HPVs difficult. In the absence of a cellular ...... 72:101-146. 22. Lutzner, M. A. 1978. ... sized squamous cell carcinoma from patients with epider- modysplasia ...
Vol. 48, No. 2
JOURNAL OF VIROLOGY, Nov. 1983, p. 340-351
0022-538X/83/110340-12$02.00/0 Copyright © 1983, American Society for Microbiology
Human Papillomaviruses Associated with Epidermodysplasia Verruciformis II. Molecular Cloning and Biochemical Characterization of Human Papillomavirus 3a, 8, 10, and 12 Genomes DINA KREMSDORF,1 STEFANIA JABLONSKA,2 MICHEL FAVRE,1 AND GERARD ORTHl* Unite de l'Institut National de la Sante et de la Recherche Medicale 190, Institut Pasteur, 75015 Paris, France,1 and Department of Dermatology, Warsaw School of Medicine, Warsaw, Poland2
Received 28 December 1982/Accepted 29 June 1983
The DNAs of four human papillomaviruses (HPVs) that were found in the benign lesions of three patients suffering from epidermodysplasia verruciformis have been characterized. The flat wart-like lesions and the macular lesions of patient 1 contained two viruses, HPV-3a and HPV-8, respectively, whose genomes had previously been only partially characterized. The flat wart-like lesions of patient 2 and the macular lesions of patient 3 each contained a virus previously considered as belonging to types 3 and 5, respectively. These viruses are shown in the present study to be different from all of the HPV types so far characterized; they have tentatively been named HPV-10 and HPV-12. The HPV3a, HPV-8, and HPV-12 DNAs and the two SalI fragments of HPV-10 DNA (94.1 and 5.9% of the genome length) were cloned in Escherichia coli after having been inserted in plasmid pBR322. The cloned HPV genomes have similar sizes (about 7,700 base pairs), but their guanine-plus-cytosine contents differ from 41.8% for HPV-12 DNA to 45.5% for HPV-3a DNA. The study of the sensitivity of the four HPV DNAs to 14 restriction endonucleases permitted the construction of cleavage maps. Evidence for conserved restriction sites was found only for the HPV-3a and HPV-10 genomes since 5 of the 21 restriction sites localized in the HPV-3a DNA seem to be present also in the HPV-10 DNA. Hybridization experiments, performed in liquid phase at saturation, showed a 35% sequence homology between HPV-3a and HPV-10 DNAs, 17 to 29% sequence homology among HPV-5, HPV-8, and HPV-12 DNAs, almost no sequence homology between the HPV-3a or HPV-10 DNA and the other HPV DNAs, and a weak homology between HPV-9 DNA and HPV-8 or HPV-12 DNA. Blot hybridization experiments showed no sequence homology between the HPV-3a, HPV-8, and HPV-12 DNAs and the DNAs of the HPVs associated with skin warts (HPV-la, HPV-2, HPV-4, and HPV-7) or with mucocutaneous and mucous membrane lesions (HPV-6b and HPV-lla, respectively). One exception was a weak sequence homology between the HPV-2 prototype and HPV-3a or HPV-10 DNA. These results show that at least the following six HPV types are associated with epidermodysplasia verruciformis: HPV-3a and HPV-10, which are associated with flat warts in the general population, and HPV-5, HPV-8, HPV-9, and HPV12, which are associated specifically with epidermodysplasia verruciformis. Recent studies have shown the great plurality of human papillomaviruses (HPVs) since 11 HPV types, most of them including subtypes, so far have been recognized on the basis of the nucleotide sequence homologies which exist among the viral genomes (4, 8-12, 15, 20, 27-36; M. Favre, S. Jablonska, 0. Croissant, S. Obalek, and G. Orth, manuscript in preparation). One of the best examples of this genetic heterogeneity is given by the analysis of the viruses present in lesions of patients suffering from
epidermodysplasia verruciformis (EV). EV is a rare disease which lasts throughout life and whose pathogenesis depends on genetic, immunological, and extrinsic factors (17-19, 22), as well as on specific HPVs (18-20, 27, 29, 31, 32, 34-36, 42). This disease is characterized by flat warts or macular lesions or both spread throughout the body and by an early development of skin cancers in a high proportion of patients (18, 22, 27). Numerous HPVs have been found associated with benign EV lesions. Some of these 340
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TABLE 1. Nomenclature of HPVs identified during previous studies of Polish patients suffering from EV
First instance of EV associated with these viruses'
Proposed name (HPV
Previous name (HPV
type)
type)
3ac
3d
JD
Almost homogeneous in warts
5b
5bd
JD
Almost homogeneous in macules; found in two carcinomas
8e
5cd
DD
Major virus in macules
gb
sad 3f, 3fg Sdd
JGh
Patient
Characteristics of isolatesb
Incidence in other patientsb
Major virus in warts of patient 1; tr amt in warts of patient 5 Minor component in macules and warts of patients 5 and 1; found in a carcinoma of patient 5 Minor component in lesions of patient 5 Tr amt in warts of patient 1
Major virus in warts and macules Almost homogeneous in warts RM Almost homogeneous in warts and macules a Patients DD, JG, RM, JD, and JK previously studied (20, 27, 29, 31) are referred to as patients 1, 2, 3, 4, and 5, respectively, in this paper. The viruses were identified by molecular hybridization experiments (27, 29, 31) or restriction enzyme analysis (27) or both of DNA preparations obtained from pooled scrapings of flat wart-like lesions or of reddish and pityriasis versicolor-like macular lesions. b From unpublished data. In addition to the HPVs mentioned, the restriction enzyme analysis of DNA preparations obtained from pooled scrapings of lesions indicates the existence of at least two additional HPVs in patients 5 and 1 (Orth et al., in preparation). ' Favre et al., in preparation. d From reference 27. e From reference 36. f From reference 29. g From reference 30. h Common warts associated with HPV-2 were also found in this patient (29, 31). 10 12
JK
viruses, such as HPV-3a, HPV-10, and related viruses, are also found associated with flat warts in the general population (13, 27, 29, 31; Favre et al., in preparation), whereas other viruses so far have been found only in lesions of EV patients (19, 20, 27, 29, 31, 32, 34, 36, 42). The first indications of the existence of specific EV viruses came from our previous studies (19, 20, 27, 29, 31). These HPVs initially had been grouped within types 3 and 5 on the basis of partial DNA sequence homologies detected by RNA-DNA filter hybridization experiments, with labeled RNAs complementary to HPV DNA isolates considered as the prototypes of types 3 and 5 (20, 27, 29, 31). The analysis of viral DNA isolates, using restriction endonucleases, had led to the identification of several HPV-3 and HPV-5 subtypes (Table 1) (27, 30). In addition, these studies had shown that most patients were infected with several viruses in variable proportions, depending on the sites of the lesions (27). Either the heterogeneity of the viral DNA preparations obtained from mixtures of lesions from the same patient or the small quantity of available DNA made the characterization of the HPVs difficult. In the absence of a cellular system which might allow in vitro replication of HPVs, cloning of the viral genomes in Escherichia coli, after insertion in plasmid pBR322 or bacteriophage X DNA, was a way to circumvent
the difficulties (6, 8, 10, 13, 15, 20, 32, 34, 36). The first step in our study, intended to characterize biochemically the viruses associated with EV, was the molecular cloning of the genome of two viruses isolated from two Polish patients (20). These two DNAs presented virtually no sequence homology and constituted two distinct HPV types, type 5 and type 9 (Table 1). In the present paper, we report the molecular cloning and biochemical characterization of the genomes of four HPVs isolated from three other previously studied Polish EV patients (Table 1) (18, 27, 29, 31). The first virus, found in the flat warts of patient 1 (27, 29, 31), corresponds to the prototype of type 3 (HPV-3a) (27, 30). The second virus, initially called HPV-5c (3, 19, 27), was isolated from the macular lesions of the same patient. By its sensitivity to several restriction endonucleases, this virus is close to the virus later identified in the lesions of an Upper Volta EV patient and called HPV-8 by Pfister et al. (36). These two viruses so far have been only partially characterized. The third virus, initially considered as belonging to type 3, was isolated from flat wart-like lesions of patient 2 (29, 31). The fourth virus, previously called HPV-5d (27), had been isolated from the macular lesions of patient 3 (27, 29, 31). Studies of DNA sequence homologies have shown that these latter two viruses are new HPV types which have tenta-
342
KREMSDORF ET AL.
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bO-
-134JO
1
I'l
o.72 -
01.7.1
it
+-0.63-
t-o0-50-
FIG. 1. Restriction endonuclease analysis of HPV sequences integrated in recombinant DNAs. Uncloned HPV-3a (lane a), pBR322/HPV-3a (lane b), uncloned HPV-8 (lanes c and d), pBR322/HPV-8 (lane e), pBR322/HPV-10 Sall fragment A (lane f), uncloned HPV-10 (lane g), pBR322/HPV-10 Sall fragment B (lane h), uncloned HPV-12 (lane i), and pBR322/HPV-12 (lanej) DNAs were cleaved with Hindll endonuclease (lanes f to h), a mixture of HindIl-Hindlll endonucleases (lanes a, b, c, i, andj), or BamHl and Hindll endonucleases (lanes d and e). Electrophoretic separation of the fragments was carried out at 95 mA for 4 h in 1.2% agarose slab gels as described in the text. HPV DNA fragments are indicated by their molecular weights x 10'. Only fragments corresponding to the major cleavage patterns are labeled in lanes c and d. The underlined molecular weights correspond to fragments adjacent to pBR322 DNA.
tively been called HPV-10 and HPV-12, tively.
respec-
5 to 21% sucrose gradients in the presence of ethidium bromide (20). Restriction enzyme analysis. The following endonucleases were used in accordance with the instructions
MATERIALS AND METHODS DNA preparation. The viral DNAs were selectively extracted (27) from pooled scrapings of hand lesions of patient 1 resembling flat warts (HPV-3a), from macular lesions of the trunk (HPV-8) found in the same patient, from pooled scrapings of flat warts of the hands of patient 2 (HPV-10), and from pooled scrapings of macular lesions of the trunk of patient 3 (HPV12). Form I DNA was purified by equilibrium centrifugation in cesium chloride gradients in the presence of ethidium bromide and by sedimentation in sucrose gradients in the presence of ethidium bromide (27). DNAs of HPV-la, HPV-2, HPV-4, and HPV-7, as well as the cloned HindIll A and B fragments of HPV-5 DNA (20), the cloned BamHl-EcoRI A and B fragments of HPV-6b DNA (8), the cloned BamHl single fragment of HPV-9 DNA (20), and the cloned BamnHI single fragment of HPV-lla (10), were prepared as previously described (20). Cloned HPV-6b and HPVlla DNAs were kindly given to us by L. Gissmann. Molecular cloning of HPV DNA in E. coli. The biological hazards associated with the experiments described in this publication had previously been examined by the French National Control Committee. After cleavage by HindIll (HPV-3a and HPV-12), BamHl (HPV-8), or Sall (HPV-10), the DNAs were inserted in plasmid pBR322, cloned in E. coli K-12 strain C600 (1), amplified, and extracted as previously described (20). After treatment of the recombinant DNAs with the same endonuclease used for insertion of the viral sequences in plasmid pBR322, the viral and plasmid sequences were separated by sedimentation in
TABLE 2. Size and G+C contents of cloned HPVla, HPV-3a, HPV-5, HPV-8, HPV-9, HPV-10, and HPV-12 DNAs Cloned DNA
pairs)'z
content
41.0 HPV-la 7,811' 45.5 7,800 HPV-3a 40.9 7,000'e HPV-5 HindIII-Ad 43.6 HPV-8 7,600 41.6 HPV-9 7,200e 44.2 7,100 HPV-10 Sall-A 41.8 HPV-12 7,800 ' The sizes of the cloned HPV DNAs and the Sall A fragment of HPV-10 DNA (94.1% of the genome length) were determined by measuring with the electron microscope thle digestion products of the recombinant DNAs treated with the same endonuclease as that used for insertion of the viral sequences as described in the text. The size of pBR322 DNA (4,362 base pairs) (40) was taken as a standard. 6 The G+C contents of the HPV DNAs were deduced from their buoyant densities (23) as described in the text. The DNA of M. lvsodeikticus (72% G+C) (2) was taken as a marker. ' As deduced from the complete nucleotide sequence of the HPV-la genome (6). d The size of the HindIll A fragment of HPV-5 DNA represents 93.4% of the genome (20). e Taken from Kremsdorf et al. (20).
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Hind III Hpa 11
Du0mFiI
63.1
Hpa I
Aval'N
AaD
C;-~l
F
inu
FIG. 2. Cleavage map of HPV-3a DNA. The unique HindlIl cleavage site was taken as the origin. The distances from the origin are given in percentages of the HPV-3a genome. EcoRI, Sacl, and Sall endonucleases do not cleave HPV-3a DNA. of the manufacturer: BgIl, BglII, EcoRI, Hindll, HpaI, Sacl, and Sall (Boehringer Mannheim Corp.); AvaI, BamHI, HindIII, PstI, and SmaI (Bethesda Research Laboratories); and Pvul and PvuII (New England Biolabs). The uncloned and cloned viral DNAs, as well as the recombinant plasmids (0.2 to 0.5 ,ug), were digested by one or a mixture of endonucleases, and the products of the digestions were separated by electrophoresis in agarose (1.2%) vertical slab gels as previously described (27). The sizes of the fragments were determined by their electrophoretic mobilities, using the HindIll digestion products of HPV-la and bacteriophage X as standards (9, 26). Electron microscopy of DNA. The sizes of the cloned HPV-3a, HPV-8, and HPV-12 DNAs and of the cloned Sall A fragment of HPV-10 DNA were determined by electron microscopy as previously described (28, 30). The cleavage products of the recombinant DNAs, treated with the same endonuclease used for insertion of the viral DNA, were measured. The sizes of the HPV DNAs and of pBR322 DNA were determined by measuring 60 molecules, with pBR322 DNA as an internal standard (40). Analytical ultracentrifugation. The guanine-plus-cytosine (G+C) contents of the HPV DNAs were evaluated from the buoyant density determined by equilibrium density gradient centrifugation in CsCl as described by Mandel et al. (23). A mixture of cloned
HPV DNA and Micrococcus iysodeikticus DNA (2 and 1 ,ug, respectively, in 10 mM Tris-hydrochloridelmM EDTA, pH 7.9) was adjusted to a density of 1.70 mg/ml by adding solid CsCl and centrifuged at 44,000 rpm at 20°C for 24 h (Beckman model E analytical ultracentrifuge with an AnF Ti rotor). The optical densities were measured with a photoelectric recorder.
Liquid-phase reassociation of DNA. The HPV DNAs were labeled by nick translation in the presence of [a32P]dCTP (2,000 to 3,000 Ci/mmol; Amersham International, Ltd.) and E. coli DNA polymerase I (grade I;
Boehringer Mannheim Corp.) and fractionated by sedimentation in alkaline sucrose gradients (5 to 20%) (20, 41). The unlabeled viral DNAs (10 ,ug/ml) mixed with calf thymus DNA (1 mg/ml) were sonicated, denatured, and hybridized with the labeled probes (2,000 cpm) as previously described (20). The percentage of radioactive hybrids was determined either by nuclease S1 (Boehringer Mannheim Corp.) digestion or by batchwise chromatography on hydroxylapatite (41). DNA blot hybridization. The HPV-3a, HPV-8, HPV10, and HPV-12 DNAs isolated from recombinant plasmids, as well as the DNAs of skin HPVs (HPV-la, HPV-2, HPV-4, HPV-5, HPV-7, and HPV-9) and of HPVs associated with condylomas (HPV-6b and HPVlla), were cleaved by one endonuclease or by a mixture of endonucleases, and the digestion products
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BamH I
I
Hpa
BgHpa
I
FIG. 3. Cleavage map of HPV-8 DNA. The unique BamHl cleavage site was taken as the origin. The distances from the origin are given in percentages of the HPV-8 genome. Mapping of the Hindll C and F fragments, indicated within parentheses, was not possible. Hindlll, PvuI, Sacl, and Sall endonucleases do not cleave HPV-8 DNA. were separated by electrophoresis in agarose vertical slab gels. The DNA fragments were denatured and transferred (39) to a Gene Screen hybridization transfer membrane (New England Nuclear Corp.) in 25 mM sodium phosphate buffer, pH 6.5. After being heated at 80°C for 4 h, the filters were prehybridized in a solution of Denhardt solution (7), 2x SSC (1 x SSC is 150 mM NaCl plus 15 mM sodium citrate), 50 mM sodium phosphate (pH 6.5), calf thymus DNA (250 p.g/ml), and 35% deionized formamide overnight at 42°C and then hybridized in the same solution with 32P-labeled viral DNA (2 x 104 cpm/cm2) during 48 h at 42°C. The filters were washed by shaking in a solution of 60 mM Tris-hydrochloride (pH 8), 2 mM EDTA, 300 mM NaCl, and 0.5% sodium dodecyl sulfate during 30 min at 50°C and then in 3 mM Tris base for 30 min at room temperature. These are the conditions described by the manufacturer, with slight modifications. The effective hybridization temperature was deduced, in each case, from the melting temperature (T,,), which was calculated from the following equation: T,, (degree Celsius) = 81.5 + 16.6 (log M) + 0.41 (percent G+C) - 0.72 (percent formamide), where M is the concentration of the monovalent salt (15, 24, 38). The hybrids were detected by autoradiography on Kodak AR X-Omat X-ray films, using Du Pont Lightning-Plus screens.
RESULTS Molecular cloning of HPV DNAs. The viral DNA preparations were obtained from the flat warts of the hands (HPV-3a) of patient 1 and from macular achromic lesions of the trunk (HPV-8) of the same EV patient, from the flat warts (HPV-10) of patient 2, and from mixtures of scrapings of macular lesions of the trunk (HPV-12) of patient 3. The detailed case reports of these three patients have been presented before (27, 29, 31). After cleavage by HindII or a mixture of Hindll and HindlIl endonucleases, the preparations of HPV-3a, HPV-10, and HPV12 were practically homogeneous (Fig. la, g, and i). In contrast, the HPV-8 DNA preparation was heterogeneous as shown by the presence of numerous minor bands, which correspond to
additional molecular species (Fig. lc). Previous studies have shown that HindIlI endonuclease cleaves the HPV-3a and HPV-12 DNAs only once, whereas BamHI endonuclease cleaves the HPV-3a and HPV-8 DNAs only once (27). Since these two endonucleases cleave the pBR322 DNA only once within the gene coding for
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Sal I
Hpa
FIG. 4. Cleavage map of HPV-10 DNA. One of the two Sall cleavage sites was taken as the origin. The distances from the origin are given as percentages of the HPV-10 DNA length. The seven PvuIl fragments of HPV-10 DNA with molecular weights 1.90 x 106, 1.20 x 106, 0.55 x 106, 0.54 x 106, 0.32 x 106, 0.25 x 106, and 0.17 x 106 have not been mapped. EcoRl, HindlIl, PvuI, and Sacl endonucleases do not cleave HPV-10 DNA.
tetracycline resistance (40), the viral genomes mitted the characterization of the recombinant were inserted in plasmid pBR322 by the HindlIl plasmids having integrated HPV-3a (Fig. la and (HPV-3a and HPV-12) or BamHI (HPV-8) cleav- b), HPV-8 DNA (Fig. ld and e), the two Sall age sites. None of the nine endonucleases which fragments of HPV-10 (Fig. lf to h), and HPV-12 cleave the pBR322 plasmid only once is a one- DNA (Fig. li and j). Size and G+C content of the cloned HPV cut enzyme for HPV-10 DNA (see below). The two fragments (94.1 and 5.9% genome length) DNAs. The sizes of the cloned HPV-3a, HPV-8, which resulted from the cleavage of HPV-10 HPV-10, and HPV-12 DNAs (Table 2) were DNA by Sall endonuclease were inserted in the determined by measuring, in an electron microsame resistance gene of plasmid pBR322. After scope, the digestion products of the recombinant transfection of the competent bacteria, the colo- DNAs after treatment with the endonuclease nies resistant to ampicillin and sensitive to tetra- used for cloning. pBR322 DNA (4,362 base cycline and potentially containing a recombinant pairs) (40) was used as an internal standard. The plasmid were selected, and the recombinant HPV-3a, HPV-8, and HPV-12 DNAs were found DNAs were extracted (20). The electrophoretic to have similar sizes (about 7,700 base pairs), mobilities of the products obtained after diges- whereas the 94.1% HPV-10 Sall A fragment tion of the recombinant DNAs, the uncloned contained 7,100 base pairs. The G+C contents DNAs, and the pBR322 DNA with a mixture of of these viral DNAs, of the HindlIl A fragment the endonucleases used for cloning and of of the HPV-5 DNA (93.4% of the genome), and HindII endonuclease were compared. Since of the HPV-9 DNA previously cloned (20) were HindII endonuclease cleaves the palindromic deduced from their buoyant densities (Table 2) sequence recognized by SalI endonuclease (37), (23). The buoyant densities were determined by the HPV-10 SalI fragment A and B recombinant equilibrium density gradient centrifugation in DNAs and the uncloned HPV-10 DNA were cesium chloride gradients, with M. lysodeikticus treated only with HindII. This comparison per- DNA as the standard (2). The G+C contents of
346
KREMSDORF ET AL.
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Hind
Ill
Ava I Sma I 82.0 N
Pvu 77.7
Sal I
FHpa
I
Sal I FIG. 5. Cleavage map of HPV-12 DNA. The unique Hindlll cleavage site was taken as the origin. The distances from the origin are given in percentages of the HPV-12 genome. BamHl endonuclease does not cleave HPV-12 DNA.
these DNAs varied from 40.9 to 45.5% (Table 2). The value obtained for HPV-la DNA, taken as a control (41%), was identical to that previously reported by Gissmann and zur Hausen (12). Construction of a cleavage map of the viral DNAs. The linear HPV-3a, HPV-8, and HPV-12 DNAs and the two Sall fragments of HPV-10 DNA excised from the plasmid sequences were used to study their sensitivities to 14 restriction endonucleases. The cleavage maps of the viral genomes were built by comparing the sizes of the fragments generated by cleavage of the DNAs by one endonuclease or by a mixture of this endonuclease with another endonuclease whose cleavage sites were used as a reference (Fig. 2 to 5). The sizes of the fragments (53.8 and 46.2% of the genome length) produced after cleavage of the uncloned HPV-10 DNA by endonuclease BglII permitted the unambiguous respective orientation of the Sall A and B fragments. The map of HPV-8 DNA and that reported by Pfister et al. for the HPV-8 of an Upper Volta patient (36) differ in the localization of some PstI restriction sites. This was demonstrated by comparing our uncloned HPV-8 DNA
isolate and an HPV-8 isolate obtained from an African patient similar to that of Pfister et al. (Fig. 6). Clearly, both isolates have distinct PstI cleavage patterns but the same HindII cleavage pattern as detected by hybridization with a cloned HPV-8 DNA probe. The presence of common restriction sites among the DNAs of HPV-3a, HPV-8, HPV-10, and HPV-12 and between these DNAs and those of HPV-5 and HPV-9 (20) was checked. No analogy was observed among the cleavage maps of the EV HPVs, with the exception of the HPV3a and HPV-10 maps (Fig. 7). Of the 21 sites mapped on HPV-3a DNA, 5 were also found on HPV-10 DNA when the BglI site, localized at 33.6 map units from the unique HindlIl site of the HPV-3a DNA (Fig. 2), was aligned with the unique BgII site of HPV-10 DNA (Fig. 4). Analysis of the sequence homologies among HPV DNAs. The sequence homologies among the HPV-3a, HPV-8, and HPV-12 genomes and between these DNAs and the DNAs of the prototypes of the HPV types associated with EV (HPV-5 and HPV-9), and with HPV-la DNA (12) taken as a control, were first examined by
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HPV-5, HPV-8, and HPV-12 were detected by measuring the fraction of nuclease Si-resistant 1.60 1.60 hybrids, whereas slightly higher values (22.7 to '1. 55 1.55" 31.6%) were observed by batchwise chromatog1.15 1.15 raphy on hydroxylapatite (Table 3). A very weak 0.80 Wi: cross-hybridization (less than 3%) or none was observed between the genomes of these two ,0.50 groups or between the genomes of these viruses 0.46W 0,46 0.46 and the HPV-la and HPV-9 DNAs. The nucleotide sequence homologies among the HPV-3a, HPV-8, HPV-10, and HPV-12 ge0.10 l10 nomes and between these genomes and those of the prototypes of the EV-associated HPV types (HPV-5 and HPV-9) or those of skin warts d a b (HPV-la, HPV-2, HPV-4, and HPV-7) were also FIG. 6. DNA sequence homology among different analyzed by blot hybridization experiments (20, HPV-8 DNA isolates. The uncloned HPV-8 DNA 41). The HPV DNAs were cleaved by one isolate (lanes a and c) used for molecular cloning and endonuclease or by a mixture of endonucleases, an HPV-8 DNA isolate obtained from an African producing four to six fragments (Fig. 8A). The patient (lanes b and d) were cleaved with Hindll (a and localization of the cleavage sites on these geb) or PstI (c and d) endonuclease. The cleavage nomes is known (Fig. 2 to 5) (9, 15, 20; Favre et products were electrophoresed at 95 mA for 4 h in al., in preparation), with the exception of the vertical 1.2% agarose slab gels. The DNA fragments HPV-7 genome. The hybridization of the DNA were denatured, transferred to a Gene Screen hybrid- fragments, immobilized on a membrane, with a ization transfer membrane, and hybridized with 32p_ labeled cloned HPV-8 DNA (specific activity, 2 x 108 32P-labeled DNA probe was performed under cpm/,ug). The hybrids were detected as described in stringent conditions, that is, at an effective hythe text. The molecular weights (x106), indicated on bridization temperature lower by 27, 26, and the right of lanes b and d, are in agreement with the 25°C than the Tm of HPV-3a, HPV-8, and HPVvalues reported by Pfister et al. (36). 12 DNAs, respectively. Under these conditions, only hybrids with at least 81% homologous bases are stable (15, 16, 20, 24, 38). No cross-hybridization could be detected bereassociation in liquid phase at saturation (20, 41). The reassociations were performed in 0.48 tween the HPV-3a DNA probe and the HPV-8 or M NaCI-1 mM EDTA (pH 6.8) at 68°C. This HPV-12 genome (Fig. 8). Reciprocally, no crosscorresponds to an effective hybridization tem- hybridization was detected between the HPV-8 perature of 25 to 27°C lower than the Tm of the or HPV-12 DNA probe and the HPV-3a DNA. different HPV DNAs as deduced from the The HPV-3a DNA probe hybridized with Schildkraut-Lifson equation (38), taking into ac- Hindll fragments A, B, and C of HPV-10 DNA count the G+C contents of the different HPV and, to a lesser degree, with Hindll fragment B DNAs (Table 2). Cross-hybridizations of about of HPV-2 DNA, but did not hybridize with the 33% between the HPV-3a and HPV-10 DNAs prototypes of types 1, 4, 5, 7, and 9. Conversely, and of 15.7 to 29.3% among the genomes of there was an appreciable degree of hybridization -W4
IV 3a S
4c
Bgl I
-l _& 5 _
(33*)
cm ,
c~ @_ a)
I 11%
0
A
Bgl I
10
:ir 100 cl
HPV 10 FIG. 7. Evidence for restriction sites common to HPV-3a and HPV-10 DNAs. The HPV-3a and HPV-10 cleavage maps were aligned, taking as origins the BglI site mapped at 33.6% from the HindIlI site on the HPV-3a DNA (Fig. 2) and the unique BglI site of HPV-10 DNA (Fig. 4). Cleavage sites differing by less than 1 map unit in their localization were considered as conserved.
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TABLE 3. DNA sequence homology among HPV-la, HPV-3a, HPV-5, HPV-8, HPV-9, HPV-10, and HPV12 as determined by cross-hybridization in liquid phase" % Hybridization with 32P-labeled HPV DNAs Unlabeled DNA
HPV-la HPV-3a HPV-5 HPV-8 HPV-9 HPV-10 HPV-12
HPV-la 100 (100) 0.7 (0.3) 1.1 (2.9) 2.2 (1.6) 1.4 (0.7) ND 1.9 (4.1)
HPV-3a 0.1 (0.6) 100 (100) 0.2 (1.3) 1.1 (0.9) 0.4 (1.2) 32.3 (36.9)
HPV-5 0.3 (3.6) 1.8 (1.3) 100 (100) 15.7 (23.7) 3.1 (4.9) ND 19.8 (31.6)
HPV-8 2.1 (0.8) 3.3 (5.4) 16.8 (22.7) 100 (100) 1.8 (0.8) ND
HPV-9 1.0 (2.8) 3.4 (4.1) 2.5 (4.0) 1.8 (3.2) 100 (100) ND
HPV-10 0.1 (1.9) 34.2 (37.2) 0.5 (1.6) 0.1 (0.9) 0.3 (0.5) 100 (100)
HPV-12 0.4 (1.5) 2.8 (6.7) 17.3 (29.7) 28.0 (30.8) 0.1 (1.9) ND
0.1 (2.0) 23.0 (28.4) 0.5 (3.9) 0.1 (0.8) 100 (100) 32P-labeled HPV DNAs (2,000 cpm) were annealed with either calf thymus DNA (25 ,ug) or unlabeled HPV DNAs (0.25 p.g) as described in the text. The specific activities of HPV-la, HPV-3a, HPV-5, HPV-8, HPV-9, HPV-10, and HPV-12 DNAs were 1.3 x 108, 9.0 x 107, 8.1 x 107, 7.9 x 107, 8.8 x 107, 2 x 108, and 5.3 x 107 cpm/jxg, respectively. The percentage of hybridization was determined by measuring the nuclease Si-resistant fractions or by batchwise chromatography on hydroxylapatite (values shown within parentheses) (20, 41). The numbers represent the values corrected for self-annealing of the probes (4 to 15%) and normalized to 100% for the homologous hybridization (75 to 95%). ND, Not determined. a
between the HPV-10 DNA probe and all of the HPV-3a DNA fragments, a very weak degree of hybridization with the Hindll A and B fragments of the HPV-2 DNA, and no hybridization with the other HPV DNAs (data not shown). The HPV-8 and HPV-12 DNA probes cross-hybridized, to variable extents, with virtually all HPV12 and HPV-8 fragments, respectively. These probes hybridized to a variable extent with PvuII-EcoRI (34.4 to 81.7 map units), HindIllPvuII (6.6 to 34.4 map units), and PvuII-HindIII (90.7 to 100 map units) fragments of the HPV-5 DNA (20) and very weakly with the Hindlll B fragment (55.5 to 91.2 map units) of the HPV-9 DNA (20) (Fig. 8). No cross-hybridization was detected between the prototype DNAs of types 1, 2, 4, and 7 with either the HPV-8 or HPV-12 DNA probe, whereas a very weak cross-hybridization previously had been reported between the HPV-5 DNA probe and some fragments of HPV-la and HPV-4 DNAs immobilized on diazotized paper (20). DNA blot hybridization experiments were performed to detect possible nucleotide sequence homologies between the genomes of EV HPVs and the genomes of HPVs associated with mucocutaneous or mucous membrane lesions (HPV-6b and HPV-1la) (8, 10, 25). The cleaved HPV-3a, HPV-5, HPV-8, HPV-9, HPV-10, and HPV-12 DNAs and the fragments generated by cleavage of HPV-6b and HPV-lla DNAs with mixtures of BamHI-EcoRI-PstI or BamHIHindll endonucleases, respectively, were hybridized with 32P-labeled HPV-6b and HPV-lla DNAs. No sequence homology was detected between the skin HPV DNAs and the HPV-6b DNA probe (Fig. 9) and HPV-lla DNA probe (data not shown), whereas cross-hybridization was observed between the genomes of HPV-6b and HPV-lla as expected (10).
DISCUSSION The biochemical characterization of the cloned DNAs of HPV-3a, HPV-8, HPV-10, and HPV-12 presented in this paper, as well as that of the HPV-5 and HPV-9 DNAs previously reported (20), shows that EV is associated with at least six types of viruses. These six types can be divided into three groups on the basis of an absence of homology or a very weak homology among the genomes of viruses belonging to different groups and of homology lower than 50% (4) among the DNAs of the prototypes of the types belonging to the same group. HPV-3a and HPV-10 belong to the former group. The genomes of these viruses present about 35% homology, similar G+C contents, and conserved restriction sites which permit the alignment of their physical maps. A very weak, but reproducible, cross-hybridization has been observed by blot hybridization between these viral DNAs and the prototype DNA of type 2, which is associated with common warts. This cross-hybridization has been estimated at 2 to 3% by experiments of hybridization in liquid medium at saturation (Favre et al., in preparation). Types 3 and 10 induce flat warts in certain patients suffering from EV and in the general population. None of the seven patients suffering from EV caused solely by HPV-3a or HPV-10, which we have previously studied, was found to carry a carcinoma (27, 29, 31). However, a virus very close to HPV-10 by its degree of homology with HPV-3a DNA and by a similar restriction endonuclease cleavage pattern has recently been identified by Green et al. (13) in benign lesions and in an in situ carcinoma of a patient suffering from EV. In addition, DNA sequences of a virus related to HPV-3 have been found in genital tumors of four patients (13).
HPVs IN EV
VOL. 48, 1983
1
2
(B)
ft>PV type
(A) 3i
4
5
-F 8
9
349
32P Iab e1ir HP>V ';D: ,DNA;
12
1~~~I0
a~~~~~~~~~~~~~~~. 1.. 3
.l
1.1O
(D)
(C)
.*1
I I
'I
FIG. 8. DNA sequence homology among HPV genomes as indicated by blot hybridization experiments. (A) HPV DNAs were cleaved with Hindll and HindIII endonucleases for HPV-la DNA (lane a); Hindll for HPV-2 DNA (lane b); Hindll for cloned HindIII/HPV-3a (lane c) DNA; BamHI, HindIII, and HpaI for HPV-4 DNA (lane d); EcoRI and PvuII for an equimolar mixture of HindlI A and B/HPV-5 cloned DNA fragments (lane e); HpaI for HPV-7 DNA (lane f); PstI and SmaI for cloned BamHI/HPV-8 DNA (lane g); EcoRI and HindlIl for cloned BamHI/HPV-9 DNA (lane h); HindII for an equimolar mixture of Sall A and B/HPV-10 cloned DNA fragments (lane i); and PstI for cloned HindIII/HPV-12 DNA (lane j). The cleavage products were electrophoresed at 95 mA for 4 h in vertical 1.2% agarose slab gels. The DNA fragments were denatured, transferred to a Gene Screen hybridization transfer membrane, and hybridized with 32P-labeled HPV-3a DNA (specific activity, 9.0 x 107 cpm/4Lg) (B), HPV-8 DNA (specific activity, 7.9 x 107 cpm/Lg) (C), or HPV-12 DNA (specific activity, 5.3 x 107 cpm/,ug) (D). The hybrids were detected as described in the text. The molecular weights (x 106), indicated on the left of the lanes, are in agreement with previous data for HPV-la, HPV-2, HPV-4, HPV-5, HPV7, HPV-9, and HPV-10 (15, 20, 27; Favre et al., in preparation).
The second group includes HPV-5, HPV-8, and HPV-12, whose DNAs present homologies from 16 to 28%, with the homologous regions being distributed virtually throughout the whole genome. Three isolates of HPV-5 and two isolates of HPV-8 have been described. Differences in the cleavage patterns by certain restriction endonucleases were demonstrated for the HPV5 isolates (20, 32, 34), and two distinct PstI cleavage patterns were similarly observed in the HPV-8 isolate characterized in the present study and in the HPV-8 isolate from an African patient (36). These two isolates have in common the 11 sites generated by four endonucleases (BamHI,
EcoRI, HindII, and HpaI) and are undistinguishable in experiments of blot hybridization; they should be considered as variant strains rather than subtypes. HPV-5, HPV-8, and HPV-12 have not been found in the general population. They are associated with reddish macules and pityriasis versicolor-like pigmented or achromic macules characteristic of EV and mostly localized on the trunk and induce flat wart-like lesions, more or less prominent, on hands, knees, and feet. The genomes of two viruses belonging to this group, HPV-5 and HPV-8, have been detected in carcinomas of patients suffering from EV (27, 32, 34; Orth et al., manuscript in prepa-
350
KREMSDORF ET AL.
J. VIROL.
mechanisms of DNA repair, by analogy with what is observed in xeroderma pigmentosum (14, 21). So far this hypothesis has been supported only by the observations of a disordered DNA synthesis in the UV-irradiated leukocytes and fibroblasts of two patients (14, 19) and of the survival of UV-irradiated herpesvirus in the irradiated fibroblasts of one of these patients (19). The multiplicity of viruses specifically associated with this rare disease is, therefore, surprising, especially since recent data obtained by our group and by other groups (34, 42; Orth et al., in preparation) indicate the existence of additional HPVs specifically associated with EV and related to either HPV-5, HPV8, and HPV-12 or to HPV-9, as well as the presence of variant strains of most of these types. The comparison of the polynucleotide sequences of HPV-la and bovine papillomavirus type 1 has suggested that the diversification of the papillomavirus genomes is probably due to two processes: an accumulation of point mutations and an exchange of genetic material between viruses and hosts or between different viruses (5). Whether a human gene defect(s) possibly involved in EV plays a role in the diversification of EV-associated viruses remains a matter of speculation. cerns
..
.,Jo
50
: ;t
1.140
:~ i
l
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FIG. 9. DNA sequence homology between EV HPV DNAs and HPV-6b DNA as checked by blot hybridization experiments. HPV-6b DNA (lanes a and b) was cleaved by a mixture of BarnHI, EcoRI. and Pstl, whereas HPV-1la DNA (lanes c and d) was cleaved by a mixture of BaimHI and Hindul. HPV-3a (lane e), HPV-5 (lane f), HPV-8 (lane g). HPV-9 (lane h), HPV-10 (lane i), and HPV-12 (lane j) DNAs were cleaved with the same endonucleases indicated in the legend to Fig. 8. After electrophoresis of the cleavage products and staining with ethidium bromide as illustrated for HPV-6b (a) and HPV-lla (c) DNAs. the fragments were denatured and transferred to a Gene Screen hybridization transfer membrane. The fragments were hybridized with 32P-labeled HPV-6b DNA (specific activity, 108 cpm/4g), and the hybrids were detected as described in the text. The molecular weights ( x 106), indicated on the left of the lanes, are in agreement with the data reported by Gissmann et al. (10) and De Villiers et al. (8).
ration). HPV-12, which
was
isolated in
a
homo-
geneous state from the lesions of a patient presenting cancers, may also have an oncogenic
potential. The third group so far consists of a single virus, HPV-9, whose genome is smaller than the genomes of the other viruses (7,200 as compared with about 7,700 base pairs). HPV-9 is associated with macular lesions and flat wart-like lesions and has not been found in the general population. EV is a rare disease. The existence of family cases, the high frequency of consanguinity in EV families, and the association of EV with mental retardation in about 8% of the patients have led to the idea that this disease is under the control of one or more rare, recessive, autosomal genes (22). The nature of the gene defect(s) remains unknown. The preferential localization of cancers in regions exposed to the sun has led to the hypothesis that the gene defect(s) con-
ACKNOWLEDGMENTS We acknowledge the help and the facilities provided by G. Riou for the determination of G+C contents of HPV DNAs. We are indebted to H. zur Hausen and L. Gissmann for their kind gift of cloned HPV-6b and HPV-lla DNAs. We are grateful to 0. Croissant for many fruitful discussions and to Y. T. Lanni for critical reading of the manuscript. We thank B. Rubat du M rac for careful typing of the manuscript. This investigation was supported by grant P.R.C. convention no. 134-030 from the Institut National de la Sante et de la Recherche M dicale.
LITERATURE CITED 1. Bachmann, B. J. 1972. Pedigree of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 44:1-56. 2. Belozersky, A., and A. S. Spirin. 1960. Chemistry of the nucleic acids of microorganisms. Nucleic Acids 3:147186. 3. Blanchet-Bardon, C., A. Puissant, M. Lutzner, G. Orth, M. T. Nutini, and P. Guesry. 1981. Interferon treatment of skin cancer in patients with epidermodysplasia verruciformis. Lancet i:274. 4. Coggin, J. R., Jr., and H. zur Hausen. 1979. Workshop on papillomaviruses and cancer. Cancer Res. 39:545-546. 5. Danos, O., L. W. Engel, E. Y. Chen, M. Yaniv, and P. M. Howley. 1983. Comparative analysis of the human type la and bovine type 1 papillomavirus genomes. J. Virol. 46:557-566. 6. Danos, O., M. Katinka, and M. Yaniv. 1982. Human papillomavirus la complete DNA sequence: a novel type of genome organization among Papovaviridae. EMBO J. 1:231-236. 7. Denhardt, D. T. 1966. A membrane filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23:641-646. 8. De Villiers, E. M., L. Gissmann, and H. zur Hausen. 1981. Molecular cloning of viral DNA from human genital warts. J. Virol. 40:932-935.
HPVs IN EV
VOL. 48. 1983 9. Favre, M., G. Orth, 0. Croissant, and M. Yaniv. 1977. Human papillomavirus DNA: physical mapping of the cleavage sites of Bacillus amnvloliquefaciens (Batnl) and Haemophilus parainfluenzae (HpaII) endonucleases and evidence for partial heterogeneity. J. Virol. 21:1210-1214. 10. Gissmann, L., V. Diehl, H. Schultz-Coulon, and H. zur Hausen. 1982. Molecular cloning and characterization of human papilloma virus DNA derived from laryngeal papilloma. J. Virol. 44:393-400. 11. Gissmann, L., H. Pfister, and H. zur Hausen. 1977. Human papilloma viruses (HPV): characterization of four different isolates. Virology 76:569-580. 12. Gissmann, L., and H. zur Hausen. 1978. Physical characterization of deoxyribonucleic acids of different human papilloma viruses (HPV). Med. Microbiol. Immunol. 166:3-11. 13. Green, M., K. H. Brackmann, P. R. Sanders, P. M. Loewenstein, J. H. Freel, M. Eisinger, and S. A. Switlyk. 1982. Isolation of a human papillomavirus from a patient with epidermodysplasia verruciformis: presence of related viral DNA genomes in human urogenital tumors. Proc. Natl. Acad. Sci. U.S.A. 79:4437-4441. 14. Hammar, H., L. Hammar, B. Lambert, and U. Ringborg. 1976. A case report including EM and DNA repair investigations in dermatosis associated with multiple skin cancers: epidermodysplasia verruciformis. Acta Med. Scand. 200:441-446. 15. Heilman, C. A., M. F. Law, M. A. Israel, and P. M. Howley. 1980. Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences. J. Virol. 36:395-407. 16. Hyman, R. W., I. Brunovskis, and W. C. Summers. 1973. DNA base sequence homology between coliphages T7 and 4 II and between T3 and 4 II as determined by heteroduplex mapping in the electron microscope. J. Mol. Biol. 77:189-1%. 17. Jablonska, S., J. Dabrowski, and K. Jakubowicz. 1972. Epidermodysplasia verruciformis as a model in studies on the role of papovaviruses in oncogenesis. Cancer Res. 32:583-589. 18. Jablonska, S., G. Orth, M. Jarzabek-Chorzelska, G. Rzesa, S. Obalek, W. Glinski, M. Favre, and 0. Croissant. 1979. Epidermodysplasia verruciformis versus disseminated verrucae planae: is epidermodysplasia verruciformis a generalized infection with wart virus? J. Invest. Dermatol. 72:114-119. 19. Kienzler, J. L., R. Laurent, J. Coppey, M. Favre, G. Orth, L. Coupez, and P. Agache. 1979. Epidermodysplasie verruciforme. Donnees ultrastructurales, virologiques et photobiologiques: a propos d'une observation. Ann. Dermatol. Venereol. 106:549-563. 20. Kremsdorf, D., S. Jablonska, M. Favre, and G. Orth. 1982. Biochemical characterization of two types of human papillomaviruses specifically associated with epidermodysplasia verruciformis. J. Virol. 43:436-447. 21. Lehmann, A. R., and P. Karran. 1981. DNA repair. Int. Rev. Cytol. 72:101-146. 22. Lutzner, M. A. 1978. Epidermodysplasia verruciformis: an autosomal recessive disease characterized by viral warts and skin cancer. A model for viral oncogenesis. Bull. Cancer 65:169-182. 23. Mandel, M., C. L. Schildkraut, and J. Marmur. 1968. Use of CsCl density gradient analysis for determining the guanine plus cytosine content of DNA. Methods Enzymol. 12:184-195. 24. McConaughy, B. L., C. D. Laird, and B. J. McCarthy. 1969. Nucleic acid reassociation in formamide. Biochemistry 8:3289-3295. 25. Mounts, P., K. V. Shah, and H. Kashima. 1982. Viral etiology of juvenile- and adult-onset squamous papilloma of the larynx. Proc. Natl. Acad. Sci. U.S.A. 79:54255429. 26. Murray, K., and N. E. Murray. 1975. Phage lambda receptor chromosomes for DNA fragments made with
27.
28. 29.
30.
31.
32.
33. 34.
35.
36.
37. 38. 39. 40.
41.
42.
351
restriction endonuclease III of Haetnoplhilis in lienzae and restriction endonuclease I of Escherichia coli. J. Mol. Biol. 98:551-564. Orth, G., M. Favre, F. Breitburd, 0. Croissant, S. Jablonska, S. Obalek, M. Jarzabek-Chorzelska, and G. Rzesa. 1980. Epidermodysplasia verruciformis: a model for the role of papillomaviruses in human cancer. p. 259-282. In M. Essex, G. Todaro, and H. zur Hausen (ed.), Viruses in naturally occurring cancers. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Orth, G., M. Favre, and 0. Croissant. 1977. Characterization of a new type of human papillomavirus that causes skin warts. J. Virol. 24:108-120. Orth, G., S. Jablonska, M. Favre, 0. Croissant, M. Jarzabek-Chorzelska, and G. Rzesa. 1978. Characterization of two types of human papillomaviruses in lesions of epidermodysplasia verruciformis. Proc. Natl. Acad. Sci. U.S.A. 75:1537-1541. Orth, G., S. Jablonska, M. Favre, 0. Croissant, S. Obalek, M. Jarzabek-Chorzelska, and N. Jibard. 1981. Identification of papillomaviruses in butchers' warts. J. Invest. Dermatol. 76:97-102. Orth, G., S. Jablonska, M. Jarzabek-Chorzelska, G. Rzesa, S. Obalek, M. Favre, and 0. Croissant. 1979. Characteristics of the lesions and risk of malignant conversion as related to the type of the human papillomavirus involved in epidermodysplasia verruciformis. Cancer Res. 39:1074-1082. Ostrow, R. S., M. Bender, M. Niimura, T. Seki, M. Kawashima, F. Pass, and A. J. Faras. 1982. Human papillomavirus DNA in cutaneous primary and metastasized squamous cell carcinoma from patients with epidermodysplasia verruciformis. Proc. Natl. Acad. Sci. U.S.A. 79:1634-1638. Ostrow, R. S., R. Krzyzek, F. Pass, and A. J. Faras. 1981. Identification of a novel human papilloma virus in cutaneous warts of meat handlers. Virology 108:21-27. Pfister, H., A. Gassenmaier, F. Nurnberger, and G. Stuttgen. 1983. Human papilloma virus 5-DNA in carcinoma of an epidermodysplasia verruciformis patient infected with various human papillomavirus types. Cancer Res. 43:1436-1441. Pfister, H., L. Gissmann, H. zur Hausen, and G. Gross. 1980. Characterization of human and bovine papilloma viruses and of the humoral immune response to papilloma virus infection, p. 249-258. In M. Essex, G. Todaro, and H. zur Hausen (ed.), Viruses in naturally occurring cancers. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Pfister, H., F. Nurnberger, L. Gissmann, and H. zur Hausen. 1981. Characterization of a human papillomavirus from epidermodysplasia verruciformis lesions of a patient from Upper Volta. Int. J. Cancer 27:645-650. Roberts, R. J. 1980. Restriction and modification enzymes and their recognition sequences. Nucleic Acids Res. 8:6380. Schildkraut, C., and S. Lifson. 1965. Dependence of the melting temperature of DNA on salt concentration. Biopolymers 3:195-208. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Sutcliffe, J. G. 1978. pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long. Nucleic Acids Res. 5:27212728. Wold, W. S. M., M. Green, and J. K. Mackey. 1978. Methods and rationale for analysis of human tumors for nucleic acid sequences of oncogenic human DNA viruses. Methods Cancer Res. 15:69-161. Yutsudo, M., T. Tanigaki, T. Tsumori, S. Watanabe, and A. Hakura. 1982. New human papilloma virus isolated from epidermodysplasia verruciformis lesions. Cancer Res. 42:2440-2443.
