13
REFERENCES 1. Orth, G. (1987) in Salzman, N.P., and Howley, P.M. (ed.), The Papovaviridae, Vol.2, Plenum Publishing Corp., New York 199-243. 2. De Villiers, E.-M. (1989) J.Virol. 63, 4898-4903. 3. Zachow, K.R., Ostrow, R.S., and Faras, A.J. (1987) Virology, 158, 251-254. 4. Fuchs, P.G., Iftner, T., Weninger, J., and Pfister, H. (1986) J.Virol., 58, 626-634. 5. Krubke, J., Kraus, J., Delius, H., Chow, L., Broker, T., Iftner, T., and Pfister, H. (1987) J.Gen.Virol., 68, 3091-3103. 6. Iftner, T. (1990) in Pfister, H. (ed.), Papillomaviruses and human cancer, CRC Press, Boca Raton (Fl.,USA), 181-202. 7. Kremsdorf, D., Favre, M., Jablonska, S., Obalek, S., Rueda, L.A., Lutzner, M.A., Blanchet-Bardon, C., van Vorst Vader, P.C., and Orth, G. (1984) J.Virol., 43, 1013-1018. 8. Gassenmaier, A., Lammel, M., and Pfister, H. (1984) J.Virol., 52, 1019-1023. 9. Kawashima, M., Favre, M., Jablonska, S., Obalek, S., and Orth, G. (1986) Virology, 154, 389-394. 10. Kremsdorf, D., Jablonska, S., Favre, M., and Orth, G. (1982) J.Virol., 43, 436-447. 11. Maniatis, T., Frisch, E.F., and Sambrook, J. (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 12. Devereux, J., Haeberli, P., and Smithies, 0. (1983) Nucleic Acids Res., 12, 387-395. 13. Jones, N.C., Rigby, P.W.J., and Ziff, E.B. (1988) Genes & Development, 2, 267-281. 14. Biedenkapp, H., Borgmeyer, U., Sippel, A.E., and Klempnauer, K.-H. (1988) Nature, 335, 835-837. 15. Gloss, B., Yeo-Gloss, M., Meisterernst, M., Rogge, L., Winnacker, E.L. and Bernard, H.-U. (1989) Nucleic Acids Res., 17, 3519-3533. 16. Stinchcomb, D.T., Mann, C.,Selker, E., and Davis, R.W. (1981) ICN-UCLA Symp. Mol. Cell. Biol. 22, 473-488. 17. Struhl, K. (1985) Proc.Natl.Acad.Sci.USA, 82, 8419-8423. 18. Li, R., Knight, J., Bream, G., Stenlund,A., and Botchan, M. (1989) Genes & Development, 3, 510-526. 19. Blessing, M., Zentgraf, H., and Jocamo, J.L. (1987) EMBO J. 6, 567-575.
k.) 1990 Oxford University Press
Epidermodysplasia verruciformis associated human papillomaviruses present a subgenus-specific organization of the regulatory genome region Armin Ensser and Herbert Pfister Institut fur Klinische und Molekulare Virologie, Universitat Erlangen-Nurnberg, LoschgestraBe 7, 8520 Erlangen, FRG Received March 6, 1990; Revised and Accepted May 30, 1990
EMBL accession nos X52059-X52062 (incl.)
ABSTRACT The regulatory regions of the human papillomaviruses (HPVs) 9, 17, 20, and 36 were mapped, sequenced, and aligned. They revealed an arrangement of putative protein binding sites specific for the epidermodysplasia verruciformis associated HPVs. Three subgroups could be differentiated.
important cis-acting regulatory elements of papillomaviruses (6) and is therefore called long control region (LCR). All ev-LCRs showed good homology in the regions of the putative binding sites of the viral, transcription-activating E2 protein, and particularly in two motifs with 33 and 29 bp, respectively, called M33 and M29 (5).
INTRODUCTION
MATERIALS AND METHODS
At least 18 human papillomavirus (HPV) types seem to be specifically associated with epidermodysplasia verruciformis (ev), a lifelong skin disease characterized by the development of multiple flat warts and macular skin lesions (1,2). In skin cancers, which occur in 30% of the patients, HPVs 5 and 8 are most prevalent; HPV 14, 17 and 20 have been found once each. The nucleotide sequences of HPVs 5, 8, 19, and 25 (3-5) revealed the typical genome organisation of papillomaviruses except for a surprisingly short noncoding region between open reading frames (ORF) Ll and E6. This genome segment contains
The cloning of DNAs of HPV 9, 17a, 20, and 36 was described previously (7-10). Standard protocols were used for all restriction enzyme digestions, preparations of plasmid and phage DNAs, molecular cloning, radioactive labelling and blot hybridisation (11). 35S-Dideoxy-plasmid-sequencing of both strands with commercially available kits (pUC-Sequencing-kit, Boehringer Mannheim and T7-Sequencing-kit, Pharmacia) followed the instructions of the manufacturers. For sequence analysis, the UWGCG-program package (version 5.3) was used (12).
HPV 9 HPV 17 HPV 20 HPV 36
B
E H
H
H
B
I
B
E
E
_I
BH H H E
E L
S
E
eiI
E
B
S
B
E B
B HP
B BE
11
/ sequenced region _ subcloned region LCR (E6- LI)
4--
Fig.l. Physical maps of restriction enzymes cleavage sites in the genomes of HPVs 9, 17, 20, and 36, and localisation of the sequenced regions and the LCRs. B = Bam HI; E = Eco RI; H = Hind III; P = Pvu II; S = Sac I.
3920 Nucleic Acids Research, Vol. 18, No. 13
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100
Hpv2O gaattcaaat atattagagg agtggcagtt aggatttgtt cctgcaccag ataatcctat ccacGAtAca TAcaGATATA TTaaTTCtgc Hpv36 gaattcttct ttattggaag attggcagtt aggttttgta cctactccag ataaccctat tcaaGAcAcc TAtcGATATA TTgaTTCatt HPV9 GAtAtc TAcaGATATA TTgcTTCaaa Cons GA-A-- TA--GATATA TT--TTC--101 Hpv2O TGtCCtGAca Hpv36 TGtCCtGAta Hpv9 TGcCCaGAtg Hpvl 7 TG-CC-GA-Cons
201 Hpv2O AATATtCTcT Hpv36 AATActCTtT Hpv9 AATATcCTtT Hpvl 7 AgTATcCccT AATAT-CT-T Cons
aaaatcctCC aAaaGAAAgA GAaGATCCtT acaaggatct aaacTTtTGG aAtGTtGAcC TatCaGAAAG aaacccccCC tAagGAAAaA GAgGATCCcT acaaggggtt aaagTTcTGG gAtGTgGAtC TtACtGAAcG ctgttgagCC tAcaGAAAaA GAaGATCCcT ttgccaaata ctcaTTtTGG &AaGTgGAtC TaACtGAAAG TaaAcC TtACatttAG --------CC -A--GAAA-A GA-GATCC-T ---------- ----TT-TGG -A-GT-GA-C T-AC-GAAAG
AGGactcAAA AGGtaGAAAg AGGtaGAAAA AGGacGAAAA
AGG--GAAAA
Ifn-RE TTctTaTTTC TTTcTaTTTC TTTcTtTTTC TTTaTtTTTC TTT-T-TTTC
401 APM Hpv2O Hpv36 Hpv9 Hpv17 Cons
ATTGTC--TA GATTT-GATC
P 0 300 AAGCaGGTTT aCAacaggcg ACcgtAAaCg GTACaAaaac tgtaTCTtca AggttaTCta ctAggGGcgt AAGCcGGcTT aCAgcagacg ACcgtAAgCg GTACaAaatc agtgTCTtat cgAgggTtca ccAgaGGaac AAGCtGGTTT gCAa ...... ACacgAAaac GlcCtAttaa aacaTCTgtt AaAacaTCt. .Aaaaatgc AgtCaGGTTT gCAg ...... gCaaggccCa GaACtAttcg gaccTCTgta AaAgtgcCc. . .AagGGtat AAGC-GGTTT -CA------- AC---AA-C- GTAC----- ---- C--- A-A---TC-- - -A- -GG- - p 1 GtTTTCGGT. GtTTTtGGT. GaTaTCGGTt GcTTTCGGTc G-TTTCGGT-
CK-OCT ..aCAATAAA g.TcAAcTTT ..aCAgattt a.TaAAcTTT tc.CAATAAA atTtAAgTTa tctCAATAAA .. cAAaTaa ---CAATAAA --T-AA-TT-
N 33 400 TACACggTat TcaagGAtg tTTaTTTAct C. TG TACACagTat TcaagGAAtg tTTgTTTAct C......TG TcCAattTgg TatgtGAAgc aTTtTTTAac CatcttcgTG acCAaaaTgg TatgtGAAgt aTTtTTTtac CatgttcgTG ...
T-CA--- T-- T ---- GAA-- -TT-TTTA-- C------- TG
PEA-2
500
CRE P 2 NFI NFl ACTAA .. cta agatAcCaAC CGCACCCGac acAT.aaAgg tgAgTtgTGt gccaaatgag gtgagttgtg agccagaaga gatcacaWcc ACTAAgtata actctaCcAa gggaAcCgAC_CGCACCCGGT acAgtcaAcg ATAcT..gct gccaatatag a...................
ACTAAaccgt acaagtCaAc acagAgCgAC CGCACCCGGT ttATctgAtt ATAaT.gTGc ACTAAa.cgt cttcgtCaAc gc.cAgaaAC CGCACCCGGT taATcagAtt ATAaT..TGc ACTAA ----- ------ C-A- ----A-C-AC CGCACCCGGT - -AT --- A- - ATA-A- -TG-
501 NF1 NF1 NF1 Hpv2O aagtcaggct tgagccaaat cagataca.c tgcgtgccag agttggctcA Hpv36 ..ctctgttt agtgccagaa catat.catc ttggaagcag atc ...... g Hpv9 . ..........A..........A Hpvl 7 .A..........A ---------- ---------- ---------- ---------- ---------A Cons .........
..........
....................
.........
...................
601
NFl NFI gg CTGCCAA... CGcttTTGG Ctc ...- ttCt ..gAtTtaat CTa.CAA... TCGctgTTGG CAa... tCgc ..AAtTtca. CTGCCAAggt TCG... TTGG .... CAg aCCg Hpvl 7 gaAAcTgg.. aGCCAAggt T . TTGG CAgaacaCCa - -AA-T- - - - CTGCCAA- - - TCG --- -TTGG CA ----- CCCons
Hpv2O Hpv36 Hpv9
AGCcACaaaa AGC -AC--- --
200 ATTGTCctTA GAaTTgGATC gTTGTCacTA GATTTgGATC ATTaTCgtTg GATcTtGATC AaTGaCtccA GATTTaGATC
I fn-RE
301 CK-OKT STOP cAAaCGaAAa CGcaaaatt aaactcGACC Hpv36 cAAgCGcAAg CGaaaacagt aatat.GACC Hpv9 tAAgaGaAgg CGa... acc . taACC Hpvl 7 tAAaCGcAAa CGt . tca . tGACC Cons -AA-CG-AA- CG-------- ------GACC Hpv2O
AGCtACtaga AGCcACtcgc
..A
NFI TtTTGGCacA TtTTGGCtgA TccTGGC. -t TcTTGGaacA T-TTGGC- -A
NF1 701 Hpv2O .cgtacaTTa Hpv36 actggaaTTt Hpv9 .... tatTT. Hpvl 7 .... actTTc -------TTCons
P36
agcttcatcg aCcGtgttcg cCtGgtgcaa cCtGgtgcga
.. * --
* -
.
...-
--
-
-
*- .
.-
.. --.-..... ............
...... ......
.......... ..........
...... ......
..........
NFI 600 TcccAAC ... ACgtTCGgaa cAggA ..... GgaAatGta .
TTgtAAC ... ACgcTCGgat tA.gAgacat TTtgAACaat ACtaTCGtgg aAtcAccgca TTttAtCact gCttTtGtgg aAgcAccgca -C-G------ TT--AAC--- AC--TCG--- -A--A----P 3
tGCcAagGaa gGCaAccGcc gGCgcccGcc -GC-A--G-P 4 700
NF1
GCagaa.GAC CGtTAACGGT AAGttt .... tTaTTtGtAt CGggcGCGGT GatagctGAC CGgTAACGGT AAGttattta aTtTatGtAc CaggtGCGGT tCaaaacGAC CGaTAACGGT AAG ....... TcTTGGCA. CGtagGtGGT GC . AC CGaTAACGGT AG. .attat aTcTTgGaAc CGtagGCGGT GC ----- GAC CG-TAACGGT AMG ------- - T-TT-G-A- CG - -- GCGGT .
NF1 CAAT N 29 P 5 800 ctcATttggt aGtTG.TTGT tGccAaCtAC cATCaa...g CATA.gCATg tttttgcctg taacgtTaTC g..gcACaGt gaTteaTAta cacAataaTa a.tTG.TTGT tGccAACtAC cATtgc.... tATAttCAag tttttgcctg tatcgtTtTC gTaTcAtgtg a.ataaTAta ..gATcgtTg gGaTGaTTGT gGttAACaAC aATCta .... CATAcaCAT . TtTC aTaTgACcGc ctTcgtTA.. tgatTggtTt gGcTGaTaGT aGttACaAC aATCttctct CATAaatAT .aTgTgACcGc ctTcgtT... ---AT ---T- -G-TG-TTGT -G--AAC-AC -ATC------ CATA--CAT- ---------- ------T-TC -T-T-AC-G- --T---TA-......
801 TATA CK-OKT/rev Hpv2O tatatataTA TATAtAtATA tATAtATATA Tatatatata tataaataga tacataTag. Hpv36 ctgtAtagTA TAaAtAaATA aATAaATATA Tatatatat. gcttcaaagg ttggGtTttt Hpv9 .ataAgctTA TATAgAcATA aATAtATAaA .................... GgTgcc Hpvl 7 AcctTA MAT.gAtcTA cATAcA.ATA T .Gagagc - -- -A- - - TA TATA-A-ATA -ATA-ATA ------------ ---------- --- G-T --Cons ....
....
START 900 acagAtaTca tAGAGCtaat gcagagagtg caggctcatg
taaTAaTT........... .aag atgTAtTTaa cAGAGCagat t................... ..........
tctTAcTTa. --- TA-TT- -
901
AGAGCat.. *
-AGAGC- - - START
.
...*.-.--.-.-
1000
Hpv2O gctacacctc cttcttcaga agacagcgct gatgaaggac catccaatat tggagagaca aaacctttta ttcTaGAgcc aCCAttgCCT gcAACAaTct Hpv36 gcagagcaag cctcc... ga acagcag............ tacagaaaaa gaa ....... .aatat aAaga aCagctgCCT ttAACtaTtA .....
Hpv9 Hpvl7
..........
..........
............. .......... ....... ..... . . . . . . . . . .
..........
..........
............. .......... ....... ..... .. ..
Cons
----------
Hpv2O Hpv36 H pv9 Hpvl 7
1001 gtGgcCTaGC aGGgcCTgtC aGGaaCTaGC gGGagCT tGC -GG- -CT-GC
Cons
.....
gaAccttTTa GaaATaCCgc aGAatCaTTa GgcATTCCgT aGAcaCtcTt GtgATTCCtT tGAtaCcTTg tgtATTCCaT -GA--C-TT-
Fig.2. Alignment of the newly determined
.
. . . . .
a gAA CA g T aA T..g .a.. T.g...G g gCCAaaaCC T c aAA CA gTgA T-GA-CCA- - -CCT - -AACA-T-A
. . . . . . . . . . GA ca g g CCA aa a C C T ... . . .
. . .. ....... .. . . . .. Ata ---------- ---------- ---------- ----
G--ATTCC-T
..
..
TAGatGATTg TTTgATACCT TGTAAcTTcT TtGTaGAcTg TcTaATACCT TGTAAcTTTT TAaTaGATTt gTTgATACCT TGTAAaTTTT TAGTgGATat TTTatTACCT TGcAgaTTTT TAGT-GATT- TTT-ATACCT TGTAA-TTTT
.
GcggTAatTT GtggcAaaTT GcaaTAgaTT GtaaTAggTT
.
1100 TcTtaCacAT TTAGAAgTtt gtgagTTTGA
TTTAgaTTAT TTAGAAgctt
..........
TTTAtCTTAT TTtGAgcTac ttaatTTTGA TTTAgCTTAc aTAGAAtTgg tggcgTTTGA G --- TA--TT TTTA-CTTAT TTAGAA-T-- -----TTTGA
sequences of HPVs 20 and 36 and HPVs 9 and 17. Putative functionally important elements and protein binding sites indicated in bold letters or underlined. Ifn-RE=Interferon responsive element. CK-OKT=Cytokeratin-octamer (19). PO-5,P36=E2-protein binding sites. STOP=Translation stop codon of ORF LI. CRE=cAMP responsive element. PEA-2=Polyomavirus enhancer element. v-myb= Binding site for v-myb protein. NFl =Nuclear Factor 1 binding site. M33,M29=conserved motifs of 33 and 29bp respectively. CAAT= putative CAAT-box. TATA=stretch of A and T nucleotides. START=Translation start codon of ORF E6.
are
Nucleic Acids Research, Vol. 18, No. 13 3921
PO Pt M33 AP1 P2 HPV 5
*O
HPV 8
O 0
Nfl J0 0 0 ]0 00
HPV 36
* 0
]0 0
HPV 19 HPV 20 HPV 25
*u
P3 0
P3 P4 NFl CAAT M29 P5 TATA 00 000 C]m 0000 00 0-] 0 00 000 a C]
NFl
IIoo-oo_000 0.0*0o00000 0o a00 o ?
+
o
o -o *C]*.00
C
-00 0.-
C
00 000
=
HPV 9 HPV 17
Fig.3. Schematic distribution pattern of conserved, putative regulatory elements in the LCR sequences of ev-associated HPV 5 (3), HPV 8 (4), HPVs 19 and 25 (5), and HPVs 9, 17, 20, 36 (this paper). PO-5, P36 = E2 protein binding sites: 0 = ACCGN4CGGT; (3 =ACCGN4CGTT; 0 = ACCN6GGT; a= degenerated palindrome. * =AP1 binding site. a = NFl binding site. af = M33, M29 conserved motifs of 33 and 29bp, respectively. *= CAAT box. TATA = TATAstretch: +++ = -SObp; + + = -20bp; + = TATA-like sequence in HPV 17. ? in HPV 25: DNA-sequence not available.
RESULTS AND DISCUSSION The LCRs in the genomes of HPVs 9, 17, 20 and 36 were localized by Southern blot analysis using the homologous HPV 8 DNA as probe (Fig. 1). The cross-hybridising fragments were subcloned into the Bluescribe vector (Vector Cloning Systems) and sequenced. The lengths of the LCRs from the translation stop codon of LI to the start codon of E6 were 513 bp in HPV 20, 480 bp in HPV 36, 387 bp in HPV 9, and 379 bp in HPV 17. The aligned nucleotide sequences and the locations of conserved elements and putative recognition sites for DNA binding proteins (13) are shown in Fig.2. A comparison with published LCR sequences of ev-associated HPVs confirmed the following, highly conserved landmarks (Fig.3): 1. Four palindromic E2 binding sites ACCGN4CGGT or slightly degenerated versions thereof (P1-4). Palindrome P3 is the most conserved among them and includes a putative binding site for the v-myb protein (14). 2. One binding site for the transcription factor jun/API (13). 3. A cluster of direct or inverted repeats TGCCAA and related sequences, which were shown to bind the transcription factor NF1 (15). 4. A 'CAATmotif', which is generally conserved among papillomaviruses (5). HPVs 5, 8, 19, 20, 25, and 36 (group A) share the previously described conserved blocks with 33 and 29bp, respectively, an additional palindrome PO within the 3'-part of ORF LI, and a striking stretch of about 50 A and T nucleotides. HPVs 5, 8, and 36, on the one hand, and HPVs 19, 20, and 25, on the other hand, form two slightly different subgroups Al and A2. In A2-viruses, palindromes P2 and P4 are incomplete, and 12bp are deleted between API and P2; the AT rich stretch consists of monotonously alternating A and T, in contrast to subgroup Al viruses, which show two to four TAAA blocks followed by a shorter AT run. This AT stretch is without precedent among papillomaviruses, but similar motifs were identified within the autonomously replicating sequences (ARS) and promoter regions of yeast. Promoter activity was there shown to improve with increasing length of this element (16,17). Against the background
of a rather uniform organization of group A virus LCRs, an apparently recent duplication can be noted in the case of HPV 20, which has a 21bp direct repeat with 3 mismatches between pos. 451 and 492 (Fig.2). The same duplication was observed in a second HPV 20 clone from another patient, which renders it unlikely to represent a cloning artefact. The sequence ACCGN4CGTT, which was recently shown to bind E2 of bovine papillomavirus 1 (18), occurs once in HPV 36 (P36). In HPV 9 and 17 (group,B) no M29 and only a slimed down M33 could be detected. The AT stretch is shortened in HPV 9 to about 20bp and interrupted by a few other nucleotides. No classical TATA-box was found in HPV 17 in this region, but TATA-like sequences TTAAATGA and TACATACAATAT are located at pos. -36 and -22 relative to the putative E6-protein translation start codon. The group B virus sequences thus indicate that the characteristic TATA stretch and the M29 motif discussed before are not consistent features of ev-associated HPVs, and can therefore not account for their host-specificity. Despite good general homologies with group A viruses in the 3'-part of ORF LI, group B viruses display no P0 in this region, but they contain an ACCGN4CGTT following the CAAT-box (P5). No obvious correlation exists between the LCR classification into subgroups Al, A2, and B and the assumed oncogenic potential of ev-HPVs. This analysis provides another example, however, that LCRs of papillomaviruses, which belong to the generally less conserved genome segments, show a subgenousspecific organization pattern in viruses with similar tropism and pathogenic properties as observed for viruses associated with anogenital lesions.
ACKNOWLEDGEMENTS We are indebted to Dr. G. Orth who provided cloned DNAs of HPVs 9, 17a, and 36. We gratefully acknowledge helpful discussions with Dr. P.G. Fuchs. This work was supported by the Deutsche Forschungsgemeinschaft (PF 123/3 -2).
3922 Nucleic Acids Research, Vol. 18, No. 13
REFERENCES 1. Orth, G. (1987) in Salzman, N.P., and Howley, P.M. (ed.), The Papovaviridae, Vol.2, Plenum Publishing Corp., New York 199-243. 2. De Villiers, E.-M. (1989) J.Virol. 63, 4898-4903. 3. Zachow, K.R., Ostrow, R.S., and Faras, A.J. (1987) Virology, 158, 251-254. 4. Fuchs, P.G., Iftner, T., Weninger, J., and Pfister, H. (1986) J.Virol., 58, 626-634. 5. Krubke, J., Kraus, J., Delius, H., Chow, L., Broker, T., Iftner, T., and Pfister, H. (1987) J.Gen.Virol., 68, 3091-3103. 6. Iftner, T. (1990) in Pfister, H. (ed.), Papillomaviruses and human cancer, CRC Press, Boca Raton (Fl.,USA), 181-202. 7. Kremsdorf, D., Favre, M., Jablonska, S., Obalek, S., Rueda, L.A., Lutzner, M.A., Blanchet-Bardon, C., van Vorst Vader, P.C., and Orth, G. (1984) J.Virol., 43, 1013-1018. 8. Gassenmaier, A., Lammel, M., and Pfister, H. (1984) J.Virol., 52, 1019-1023. 9. Kawashima, M., Favre, M., Jablonska, S., Obalek, S., and Orth, G. (1986) Virology, 154, 389-394. 10. Kremsdorf, D., Jablonska, S., Favre, M., and Orth, G. (1982) J.Virol., 43, 436-447. 11. Maniatis, T., Frisch, E.F., and Sambrook, J. (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 12. Devereux, J., Haeberli, P., and Smithies, 0. (1983) Nucleic Acids Res., 12, 387-395. 13. Jones, N.C., Rigby, P.W.J., and Ziff, E.B. (1988) Genes & Development, 2, 267-281. 14. Biedenkapp, H., Borgmeyer, U., Sippel, A.E., and Klempnauer, K.-H. (1988) Nature, 335, 835-837. 15. Gloss, B., Yeo-Gloss, M., Meisterernst, M., Rogge, L., Winnacker, E.L. and Bernard, H.-U. (1989) Nucleic Acids Res., 17, 3519-3533. 16. Stinchcomb, D.T., Mann, C.,Selker, E., and Davis, R.W. (1981) ICN-UCLA Symp. Mol. Cell. Biol. 22, 473-488. 17. Struhl, K. (1985) Proc.Natl.Acad.Sci.USA, 82, 8419-8423. 18. Li, R., Knight, J., Bream, G., Stenlund,A., and Botchan, M. (1989) Genes & Development, 3, 510-526. 19. Blessing, M., Zentgraf, H., and Jocamo, J.L. (1987) EMBO J. 6, 567-575.
