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VIROLOGY 118, 254-259 (1982) Molecular Cloning and Partial Nucleotide Sequence of Human Papillomavirus Type la DNA ANDREAS CLAD, LUTZ GISSMANN, BERTHOLD MEIER,* ULRICH KARL FREESE, AND ELISABETH SCHWARZ’ Institut fiir Virokqie, Zen&urn fiir Hygiene, Universit(lt Freiburg, Hermann-Herder-Se 11, 7800 Freiburg, and *Institut fiir medizinische Llokumentation und Statistik, Z&rum Pathdqie, Stephan-Meier-Stmsse 26, 7800Freibury, Federal Republic of Germany Received November 18, 1981; accepted December 16, 1981 Papilloma viruses contain a DNA genome of about 5 X 106.They belong in the papo- vavirus group and induce epithelial proliferation in a variety of animal species and in man. To date, eight types of papillomaviruses have been identified in different human papillomas, some of which show an increased tendency for malignant conversion, Due to the inability to propagate papillomaviruses in tissue culture, almost nothing is known about their genetic organization and gene expression. The use of recombinant DNA tecb- niques now renders it possible to study in detail the molecular biology of papillomaviruses. Here we report the molecular cloning of the DNA of human papillomavirus type la (HPVla) in Escherichia eoli using pBR 322 as vector and the nucleotide sequence of part of the HPVla genome. The region of 1819 base pairs contains one of two pairs of short inverted repetitive sequences detected earlier by electron microscopic studies. Within the DNA sequence, three potential coding regions of only one orientation have been observed which allows prediction of the direction of transcription in this part of the HPVla genome. Human papillomavirus type 1 is found in high particle concentration in deep plantar warts (1,2,4). The DNA has been physically mapped by cleavage with var- ious restriction endonucleases (5-r). Due to some sequence variations detected in the restriction fragment patterns of HPVl DNA obtained from different wart iso- lates, HPVl has been divided into three subtypes, a, b, and c (8). We have used the DNA of HPVla for molecular cloning and sequence analysis. The EcoRI and BumHI cleavage sites were employed for construc- tion of recombinant HPVla/pBR 322 DNA molecules which were then used for trans- formation of Escherichia coli strain 490A (see Fig. 1). According to the guidelines of the German committee for biological safety (ZKBS), the cloning experiments were performed only with fragmented papillomavirus DNA. Molecular cloning of total HPVla DNA in Escherichiu coli has been reported by others (18, 14). Three of ’ To whom reprint requests should be addressed. the recombinant plasmids were used for DNA sequence analysis: KLuGl and KLuG2 containing the 4.3- and 2.3-kb BamHI-EcoRI restriction fragments, re- spectively, and KESl containing the 6.5- kb EcoRI fragment (Fig. 1). As a distinct structural feature HPVla DNA contains two pairs of short inverted repetitive sequences which have been demonstrated in the electron microscope after reassociation of single-stranded DNA (8). Similar structures have also been ob- served in the DNA of other human pap- illomavirus types as well as in bovine pap- illomavirus DNA (Gissmann, unpublished data), indicating that they may constitute a common feature in papillomavirus ge- nome structure. The two halves of one pair of these inverted repetitive sequences are located just to the left and further apart to the right of the single BarnHI site in HPVla DNA (8). Thus, we focused our ini- tial DNA sequence analysis on this part of the HPVla genome which is covered by the two Hind111 restriction fragments B 0042-6822/82/050254-06$02.00/O Capyrkht @ 19&? by Academic Press, Inc. All righta of reproduction in any form reared. 254

Molecular cloning and partial nucleotide sequence of human papillomavirus type 1a DNA

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VIROLOGY 118, 254-259 (1982)

Molecular Cloning and Partial Nucleotide Sequence of Human Papillomavirus Type la DNA

ANDREAS CLAD, LUTZ GISSMANN, BERTHOLD MEIER,* ULRICH KARL FREESE, AND ELISABETH SCHWARZ’

Institut fiir Virokqie, Zen&urn fiir Hygiene, Universit(lt Freiburg, Hermann-Herder-Se 11, 7800 Freiburg, and *Institut fiir medizinische Llokumentation und Statistik, Z&rum Pathdqie,

Stephan-Meier-Stmsse 26, 7800 Freibury, Federal Republic of Germany

Received November 18, 1981; accepted December 16, 1981

Papilloma viruses contain a DNA genome of about 5 X 106. They belong in the papo- vavirus group and induce epithelial proliferation in a variety of animal species and in man. To date, eight types of papillomaviruses have been identified in different human papillomas, some of which show an increased tendency for malignant conversion, Due to the inability to propagate papillomaviruses in tissue culture, almost nothing is known about their genetic organization and gene expression. The use of recombinant DNA tecb- niques now renders it possible to study in detail the molecular biology of papillomaviruses. Here we report the molecular cloning of the DNA of human papillomavirus type la (HPVla) in Escherichia eoli using pBR 322 as vector and the nucleotide sequence of part of the HPVla genome. The region of 1819 base pairs contains one of two pairs of short inverted repetitive sequences detected earlier by electron microscopic studies. Within the DNA sequence, three potential coding regions of only one orientation have been observed which allows prediction of the direction of transcription in this part of the HPVla genome.

Human papillomavirus type 1 is found in high particle concentration in deep plantar warts (1,2,4). The DNA has been physically mapped by cleavage with var- ious restriction endonucleases (5-r). Due to some sequence variations detected in the restriction fragment patterns of HPVl DNA obtained from different wart iso- lates, HPVl has been divided into three subtypes, a, b, and c (8). We have used the DNA of HPVla for molecular cloning and sequence analysis. The EcoRI and BumHI cleavage sites were employed for construc- tion of recombinant HPVla/pBR 322 DNA molecules which were then used for trans- formation of Escherichia coli strain 490A (see Fig. 1). According to the guidelines of the German committee for biological safety (ZKBS), the cloning experiments were performed only with fragmented papillomavirus DNA. Molecular cloning of total HPVla DNA in Escherichiu coli has been reported by others (18, 14). Three of

’ To whom reprint requests should be addressed.

the recombinant plasmids were used for DNA sequence analysis: KLuGl and KLuG2 containing the 4.3- and 2.3-kb BamHI-EcoRI restriction fragments, re- spectively, and KESl containing the 6.5- kb EcoRI fragment (Fig. 1).

As a distinct structural feature HPVla DNA contains two pairs of short inverted repetitive sequences which have been demonstrated in the electron microscope after reassociation of single-stranded DNA (8). Similar structures have also been ob- served in the DNA of other human pap- illomavirus types as well as in bovine pap- illomavirus DNA (Gissmann, unpublished data), indicating that they may constitute a common feature in papillomavirus ge- nome structure. The two halves of one pair of these inverted repetitive sequences are located just to the left and further apart to the right of the single BarnHI site in HPVla DNA (8). Thus, we focused our ini- tial DNA sequence analysis on this part of the HPVla genome which is covered by the two Hind111 restriction fragments B

0042-6822/82/050254-06$02.00/O Capyrkht @ 19&? by Academic Press, Inc. All righta of reproduction in any form reared.

254

SHORT COMMUNICATIONS 255

and C (see Fig. 1). The strategy for se- verted orientation from position 933-973 quence analysis of the HPVla HindIII-B/ C region and the resulting DNA sequence

as the only candidate for the expected in-

are shown in Fig. 2. Search for inverted verted repeat structure. The perfectly

repeats in the DNA sequence revealed an matched “core” sequence may be extended by 6 and 8 nucleotides, to yield the follow-

11-nucleotide sequence located at position ing structure: 308-318 which is exactly repeated in in-

302 318 5’-AGCTG *TTGGCACCCCCS 3’-T C GAC AACCGTGGGGGS’

AAA 991

The 11-bp sequence is particularly rich in G-C pairs, a fact which may account for the formation of stable duplex structures as seen in the electron microscope despite the shortness of the repeated sequence.

Similar short inverted repeat sequences have been detected in other DNA mole- cules, in particular at the termini of pro- karyotic insertion sequence (IS) and transposon DNAs, for example a 16-bp in- verted repeat at the ends of IS 5 at a dis- tance of 1195 bp (17, 18). The function of the inverted repeats in the papillomavirus DNAs is presently unknown.

The sequence 5’ GCTGGGGG3’ located at position 970-977 and overlap- ping partially with the 11-bp inverted re- peat sequence resembles remarkably the octamer sequence 5’ G C T G G T G G 3’ found at active Chi loci in mutant X and pBR 322 DNA (19). Chi sequences or ele- ments of Chi are also found in the recom- bination regions of immunoglobulin genes (for references see Ref. (20)) leading to the speculation that Chi sequences may par- ticipate in the rearrangements of immu- noglobulin genes (20). Whether the Chi- like sequence in HPVla DNA is involved in any kind of recombination reaction is totally unknown.

Up to now, almost nothing is known about the proteins encoded by human pap- illoma virus DNAs. In HPVla a single major capsid protein with a molecular weight of approximately 57,000 has been identified (7), but no further information is available about structural proteins or particularly about early viral proteins.

973

The DNA sequence can be used to locate potential genes in the HPVla genome. Because the sequenced section of 1819 bp

BarnIl I

ECORI

FIG. 1. Physical map of HPVla DNA. The cleavage, sites of the restriction enzymes HMIII, EcoRI, and BornHI are indicated. The H&d111 fragments are la- beled according to size. Mapping data are taken from Ref. 6. For molecular cloning of HPVla DNA, frag- ments were generated by cleaving DNA isolated from warts with EcoRI or with RamHI followed by a lim- ited digest with EcoRI. Ligation of the fragments to the vector pBR 333 cut with the appropriate restric- tion enzymes was done as described (9). After trans- formation of competent Edwrichia cdi 436A bac- teria (JO), ampicillin-resistant colonies were screened for the presence of HPVla sequences by colony filter hybridization (11) using HPVla [8PPjDNA labeled by nick translation (1%). Recombinant plasmids were further characterised by restriction endonuclease analysis. The different parts of HPVla DNA con- tained within individual clones are indicated.

256 SHORT COMMUNICATIONS

SHORT COMMUNICATIONS 257

HINDIll

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represents about one-quarter of the total HPVla genome, one can expect that it con- tains the information for at least one gene product. In order to assign possible coding regions, the complementary sequences of the two DNA strands were screened for translational termination codons. The re- sults are presented in Fig. 3. In clockwise direction, termination codons are scat- tered throughout the sequence in all three possible reading frames (Fig. 3a). In counterclockwise direction, however, long stretches uninterrupted by termination triplets are detected in two of the three reading frames: in the first reading frame a region Ib extends from position 618-88 (531 bp) and a region Ia occupies a section distal of position 1233 up to the Hind111 site at position 1819 and therefore extends for at least 586 bp (Fig. 3b). According to preliminary information of the adjacent 300 bp part of fragment HindIII-A this sketch can be assigned to reading frame Ia. In the second reading frame a third possible coding region covers 990 nucleo- tides from position 1313-324 (Fig. 3b). Re- gions Ib and IIa which are totally included in the HindIII-C/B sequence, were further screened for ATG (AUG) codons which may serve as initiation signals for trans- lation. Interestingly, the only AUG codon found within region Ib is located 430 nu-

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either does not serve as a coding region or that it represents an interior exon of a split gene. In region IIa, possible AUG initiation codons are located at positions 1289-87, 1244-42, 1136-34, and 1067-65.

I The first would give rise to a protein of 322 amino acid residues. It should be men-

FIG. 2. Nucleotide sequence of the W&II-C/B re- gion of HPVla DNA. (a) Sequence analysis was done using the method of Maxam and Gilbert (15). The internal Hind111 and the BamHI recognition site are indicated. In the lower DNA strand, the termination codons bordering the three possible coding regions Ia, Ib, and IIa (see Fig. 3) are underlined. The two halves of the inverted repeat discussed in the text

tioned that one-half of the inverted repeat

are boxed. The computer programs of Staden (16) were used to exploit the sequence data. (b) Restric- tion endonuclease cleavage sites used for sequencing are indicated. Restriction fragments were labeled at their B-ends using [-y-rsPjATP and T4 polynucleotide kinase and were fractionated on 5% polyacryamide strand separation gels (25). Each arrow pointing from 5’ to 3’ represents a 5’ terminally labeled DNA single strand with the sequenced part set off by a solid line.

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described above is located just behind the end of region IIa. Whether this close prox- imity points to any functional connection remains open.

The clearly asymmetric distribution of the translational termination codons in the two DNA strands allows one to deduce the possible direction of transcription and translation in this part of the HPVla ge- nome. With regard to the conventional orientation of the HPVla physical map as shown in Fig. 1, transcription should pro- ceed in counterclockwise direction.

The question as to which of the three coding regions are actually expressed in HPVla has to be answered in the future by the analysis of the products of tran- scription and translation. The arrange- ment of region IIa relative to the two other regions Ia and Ib leaves the possibility that genes are arranged in overlapping fashion in the HPVla genome, as is the case for example in the genomes of the papovaviruses SV40 and polyoma (21).

A first hint to the possible function of the encoded gene product(s) may come from studies of viral transcription in ham- ster tumors induced by bovine papillo- mavirus type 1 (BPVl). A virus-specific transcript of 1300 nucleotides has been isolated from such tumors devoid of any detectable virus particle synthesis and has been mapped on the BPVl genome (22). Hybridization experiments performed un- der nonstringent conditions could dem- onstrate a sequence relationship between the transcribed BPVl region and the HindIII-B fragment of HPVla (22). Thus in analogy to BPVl it may be speculated that the HPVla HindIII-C/B region more likely codes for early viral functions rather than for structural proteins.

ACKNOWLEDGMENTS

We would like to thank Drs. H. zur Hausen and G. Hobom for helpful suggestions and Mrs. G. Bren- de1 and Miss C. WSlfle for skillful technical assis- tance. This work was supported by the Deutsche For- schungsgemeinschaft SFB 31 (Mediiinische Virologie: Tumorentstehung und-Entwicklung).

SHORT COMMUNICATIONS 259

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HOWLEY, P. M., J. Vi& 36,395-407 (1989). 15. MAXAM, A. M., and GILBERT, W., In “Methods in

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16. STADEN, R., Nuc!eic Acids Res. 9,309-321 (1980). 17. CHOW, L. T., and BROKER, T. R., J. BacterioL 133,

1427-1436 (1978). 18. LUSKY, M., KRUGER, M., and HOBOM, G., Cold

Spring Harbor Synp. Quad BioL 45,173-176 (1981).

19. SMITH, G. R., KUNES, S. M., SCHULZ, D. W., TAY- LOR, A., and TRINAN, K. L., Cell 24, 429-436 (1981).

ao. KENTER, A. L., and BRISHSTEIN, B. K., Nature (Londan) 293,402-404 (1981).

21. GRIFFIN, B. E., In “Molecular Biology of Tumor Viruses,” Part 2, “DNA Tumor Viruses” (J. Tooze, ed.), pp. 61-123. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.

22. FREESE, U. K., SCHULTE, P., and PFISTER, H., virdogy 117,257-261 (1982).