VIROLOGY
185,424-427
( 199 1)
Human Papillomavirus
Type 58 DNA Sequence
YASUYUKI KIWI,* SEI-ICHI IWAMOTO, * AND TOSHIHIKO
MATSUKURAt
”
* Kanebo Institute for Cancer Research, Miyakojima-ku. Osaka 534 and t Department of Enteroviruses. National Institute of Health, Shinagawa-ku, Tokyo 14 1, Japan Received May 20, 199 1; accepted July 25, 799 1 The complete nucleotide sequence of human papillomavirus type 58 (HPV 58) DNA cloned from an invasive cervical carcinoma was determined. The HPV 58 genome consists of 7824 nucleotides, containing 37.9% of GC residues, and has a similar genome organization of other HPVs. On the nucleotide sequence level, it conserves the signal sequences for regulation of gene expression as with other genital HPVs and exhibits an extensive homology with HPV 33 (77%). Comparative analysis of amino acid sequences reveals that HPV 58 is closely related with HPVs 16,31, and 33, and is more distantly related with HPVs 6,11,18, and 39. HPVs 58,16,31, and 33 can be regarded as a group in HPV. Q 1s~ Academic
Press, Inc.
termination codons in the three reading frames of both strands is shown in Fig. 1A. All potential major open reading frames (ORFs) of HPV 58 are located on one strand as with other HPVs. On the other hand, the distribution of GC-content in HPV 58 DNA containing 37.9% of GC residues was analyzed using various biases. The representative pattern shown in Fig. 1 B reveals clearly that the GC-content is not evenly distributed throughout the genome and the GC-rich regions are clustered at around the sequences encoding ORFs E7, E2, and L2. Since other HPVs as well as animal PVs show a similar multimodal pattern but not polyoma virus (data not shown), it might be noted that the uneven distribution of GC-content in a genome is an additional common character of Papillomavirus. The exact positions and coding capacity of amino acids of each ORF are summarized in Table 1 and the complete sequence of the coding strand is shown in Fig. 2. HPV 58 has ORFs comparable in size and at similar positions as other genital HPVs, including the overlaps between ORFs El and E2 and between ORFs L2 and Ll and the inclusion of E4 ORF within E2 ORF. The E7 ORF of HPV 58 is located at immediately upstream of El ORF as E7 ORF in HPVs 16, 18,33, and 39, and the E4 ORF of HPV 58 has no initiation codon as with HPVs 16, 31, 33, and 39. The long control region (LCR) of HPV 58 consisting of 794 nucleotides (nt 7140-l 09) has a GT-rich region and conserved signal sequences at positions similar to other genital HPVs described earlier (6, 7, 9), such as glucocorticoid response element: TTGTGTACATGlTCT (nt 7246)) polyadenylation signal: AATAAA (nt 7296), PV-specific palindrome element: ACCN,GGT
Human papillomavirus (HPV), a member of genus Papillomavirus of the Papovaviridae family, is classified into more than 60 distinct types on the basis of their DNA homology now ( 7). Of these, 25 types have been isolated from or associated with genital lesions. Since the association of the specific types with the specific lesion, such as condyloma, intraepithelial neoplasia, and invasive carcinoma, has been reported ( 7, 2), the common biological functions might be reflected at the nucleotide level. However, only seven genital HPVsaresequencedsofar(HPVs6(3), 11 (4), 16(5), 18(6),31 (7),33(8),and39(9)).HPV58wascloned from an invasive cervical carcinoma and has been detected in cervical intraepithelial neoplasias as well as other invasive cervical carcinomas but not condyloma (70). In order to obtain additional information for understanding the pathogenesis of genital HPV, we determined the nucleotide sequence of HPV 58. HPV 58 DNA purified from the original clone ( 10) was subcloned in pUC 118 and 119 into four fragments using &/II, HindIll, and Xbal restriction sites. Sets of nested deletion clones were prepared with Exonucleaselll digestion and sequenced by the modified dideoxy chain method ( 17). The 7824 nucleotide sequence of HPV 58 DNA genome was obtained and aligned with the nucleotide sequences of HPVs 16 (5) and 33 (8) using the program of Lipman and Pearson ( 12). The distribution of Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under Accession No. D90400. ’ To whom requests for reprints should be addressed.
0042-6822/91
$3.00
Copyright Q 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
424
SHORT COMMUNICATIONS
425
FIG. 1. (A) Distribution of translational termination codons in the two strands of HPV 58 DNA sequence. Each vertical bar represents one termination codon in respective reading frame. The major open reading frames are located on one strand and are designeted in anaiogy to the organization of other HPV sequences. The scale corresponds to the linearized 7824 nucleotide sequence with the putstfve promoter sequence (TATA&?*) and polyadenylation signals (AAT.WA:O). (B) Distribution of GC-content in HPV 58 DNA sequenoe. The distribution of GC-content (%) was plotted for each successive 400 nucleotides. The GC-rich regions compared to a total GC-content (shown by a horltont& her) are bfeck boxed.
TABLE 1 HPV58 DNA
OPENREADINGFRAMESIN
Nucleotide position Open reading frame E6 E7 El E2 E4
E5 L2 Ll
Start ORF
First start codon
First stop codon
Amino acids coding capacity in dalton9
77 544 871 2732 3330 3856 4232 5559
110 574 883 2753 -
557 868 2815 3827 3603 4120 5660 7137
10819 72240 40779 10385 8953 50940 59035
3892 4244 5565
17793
’ Calculated from the first ATG where this exists or from the start of the open reading frame.
(nt 7487, 7780, 40, 55), nucleer factor I:TGCCA (nt 7723, 12), AP-1 :TGGACTAA (nt 7773), and promoter element for TFII:TATAAA (nt 72). The E6 ORF of HPV 58 has the consensus sequence for the E6 * ORF (7) and another poJy~~~tion signal is found at nt 4209. At the amino acid ~~~uertce level, E6 and E7 ORFs of HPV 58 has four end two sets of the Cys-X-X-Cys motif with conserved number of intervening amino acids, respectively (9). The Thr-Thr/AspPro-Ala-HeNal-lle/Leu motif found in mucosal H?V ( 13) is present in the L2 ORF of HPV 58. By comparative analysis of the total nucleotide sequence ( 12), HPV 58 shows the high homcbgy with HPVs 33 (77%) and 16 (64%). A comparkan of HPV 58 ORFs to those of seven genital HPVs as well as othersixHPVs(HPVs1(14),2(13),5(15),8(76),47 ( 17), and 57 ( 13)) was analyzed using the program of
426
FIG. define
SHORT
COMMUNICATIONS
was
SHORT
COMMUNICATIONS
Needleman and Wunsch ( 18). It reveals that HPV 58 is closely retated with HPVs 16, 31, and 33, and moderately related with HPV 6, 11, 18, and 39, and more distantly related with the remaining nongenital HPVs (data not shown). Thus, HPVs 58, 16,31, and 33 can be regarded as a group in HPV. ACKNOWLEDGMENTS We thank S. Shiotani for technical Sugase for encouraging discussions, reading of this manuscript.
assistance. H. Honda and M. and H. Shimojo for critical
REFERENCES 1. DE VIUIERS, E-M., J. Vkol. 63, 4898-4903 (1989). 2. SUGASE, M., MORIYAMA, S., and MATSUKUFIA. T., J. Med. Virol. 34, l-6 (1991). 3. SCHWARZ, E., DORST, M., DEMANKOWSKI, C., LATTERMANN, O., ZECH, R., WOLFSPERGER, E., SUHAI, S., and ZUR HAUSEN, H., EMBOJ. 2,2341-2348 (1983). 4. DARTMANN, K., SCHWA=, E., GISSMANN, L., and ZUR HAUSEN, H., Virology 151, 124-130 (1986).
5. 6. 7. 8. 9. 10. 11. 72. 13. 14. 15. 76. 17. 18.
427 SEEDORF, K., K~MMER, G., DORST, M., SUWAI, S., and RCIWEKAMP, W. G., virology 146, 181-185 (1985). COLE, S. T., and DANOS, O., 1. Mol. Biol. 193, 599-608 ( 1987). GOLDSBOR~%H, M. D., DISILVESTRE, D., TEIJEP~, G. F.. and LORINCZ, A. T., Virology 171, 306-311 ( 1989). COLE, S. T., and STF~EECK, R. E.. J. Viral. 68, 991-996 (1986). VOLPERS, C., and STREECK. R. E., Virolclgy 181.419-423 (1991). MATSUKURA, T., and SUGASE, M., virokrgy 177,833-836 (1990). TABOR, S., and RICHARDSON, C. C., Proc. Natl. Aced. Sci. USA 84,4767-4771 (1987). LIPMAN, D. J., and PEARSON, W. R., Science 227, 1435-1441 (1985). HIRSCH-BEHNAM. A., DELIUS, H., and DE VILLIERS. E-M., Virus Res. 18, 81-98 (1990). DANOS, O., KATINKA, M., and YANIV. M., EM&O J. 1, 231-236 (1982). ZACHOW, K. R., OSTROW, R. S., and FARAS, A., J. Vmx. 158,25 l254(1987). FUCHS, P. G., IFTNER, T., WENINGER, 1.. and PFIsTER, H., 1. Virol. 58, 626-634 ( 1986). KIYONO, T., ADACHI, A., and ISHIBASHI, M., Virology 177, 401405 (1990). NEEDLEMAN, S. B., and WUNSCH, C. D., 1. Mol. Biol. 48,443-453 (1970).
185,424-427
( 199 1)
Human Papillomavirus
Type 58 DNA Sequence
YASUYUKI KIWI,* SEI-ICHI IWAMOTO, * AND TOSHIHIKO
MATSUKURAt
”
* Kanebo Institute for Cancer Research, Miyakojima-ku. Osaka 534 and t Department of Enteroviruses. National Institute of Health, Shinagawa-ku, Tokyo 14 1, Japan Received May 20, 199 1; accepted July 25, 799 1 The complete nucleotide sequence of human papillomavirus type 58 (HPV 58) DNA cloned from an invasive cervical carcinoma was determined. The HPV 58 genome consists of 7824 nucleotides, containing 37.9% of GC residues, and has a similar genome organization of other HPVs. On the nucleotide sequence level, it conserves the signal sequences for regulation of gene expression as with other genital HPVs and exhibits an extensive homology with HPV 33 (77%). Comparative analysis of amino acid sequences reveals that HPV 58 is closely related with HPVs 16,31, and 33, and is more distantly related with HPVs 6,11,18, and 39. HPVs 58,16,31, and 33 can be regarded as a group in HPV. Q 1s~ Academic
Press, Inc.
termination codons in the three reading frames of both strands is shown in Fig. 1A. All potential major open reading frames (ORFs) of HPV 58 are located on one strand as with other HPVs. On the other hand, the distribution of GC-content in HPV 58 DNA containing 37.9% of GC residues was analyzed using various biases. The representative pattern shown in Fig. 1 B reveals clearly that the GC-content is not evenly distributed throughout the genome and the GC-rich regions are clustered at around the sequences encoding ORFs E7, E2, and L2. Since other HPVs as well as animal PVs show a similar multimodal pattern but not polyoma virus (data not shown), it might be noted that the uneven distribution of GC-content in a genome is an additional common character of Papillomavirus. The exact positions and coding capacity of amino acids of each ORF are summarized in Table 1 and the complete sequence of the coding strand is shown in Fig. 2. HPV 58 has ORFs comparable in size and at similar positions as other genital HPVs, including the overlaps between ORFs El and E2 and between ORFs L2 and Ll and the inclusion of E4 ORF within E2 ORF. The E7 ORF of HPV 58 is located at immediately upstream of El ORF as E7 ORF in HPVs 16, 18,33, and 39, and the E4 ORF of HPV 58 has no initiation codon as with HPVs 16, 31, 33, and 39. The long control region (LCR) of HPV 58 consisting of 794 nucleotides (nt 7140-l 09) has a GT-rich region and conserved signal sequences at positions similar to other genital HPVs described earlier (6, 7, 9), such as glucocorticoid response element: TTGTGTACATGlTCT (nt 7246)) polyadenylation signal: AATAAA (nt 7296), PV-specific palindrome element: ACCN,GGT
Human papillomavirus (HPV), a member of genus Papillomavirus of the Papovaviridae family, is classified into more than 60 distinct types on the basis of their DNA homology now ( 7). Of these, 25 types have been isolated from or associated with genital lesions. Since the association of the specific types with the specific lesion, such as condyloma, intraepithelial neoplasia, and invasive carcinoma, has been reported ( 7, 2), the common biological functions might be reflected at the nucleotide level. However, only seven genital HPVsaresequencedsofar(HPVs6(3), 11 (4), 16(5), 18(6),31 (7),33(8),and39(9)).HPV58wascloned from an invasive cervical carcinoma and has been detected in cervical intraepithelial neoplasias as well as other invasive cervical carcinomas but not condyloma (70). In order to obtain additional information for understanding the pathogenesis of genital HPV, we determined the nucleotide sequence of HPV 58. HPV 58 DNA purified from the original clone ( 10) was subcloned in pUC 118 and 119 into four fragments using &/II, HindIll, and Xbal restriction sites. Sets of nested deletion clones were prepared with Exonucleaselll digestion and sequenced by the modified dideoxy chain method ( 17). The 7824 nucleotide sequence of HPV 58 DNA genome was obtained and aligned with the nucleotide sequences of HPVs 16 (5) and 33 (8) using the program of Lipman and Pearson ( 12). The distribution of Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under Accession No. D90400. ’ To whom requests for reprints should be addressed.
0042-6822/91
$3.00
Copyright Q 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
424
SHORT COMMUNICATIONS
425
FIG. 1. (A) Distribution of translational termination codons in the two strands of HPV 58 DNA sequence. Each vertical bar represents one termination codon in respective reading frame. The major open reading frames are located on one strand and are designeted in anaiogy to the organization of other HPV sequences. The scale corresponds to the linearized 7824 nucleotide sequence with the putstfve promoter sequence (TATA&?*) and polyadenylation signals (AAT.WA:O). (B) Distribution of GC-content in HPV 58 DNA sequenoe. The distribution of GC-content (%) was plotted for each successive 400 nucleotides. The GC-rich regions compared to a total GC-content (shown by a horltont& her) are bfeck boxed.
TABLE 1 HPV58 DNA
OPENREADINGFRAMESIN
Nucleotide position Open reading frame E6 E7 El E2 E4
E5 L2 Ll
Start ORF
First start codon
First stop codon
Amino acids coding capacity in dalton9
77 544 871 2732 3330 3856 4232 5559
110 574 883 2753 -
557 868 2815 3827 3603 4120 5660 7137
10819 72240 40779 10385 8953 50940 59035
3892 4244 5565
17793
’ Calculated from the first ATG where this exists or from the start of the open reading frame.
(nt 7487, 7780, 40, 55), nucleer factor I:TGCCA (nt 7723, 12), AP-1 :TGGACTAA (nt 7773), and promoter element for TFII:TATAAA (nt 72). The E6 ORF of HPV 58 has the consensus sequence for the E6 * ORF (7) and another poJy~~~tion signal is found at nt 4209. At the amino acid ~~~uertce level, E6 and E7 ORFs of HPV 58 has four end two sets of the Cys-X-X-Cys motif with conserved number of intervening amino acids, respectively (9). The Thr-Thr/AspPro-Ala-HeNal-lle/Leu motif found in mucosal H?V ( 13) is present in the L2 ORF of HPV 58. By comparative analysis of the total nucleotide sequence ( 12), HPV 58 shows the high homcbgy with HPVs 33 (77%) and 16 (64%). A comparkan of HPV 58 ORFs to those of seven genital HPVs as well as othersixHPVs(HPVs1(14),2(13),5(15),8(76),47 ( 17), and 57 ( 13)) was analyzed using the program of
426
FIG. define
SHORT
COMMUNICATIONS
was
SHORT
COMMUNICATIONS
Needleman and Wunsch ( 18). It reveals that HPV 58 is closely retated with HPVs 16, 31, and 33, and moderately related with HPV 6, 11, 18, and 39, and more distantly related with the remaining nongenital HPVs (data not shown). Thus, HPVs 58, 16,31, and 33 can be regarded as a group in HPV. ACKNOWLEDGMENTS We thank S. Shiotani for technical Sugase for encouraging discussions, reading of this manuscript.
assistance. H. Honda and M. and H. Shimojo for critical
REFERENCES 1. DE VIUIERS, E-M., J. Vkol. 63, 4898-4903 (1989). 2. SUGASE, M., MORIYAMA, S., and MATSUKUFIA. T., J. Med. Virol. 34, l-6 (1991). 3. SCHWARZ, E., DORST, M., DEMANKOWSKI, C., LATTERMANN, O., ZECH, R., WOLFSPERGER, E., SUHAI, S., and ZUR HAUSEN, H., EMBOJ. 2,2341-2348 (1983). 4. DARTMANN, K., SCHWA=, E., GISSMANN, L., and ZUR HAUSEN, H., Virology 151, 124-130 (1986).
5. 6. 7. 8. 9. 10. 11. 72. 13. 14. 15. 76. 17. 18.
427 SEEDORF, K., K~MMER, G., DORST, M., SUWAI, S., and RCIWEKAMP, W. G., virology 146, 181-185 (1985). COLE, S. T., and DANOS, O., 1. Mol. Biol. 193, 599-608 ( 1987). GOLDSBOR~%H, M. D., DISILVESTRE, D., TEIJEP~, G. F.. and LORINCZ, A. T., Virology 171, 306-311 ( 1989). COLE, S. T., and STF~EECK, R. E.. J. Viral. 68, 991-996 (1986). VOLPERS, C., and STREECK. R. E., Virolclgy 181.419-423 (1991). MATSUKURA, T., and SUGASE, M., virokrgy 177,833-836 (1990). TABOR, S., and RICHARDSON, C. C., Proc. Natl. Aced. Sci. USA 84,4767-4771 (1987). LIPMAN, D. J., and PEARSON, W. R., Science 227, 1435-1441 (1985). HIRSCH-BEHNAM. A., DELIUS, H., and DE VILLIERS. E-M., Virus Res. 18, 81-98 (1990). DANOS, O., KATINKA, M., and YANIV. M., EM&O J. 1, 231-236 (1982). ZACHOW, K. R., OSTROW, R. S., and FARAS, A., J. Vmx. 158,25 l254(1987). FUCHS, P. G., IFTNER, T., WENINGER, 1.. and PFIsTER, H., 1. Virol. 58, 626-634 ( 1986). KIYONO, T., ADACHI, A., and ISHIBASHI, M., Virology 177, 401405 (1990). NEEDLEMAN, S. B., and WUNSCH, C. D., 1. Mol. Biol. 48,443-453 (1970).
