MARIE-CHRISTINE DEAU,1 MICHEL FAVRE,1 STEFANIA JABLONSKA,2. LUIS-ALFREDO RUEDA,3 ADGDERARD ORTHl*. Unite des Papillomavirus, Unite ...
Vol. 31, No. 11
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1993, p. 2918-2926
0095-1137/93/112918-09$02.00/0 Copyright © 1993, American Society for Microbiology
Genetic Heterogeneity of Oncogenic Human Papillomavirus Type 5 (HPV5) and Phylogeny of HPV5 Variants Associated with Epidermodysplasia Verruciformis MARIE-CHRISTINE DEAU,1 MICHEL FAVRE,1 STEFANIA JABLONSKA,2 LUIS-ALFREDO RUEDA,3 ADGDERARD ORTHl* Unite des Papillomavirus, Unite de l'Institut National de la Sante et de la Recherche Medicale 190, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France'; Department of Dermatology, Warsaw School of Medicine, Warsaw, Poland2; and Centro Dermatologico F. Lleras A., Bogota, Colombia3 Received 8 March 1993/Returned for modification 27 April 1993/Accepted 3 August 1993
Variants of oncogenic human papiliomavirus type 5 (HPV5), specifically associated with epidermodysplasia verruciformis, were recognized on the basis of the genetic heterogeneity of the E6 open reading frame (ORF). To further evaluate the genetic heterogeneity of HPV5, we sequenced the long control region (LCR), the E7 ORF, and the terminal parts of the E2 ORF of five previously characterized HPV5 variants and compared the data with the published HPV5al and HPV5b sequences. Alignment of the variants showed 140 (7.6%) variable nucleotides of 1,854 sequenced. Nucleotide substitution rates varied from 3.6% in the E7 ORF to 11% in the E6 ORF. By sequencing the variable region encompassing the LCR 3' part and the E6 ORF of isolates from six additional epidermodysplasia verruciformis patients, we identified three new variants and three already known variants, indicating the stability of HPV5 variants. This stability was further demonstrated by the identity of isolates obtained years later from benign and malignant lesions of three patients. Phylogenetic analysis of the 10 HPV5 variants distributed them into three groups, tentatively defining subtypes a, b, and c. The phylogenetic grouping shows no geographical dependence, a fact that may be related to the host restriction that characterizes HPV5 infections. No differences in the enhancer potential of the LCR or in the transactivating properties of the E2 protein assayed in vitro were observed among HPV5 variants. Whether HPV5 variants possess distinct biological properties in vivo remains to be determined. a maximal sequence divergence of 8.7%. In the E6 gene of three HPV8 variants, 3.2% variable nucleotide positions, corresponding to 4.5% amino acid changes, have been identified (7, 11). Furthermore, the same HPV5 or HPV8 E6 sequences were determined for isolates from benign or malignant lesions of patients (7). In view of the small number of HPVS and HPV8 isolates characterized so far, because of the rarity of EV patients, it is striking that the variability observed for oncogenic EV HPVs is as extensive as that described for the widespread HPV16 variants. Because EV is a rare experiment of nature (27), the potentially oncogenic EV HPVs should be a useful model for understanding the variability and evolution of HPVs. Our first aim was to further analyze five HPVS variants cloned by our group (7, 19) by sequencing genomic regions other than the E6 ORF, namely, the LCR, the E7 ORF, encoding a putative oncoprotein, and the 5' and 3' domains of the E2 ORF, encoding the functional domains of a transregulating protein. In an attempt to further define the genetic variability of HPVS, we characterized isolates from six additional patients of different geographical origins by sequencing the variable region encompassing the 3' end of the LCR and the E6 ORF. We addressed the question of the stability of HPVS variants by comparing isolates taken from benign and malignant lesions of four patients at various intervals of time (up to 12 years). We established a phylogenetic classification of the 10 HPVS variants identified so far, as related to their geographical origin, to obtain some insight into the evolution of HPVS. Finally, to determine whether the genetic variability of HPVS affects its biological properties, we assayed in vitro the enhancer activity of the
Human papillomaviruses (HPVs) constitute a large group of DNA viruses that are associated with benign and malignant proliferations of squamous epithelia. At least 66 types have been identified so far (38) on the basis of less than 50% cross-hybridization under most stringent hybridization conditions (5), while subtypes share greater than 50% crosshybridization (5, 18). In addition to this great multiplicity of types and subtypes, the existence of stable DNA sequence variants has been disclosed for oncogenic HPV type 16 (HPV16) (3, 9, 15), the most frequently encountered HPV in anogenital cancers (46), and for the HPVS and HPV8 (7, 44), associated with skin cancers of epidermodysplasia verruciformis (EV), a rare disease characterized by a genetically determined, abnormal susceptibility to these viruses (27). The sequences of a 364-bp fragment of the long control region (LCR) of 118 HPV16 isolates revealed 38 molecular variants with 11.5% variable nucleotide positions and a 5% maximal divergence (3). Phylogenetic trees disclosed Eurasian and African evolutionary lineages (3). The complete nucleotide sequences of prototypical HPV5a1 (45) and a variant designated HPV5b (44) revealed a nucleotide divergence of 5% in the LCR and amino acid changes of 1.8 to 6% in the viral proteins (44). A comparison of four HPVS variants obtained from three EV patients (7) with prototypical HPVS (45) disclosed 10.4% variable nucleotide positions in the E6 open reading frame (ORF), corresponding to 10.4% amino acid substitutions in the E6 putative oncoprotein, and
*
Corresponding author. 2918
PHYLOGENY OF HPV5 VARIANTS
VOL. 31, 1993
2919
TABLE 1. Origin and classification of HPV5 isolates Classification Patient
Nationality
Polish
1
3
Dutch Algerian
4
Colombian
5c 6
Colombian French
7
Polish
8
Polish
2
9
l0d
Algerian Polish
Sequenced regionsb
Lesions
Benign Malignant Benign Benign Metastatic Benign Malignant Benign Benign Malignant Benign Malignant Benign Benign Benign
Chronologica
Phylogenetic
5a2 5a2
5a2 5a2
3' LCR, E6
5a2
5a2
3' LCR, E6
5a3 5a3
5b3 5b3
LCR, E6, E7, E2 LCR, E6, E7, E2
5a4 5a4
5b2 5b2
5a4
5b2
LCR, E6, E7, E2 LCR, E6, E7, E2 LCR, E6, E7, E2
5a5 5a6
5a3 5cl
5a7 5a7
5a4 5a4
5a8 5a9 5b
Sa5 5b4
5bl
LCR, E6, E7, E2
LCR, E6, E7, E2 LCR, E6, E7, E2 3' LCR, E6 3' LCR, E6 3' LCR, E6 3' LCR, E6 3' LCR, E6
a By comparison with prototypical HPV5al and HPV5b, both cloned from Japanese patients (44, 45). b Only the 5'- and 3-terminal 300 nt of the E2 ORF were determined. c Sister of patient 4. d Sister of patient 8.
LCR and the transactivating properties of the E2 proteins of different HPVS variants.
MATERIALS AND METHODS Tissue specimens. Scrapings were taken from skin flat warts and macules of 10 EV patients originating from Europe, North Africa, and South America (Table 1). Biopsies were obtained from cancers of five of these patients (patients 1, 3, 4, 5, and 7), i.e., an in situ squamous cell carcinoma of the forehead (patient 5), invasive squamous cell carcinomas located on the forehead (patients 1 and 4) or in the retroauricular region (patient 7), and a cervical lymph node metastatic tumor of an invasive carcinoma of the forehead (patient 3) (Table 1). Samples from benign lesions and cancers were obtained on the same day (patients 4 and 5) or at time intervals of 2 years (patient 7), 3 years (patient 3), and 12 years (patient 1). Case reports have been described elsewhere for patients 1 and 8 (19), patients 2, 4, and 7 (patients 5, 6, and 1, respectively, in reference 18), patients 3 and 9 (patients 2 and 1, respectively, in reference 23), patient 6 (patient 3 in reference 21), and patient 10 (28). Patients 3, 4, and 6 corresponded to patients A, B, and C respectively, in reference 7. HPV5 isolates. The molecular cloning of HPV5a2, HPV5a3, HPV5a4, HPV5a5, and HPV5a6 variants from benign or malignant lesions of four HPV5-infected EV patients has been reported elsewhere (7, 19). Prototypical HPV5al (45) was kindly provided by R. Ostrow (University of Minnesota, Minneapolis). The fragments containing the 3' part of the LCR and the E6 ORF (nucleotides [nt] 7716 to 690) of isolates from patients 1 and 2 (5a2), patient 5 (5a4), patient 7 (5a7), patient 8 (5a8), patient 9 (5a9), and patient 10 (Sb) were amplified by the polymerase chain reaction (PCR) technique (33) with primers located between nt 7716 to 7735 and nt 690 to 674. PCR reaction mixtures contained 50 to 100
ng of DNA, 50 mM KCl, 10 mM Tris HCl (pH 8.3), 100 ,uM each deoxynucleoside triphosphate, 1.5 mM MgCl2, 25 pmol of each primer, and 2 U of thermostable Thernus aquaticus (Taq) DNA polymerase (Perkin-Elmer Cetus Instruments, Emeryville, Calif.). Mixtures were incubated for 5 min at 94°C for DNA denaturation. Subsequently, 30 cycles of amplification were performed with a PCR processor (Hybaid, Ltd). Each cycle included a denaturation step at 92°C for 30 s, an annealing step at 55°C for 1 min, and a chain elongation step at 72°C for 2 min. The final elongation step was prolonged for another 7 min. After end filling with the Klenow fragment of DNA polymerase I (34), the fragments were inserted by blunt ligation at the unique SmaI site of a
pBluescript plasmid (Stratagene). DNA sequencing. The nucleotide sequences of the LCR, the E7 ORF, and the 5' and 3' parts of the E2 ORF of HPV5a2 to HPV5a6 variants were determined with the oligonucleotide primers described in Table 2. The nucleotide sequence of the E6 ORF of HPV5a2 was determined as previously described (7). The nucleotide sequences of fragments corresponding to the 3' part of the LCR and the E6 ORF were determined with M13 oligonucleotide primers complementary to sequences flanking the insert and an internal E6 primer (nt 438 to 454) in both orientations. Sequencing was performed on both strands of recombinant plasmids by the dideoxy chain termination method (35). Phylogenetic trees. Multiple alignments of the nucleotide sequences were performed by use of the fast method of Wilbur and Lipman (43), which compares pairs of sequences and provides a similarity score for each comparison. The similarity scores were used to construct a phylogenetic tree by unweighted pair-group maximum-average (UPGMA) analysis (37), which yields clusters of similar sequences. The UPGMA method was executed with the CLUSTAL V option (14). The results of the multiple sequence alignments were also used to construct phylogenetic trees by the max-
2920
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imum parsimony method (DNAPARS) with the PHYLIP 3.4 programs (Phylogeny Inference Package) (10). To root the trees, the HPV47 sequence (17) was used as an outgroup. Bootstrapping was used to estimate the confidence limits of tree groupings (10), which were significant at the 95% level. CAT assays. Fragments containing the LCR of HPV5al, HPV5a3, HPV5a4, and HPV5a6 (nt 7427 to 454) (45) or the E2 gene of HPV5al, HPV5a3, HPV5a5, and HPV5a6 (nt 2716 to 4283) were amplified by the PCR technique (33). The LCR PCR products were inserted in a chloramphenicol acetyltransferase (CAT) reporter plasmid (pTKM vector) 5' to the enhancerless promoter of the herpes simplex virus thymidine kinase gene (39). The E2 PCR products were inserted in the pRSV vector downstream of the Rous sarcoma virus long terminal repeat (12). The nucleotide sequences of the cloned LCR and E2 genes were verified. Constructs were transfected in human SW13 cells (20) as previously described (39), with the pRSVcat vector (12) and the pTKM vector as positive and negative controls, respectively. Transfection was performed by a calcium phosphate DNA precipitation technique (4). Two days after transfection, CAT assays were performed as described previously (32). Nucleotide sequence accession numbers. The nucleotide sequence data reported here have been deposited in EMBL data bases under accession numbers X7 4618 (HPV5a2, LCR-E6-E7), X7 4619 (HPV5a4, LCR-E6-E7), X7 4620 (HPV5a3, LCR-E6-E7), X7 4621 (HPV5a5, LCR-E6-E7), X7 4622 (HPV5a6, LCR-E6-E7), X7 4641 (HPV5a7, LCR-E6), X7 4642 (HPV5a8, LCR-E6), X7 4643 (HPV5a9, LCR-E6), X7 4644 (HPV5a2, 5' E2), X7 4645 (HPV5a2, 3' E2), X7 4646 (HPV5a3, 5' E2), X7 4647 (HPV5a3, 3' E2), X7 4648 (HPV 5a4, 5' E2), X7 4649 (HPV5a4, 3' E2), X7 4650 (HPV5a5, 5' E2), X7 4651 (HPV5a5, 3' E2), X7 4652 (HPV5a6, 5' E2), and X7 4653 (HPV5a6, 3' E2).
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RESULTS Sequence variation in different genomic regions of known HPV5 variants. We previously cloned seven HPV5 isolates from benign and malignant lesions of four EV patients (patients 1, 3, 4, and 6) and showed that they corresponded to five variants (HPV5a2 to HPV5a6) on the basis of their restriction maps (7, 19) and the nucleotide sequence of the E6 gene for HPV5a3 to HPV5a6 (7) (Table 1). We have now determined the nucleotide sequence of the HPV5a2 E6 gene, which shows six nucleotide changes in comparison with the most closely related E6 gene sequence, that of HPV5a5 (Fig. 1). Our aim was to determine to which extent this genetic heterogeneity affected other genomic regions of the HPV5 variants. We determined the nucleotide sequences of the LCR, of the E7 gene, which likely encodes an oncoprotein (24, 25), and of the 5'- and 3'-terminal 300 nt of the E2 gene, which encodes a transregulating protein (13). As a control, we sequenced the same regions of prototypical HPV5al, provided by R. Ostrow (29, 45), and found differences at five nucleotide positions in comparison with published data (45) (Fig. 2). We shall refer thereafter to this sequence as the corrected sequence. We compared the nucleotide and deduced amino acid sequences of our isolates with the sequences of HPV5al and HPVSb (44). The nucleotide substitution rates ranged from 3.6% for the E7 ORF to 11% for the E6 ORF (Table 2). Altogether, including the E6 ORF, 140 (7.6%) nt of the 1,854 nt sequenced were found variable in the HPV5 sequence (Table 2), with a maximal divergence of 3% between variants. A comparison of the LCRs of the seven HPV5 variants
1
773? Sal
2921
PHYLOGENY OF HPV5 VARIANTS
VOL. 31, 1993
?AAAGACCGTTAACGGTAAGTTGCAATTTCCTT0TACCAG4
TGCGGTATTOGGATTTCACAATTATAA TOGTTGTTGCCAACTACCATAGGCATATTCAAGTTTTTGCCTGTATC
a7 ...-.. .A.-.-------. T-.0 -AA -
a8
b
a4
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c --------G a--------------------------------------G ----******--- -- ---- TG ----- G -----------------------s----- C---- e----------------G-----------s----A---- G - [= =ss ------G --------------- C v-*-G ----------------- A--- G......................... AA -----^TaG.......... -
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a8 ... C.A
a6
.0.... G ----- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -b-. A--T----T------------*------ c --------------a- -G TO . -G--------- ------0.C. -G.A~~~...............%.-...........
105 Sal a2 aS a7 as
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E6
d'.;-' GTTTTCGTATCCTG -------------- -- -- -- -- -- --- -- -- -- -- - - --- -- -- -- -- -- - --
A.Ti*,,:,,T,#.T.*.T"'i'''''"F:.GGTTCGATAGCEEGTAGACGA
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T ---C-------- -------- T--T------- -------------- -------- G--------------------------- -- --------------T---C ----------------- T--------w------------------------ G-*- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- ---T- --C----------------- T----------------------- -------- a- ..-.-.-.-.--.-- -- -- -..- -- -- -- -- -- -- -- -- -- -- -- ---T ---C------T-------*---T----*----------------------------G-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
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b *- -- *-- -- ----- . T ---C ----------aG - ----- T -------------------------.-----------------A ----------------------- ---------T --C-----------------T------------------------A--C---------------A----------------------.----------a4 ---A----------TO -GA A,,,,,,,,-.0...---------------.......... -- --0 T----------------------------..a3 ---------A- ------------TA O.0.C.......... ................... T0-------------------------------------..a9 -------------- A------. ---0.-------------- GC--A------T-------------. a6 -------------- T-- C----A ------------TT.C -
219 Sal ACCAACAGAAACTGACAG^AvAAAAGTAAGGCAGAATTACCTTTAAGTATTAGAGACTTAGCTGAAGCCTTAGGCATCCCTGTGATTGATTGTTTAATACCTTGCAATTTCTGTGG
CA- ----------************-*--.-*-.*-.-*--.-.- T----C.------------G-- -------A----*----------C ------------------------
a2
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aS
*------------ C----- ---------------------------cA ---------------------------------- C------.------G----^----- aT CA --------------.---... ---..........--c-C-- -- ...------ -G--------- A-T ------------ C------- --------------------------.- -------.-..----T------------C ---------------- ------------ ----- -- - -- CA -- -T--------------- -- ---- --------C------------ aG-- -- -- - -- A-T
a7 a8
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C-c-C a4 -C- C a3 a9 a6 ---------------------------..........-----..--c- .c-C .---
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-G------A -------------- C-T --------- A -----C T. .A -------------A G-A-------------- C-T --------- A-
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----------------------T-------------------------- T-----
334 t iIt It t Sal CIAACTTTCTAAATTATTTGGAAGCTTGTGAATTCGACTACAAAAGGCTTAGTCTAATTTGGAAAGATTATTGTGTGTTTGCGTGCTGTCGCGTATGCTGTGGCGCCACTGCAACT a2 aS
----- ------- ---------------------------C AA------------A-------------------- --A---------------------------C ***.A-----------------------C -------- ------- AA -------------------------- ------------------------------ ------- ---
----------------------------******-*----c-CT ------------------------ --C - -- -- -- -- -- ---- -AA ------ --A- ----- -- --**-**- -- -*- -- ---cb T--A---T -------- --------------.--------- AA----- C-------.------.-------------------T------------------------------A a4 T--A --- T-- ----.---.------C ------------------- AA ----- C--.------------------------------T----------------------------- A a3 T--A---T ------------ ----C -- ------T------ ---- AA----- C----- ---------------------------T------------- ----------------
as
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a9 T. -A---T ---------------- C--------T ---------- AA----- C----------------- ---------------T---------------- ------------a6 T---- -T---- ------------- --------T ---------- -A----- C---------- G-GA ------- --- A --A-A-T- - ---- ----- --- ---- T-----------A
449 a2 aS
TATGAATTTAACCAATTTTATGAGCAGACAGTGTTAGGAAGAGATATTGAATTAGCTTCAGGACTTTCAATATTTGATATTGATATCAGGTGTCAAACTTGCTTAGCATTTCTTG ---- -- -- -- -- -- -- -- -*****- -- -- ------ T----- -C--e-------- G----- ------------G-TT------- ----------------------------------C - -- -- ---- -G--- -----------G--G--T -------------------------------------------- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --c
a7 as
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b a4 a3 a9 a6
C --G------G--G--T -------------------------------------------------------C --- -------G ---------------- G--T ------------------------------- ------ ----C -------- T------ --------------------------------G -------------- C- G--T ----------------------------------- ---------C -------- T------------- -------------------------G -------------- C--G- T --------------------------------------------C -------- T-- ---..------.-------------------------G ---.-----.-.--.-C--G--T ------------------- -------------------------C -------- T-------------------------------------- G------------ -- C--G--T --------------------------------------------C --****----- T-----..---.----G------- ---.-------------G ---- -------T.-C--T.-T -------------------------- G----------------
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Sal ACATTATAGAAAAGTTAGATTGCTTTGGCAGAGGCCTTCCCTTTCATAAGGTGAGGAACGCCTGGAAGGGAATCTGTAGGCAGTGTAAGCATTTTTATCATGATTGG aS
. . ..--------------C----. T- TA.-----------------------------------------------------------------.--T.0. a7 -----
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T--------------------A-----------
FIG. 1. Comparison of the nucleotide sequences of the 3' part of the LCR and the E6 ORF of 10 HPV5 variants. The nucleotide sequence of HPVSal (45) is given from nt 7736 to 673. Only substitutions observed in the variants are indicated. The initiation codon and the stop codon of the E6 gene are boxed. The E2-responsive elements and the M29 motif are indicated by thin lines and a thick line, respectively. The A/T-rich region, which contains TATA boxes, is shaded. The direct duplication of nt 18 to 24, present in the published sequence of HPVSb (44) (hatched box), was not found in our HPVSb isolate. Nucleotide changes leading to amino acid changes in the E6 amino acid sequence deduced from the nucleotide sequence are shown by arrowheads. The three pairs of overlined arrowheads correspond to a single codon.
2922
DEAU ET AL.
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amino-terminal transactivation domain, a proline-rich "hinge" region, and a carboxy-terminal domain required for sequencespecific DNA binding and dimer formation (13). In the 5' end of E2, 14 of 300 nt positions were found variable (Table 2), and 4 of them resulted in amino acid changes. In the 3' end of E2, 22 of 300 nt positions were found variable, leading to six amino acid changes (Table 2). Only the Asp-13--3Glu and Thr-487-*Ser substitutions correspond to conservative
T
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.....Search for new HPV5 variants. To further define the genetic heterogeneity of HPV5 and/or to evaluate the stability of the known variants, we sequenced the most variable genomic region, encompassing the 3' part of the LCR and t.... T the E6 ORF (nt 7736 to 673), of HPV5 isolates obtained from A A six additional EV patients of different geographical origins T14 0 A (patients 2, 5, 7, 8, 9, and 10) (Table 1). A comparison of the C nucleotide sequences (Fig. 1) showed that three isolates A ......corresponded to new variants, referred to as 5a7, 5a8, and 5a9 (Table 1). In contrast, the other three isolates correG4058->T /T c sponded to variants already described, 5a2 (patient 2), 5a4 T4 0 07 5 .->C \ A4056->C C (patient 5), and Sb (patient 10) (Table 1 and Fig. 2), demonA4055->1 A strating further the stability of the HPV5 variants. It should A. be stressed that the short direct duplication reported for the LCR (44) was not found in our HPV5b isolate (Fig. AAT .....HPVSb T 1). As for the three new variants, five additional variable FIG. 2. Genetic heterogeneity of HPV5. Con positions (three in the 3' part of the LCR and two in the E6 of corrected HPV5al and HPV5a3 nucleotide sequences The nucleotide ORF) were found in the 684-nt variable fragment. On the sequences of HPV5al and HPVSa3 in the LCR (A) and in the 3' end whole, when the 10 variants were compared, 28 (13.3%) and of E2 (B) were determined as described in Materiails and Methods. 54 (11.4%) variable positions were identified in the 3' part of HPV5al nucleotides differing from the published sequence of the the LCR (211 nt, including the T insertion found in all same clone (45) are shown on the left. The nucleoti ides indicated by variants but 5al) and in the E6 gene (473 nt), respectively, a letter and a three- or four-digit number correspornd to the original with a maximal divergence of 8.8% between variants (5a6 sequence of Zachow et al. (45). The homologous versus 5a7 or 5a8). A comparison of the amino acid seotides are given on the right as an example of se quencevariation quences of the E6 proteins, deduced from the nucleotide among HPV5 variants. The additional T between p) ositions 118 and 119 (asterisk) and the changes from T to C at positi(on 121 (plus) and sequences, revealed 20 amino acid substitutions located at C to T at position 140 (plus), common to HPV5a2 to HPV5a6 and 19 (12.1%) positions of 157. Eleven changes were nonconHPV5b, are indicated. servative: Gly-4-+Glu, Gln-9--Pro, Leu-19--Phe, Leu21--Ser, Asp-25-+Gly, Asn-46--Lys, Asp-67--+Gly, Cys73--Arg, Gln-92--+Arg, Leu-134--Tyr, and His-1SS-*Asn disclosed 41 variable nucleotide positions of1 482 (Tables 2 (31). Pairwise comparisons of the E6 amino acid sequences, and 3), with a maximal divergence of 5.2% bettween variants. deduced from the nucleotide sequences, showed that the A T insertion at position 119 was found in all variants, in putative E6 proteins differed by 1 (Sa5 versus 5a8) to 16 (5a6 comparison with prototypical HPVSa1 (Fig. 2A). Furtherversus 5a8) amino acids. Identical E6 amino acid sequences more, the HPVSb LCR shows a short direct duplication (nt were found for the 5a3, 5a4, 5a9, and Sb variants and for the 18 to 24) (44). The genetic heterogeneity is mi ainly located in SaS and 5a7 variants. These results thus allowed us to the 3' part of the LCR, between nt 7736 aind 199, with a distinguish six alleles of the HPVS E6 gene. substitution rate of 11.9%, close to that (11%) of the adjacent Stability of HPV5 variants in individual patients. To further E6 gene (Fig. 1). The modifications affect ne -ither the putaanalyze the stability of the variants, we compared HPV5 tive regulatory elements, such as AP1 and P consensus isolates obtained from benign and malignant lesions of four binding sites, E2-responsive elements, anId CAAT and patients, each infected with a different variant, 5a2, 5a3, 5a4, or TATA boxes (8), nor the two motifs, of 33 and 29 nt, 5a7 (Table 1). Samples were taken at the same time (5a4) or conserved among the LCRs of HPV5-related [ types (30). after 2 years (5a7), 3 years (5a3), and 12 years (5a2). A The HPVS E7 gene is 309 nt long and enco'des a protein of comparison of the LCR, E6, E7, and E2 sequences (1,854 nt) of 103 amino acids. Eleven nucleotide positioins were found two 5a3 and 5a4 isolates and of the sequences of the 3' part of variable in the HPVS E7 ORF, resulting in f our amino acid the LCR and the E6 ORF (684 nt) of two 5a2 and 5a7 isolates substitutions in the predicted amino acid sequ ence, including showed no differences between isolates of the same variant. three nonconservative changes (Asn-45--Ile Ser-49--Phe, Phylogenetic analysis of HPV5 variants. A phylogenetic and Asn-60--.His) (31) (Tables 2 and 3). Pai: rwise comparitree was generated from the alignment of the nucleotide sons revealed none (5a3 versus 5a4) to seven (5a5 versus sequences of the 3' part of the LCR and the E6 ORF of the 5a6) nucleotide changes and none (Sal versus 5a2 and 5a3 or 10 HPV5 variants by use of a distance matrix approach 5a4 versus Sb) to four (SaS versus 5a6) amin( acid changes. (UPGMA). The variants were clustered into three groups: The Cys-X-X-Cys motifs, postulated to mediaite zinc binding Sal, 5a2, 5a5, 5a7, and 5a8; 5a3, 5a4, 5a9, and Sb; and 5a6 (1), the Leu-X-Cys-X-Glu consensus bindi site of the (Fig. 3A). The same grouping was obtained by use of plOSRb protein (24), and the amino acids coinserved among maximum parsimony analysis. For seven variants (5al, 5a2, all the papillomavirus E7 proteins (6) were fo und invariable. 5a3, 5a4, 5aS, 5a6, and 5b), a similar grouping was obtained The E2 ORF encodes a multidomain prote-in with a large from the alignment of the LCR, the E6, the E7, or the E2 Tr
-
>C A
C
A
A
~--
nparison .
v ing
.
VOL. 31, 1993
PHYLOGENY OF HPV5 VARIANTS
2923
TABLE 3. Nucleotide and amino acid substitutions among HPV5 variantsa Substitution in: Region
LCR
Nucleotide
7470 7492 7527 7552 7587 7605 7613 7640 7658 7660 7669 7670 7680 7695 7721 7725 7739 11 14 16 17 19 38 40 41 47 48 54 83
al
T T T C A A A G A A G G G T G T A T A T T C T G G C A A T
118*
E7
5' E2
122 127 133 140 169 173 176 177 180 184 187 745 770 796 804 808 839 840 909 917 935 938 2761 2800 2830 2884 2929 2935 2938 2951 2971 2974 2980 2984 2987 3018
T T A C G A A T G C C T G A
G C G A C A C T T G A T T A C C G A T G C T
a2
a5
-
Amino acid
a3
a4
b
a6
C
C
C
-
_
_ G
G
G
G
_
_
G
change
~~~G A
~~~G ~~~~A
G
G G T
-_
-
C -
T
A A T
T
-
A
G A
G A
G
G
A
A
G
G
G
-
A--
_
~~~~~~~~T
T A A
-
-
-
G
A
-_
T G G
T G G.
T G G
-
-
G
C
C-
T c
T C
T C
T C
T C
T C
T
T
T
T
T
T
G
G
_
_
-A AA
-
A
-
F-28-*Y -
T
T
T
T
N-45--)
S-49--F -
C T
C T
C T
C -
-
T
T
T
-
N-60--+H
c A
G
G
n l-1 s,I
G
A G ~~C ~~C G T T A
G
_-_-
G -C
C
C
T
C
A-88--S
-
-C
C
C
G
H-89-)D
C
I-99- T
Continued on following page
2924
DEAU ET AL.
J. CLIN. MICROBIOL.
TABLE 3-Continued Region
Substitution in:
Nucleotide al
3' E2c
3979 3980 4024 4048
4051 4056 4080 4086 4108 4111 4146 4183 4188 4192 4195 4213 4216 4225 4228 4241 4242 4255
A C C A G G A T T T G C C A G G G C A G C G
a2
a5
a3
T A
-
a4
A -
A C A C
A
C
A A T
Amino acid change change
a6
A T -
A A
C
-
T A G -
b
C-445-*Y K-453-*T
C
M-455-(Kor T
-
A G
R-473--*L
G
T-487->S G
A A
A A
A A C
A -
A G -
C G T
A-505->R
a Sequence data for HPV5al were from Zachow et al. (45) and corrected in this study; those for HPV5b were from Yabe et al. (44).-, identity. b The additional T present in all sequences except in HPV5al is designated 118* to avoid changing the numbering of the prototypical HPV5al sequence (45). c nt 4055 to 4058 in E2 were found to be different in the HPV5al sequence determined in this work and in the published sequence of the same clone (45). Asn-445 (AAT) predicted from the published sequence is changed to a Cys (TGC).
nucleotide sequences (data not shown) or from that of the whole 1,854 nt sequenced (Fig. 3B). The 10 HPV5 variants were isolated from 12 patients born and living in different areas (Europe, North Africa, South America, and Asia) (7, 18, 19, 21, 23, 28, 29, 44). Strikingly, the phylogenetic grouping did not disclose any geographical dependence (Fig. 3A). LCR enhancer and E2 transactivating activities of HPV5 variants. The LCR of papillomaviruses contains promoters and tissue-specific or inducible enhancers regulating the transcription of the viral genes (30). The viral E2 gene product is a major transactivating protein (39). Our aim was to check whether the genomic variability observed in the LCR and E2 region could lead to variable levels of expression of the early genes. To address this question, the LCR of the 5al, 5a3, 5a4, or 5a6 variant was inserted into the pTKM reporter plasmid (40), 5' to the CAT gene, and the E2 gene of the Sal, 5a3, or SaS variant was cloned in the pRSV expression vector. Attempts to clone the 5a6 E2 gene were unsuccessful. In vitro transfection assays were performed with SW13 cells, derived from a human adrenocortical carcinoma (20), previously shown to be a reliable tool for analyzing papillomavirus enhancer activities (40). The constitutive enhancer activity of the LCR of HPV5 is low (1 to 3% CAT conversion), regardless of the variant. Cotransfection of HPV5 E2 expression vectors yielded only 3- to 6-fold transactivation, compared with 21- to 48-fold transactivation by the bovine papillomavirus type 1 (BPV1) E2 protein, used as a positive control (Table 4). No obvious differences were observed for different combinations of HPV5 LCR and E2 plasmids (Table 4). DISCUSSION Variants of oncogenic HPV5 have been recognized on the basis of the genetic heterogeneity of the E6 ORF (7, 44, 45). We demonstrated in this study that the genetic heterogeneity
A
5al/al (Japan) 5a2/a2 (Poland, Netherlands) 5a5/a3 (France) 5a7 / a4 (Poland) 5a8/a5 (Poland)
5b/bl (Japan, Poland) 5a4/b2 (Colombia,Colombia) Sa3/b3 (Algeria) 5a9/bb4 (Algeria) 5a6/cl (France)
B
S5al/al 5a2/a2 5aS/a3 5b/bl
92
94
96
98
Asia, a
Europe
5a3/b3 5a4/b2
Asia, Europe, b North Africa, South America
Sa6/cl
c
Europe
100
Identity ('i)
FIG. 3. Phylogenetic tree of HPV5 variants. (A) The phylogenetic tree was generated from the alignment of the nucleotide sequences of the 3' part of the LCR and the E6 ORF of 10 variants by use of the UPGMA distance matrix method (14). A proposed classification (italic characters) and the geographical origin of isolates are indicated. The HPV47 nucleotide sequence (17) was used as an outgroup (data not shown). (B) The phylogenetic tree was obtained from the alignment of the 1,854 nt corresponding to the sequences of the LCR, the E6 ORF, the E7 ORF, and the 5' and 3' parts of the E2 ORF of 7 of the 10 HPV5 variants.
PHYLOGENY OF HPV5 VARIANTS
VOL. 31, 1993 TABLE 4. Transactivation of HPV5 LCR CAT plasmids by HPV5 variant E2 proteins Transactivation by the following E2 protein":
LCR CAT plasmid
HPV5al
HPV5a3
Sal 5a3 5a4 5a6
6.5 4.5 5.2 2.3
4.3
3.5
38
3.0
2.5
48
HPV5a5
BPV1
24 21 a Transactivation corresponds to the ratio of the CAT conversion observed 5.6 4.3
5.8 3.1
in the presence of a given E2 protein to that (1 to 3%) observed in the absence of an E2 protein (pBluescript control). The BPV1 E2 expression vector (40) was used as a positive control. The values correspond to the mean of two duplicate transfection experiments. Cotransfections were done as described in Materials and Methods.
found in the E6 gene also affects other regions of the genome by comparing the nucleotide sequences of the LCR and the E7 and E2 genes of five previously characterized HPV5 variants (HPV5a2 to HPV5a6) and of prototypical HPV5al (45) and HPV5b (44). On the whole, 7.6% of the 1,854 nt sequenced were found variable when the seven HPVS variants were compared. The frequency of nucleotide substitutions varied from 3.6% (E7) to 11% (E6). The apparent genetic stability of the HPV5 E7 gene as compared with the E6 gene is intriguing and could reflect some specific functional constraints. To analyze further the genetic heterogeneity of HPVS, we sequenced the most variable region, encompassing the 3' part of the LCR and the E6 gene, for isolates obtained from six additional EV patients. We identified three new variants, together with the three already known 5a2, 5a4 and 5b variants. The same variants were found in two sisters (5a4), in two patients from European countries (5a2), and in two patients from Europe and Japan (Sb) (Table 1), emphasizing that variants constitute stable entities. This result was further demonstrated by the identity of the sequenced genomic regions of isolates obtained at various intervals of time (up to 12 years) from either benign or malignant lesions of four patients infected with different variants. Point mutations in the viral genome are thus unlikely to be involved in tumor progression. Phylogenetic trees constructed from alignments of the nucleotide sequences of the 3' part of the LCR and the E6 gene, available for 10 variants, or of the LCR, the E6 and E7 ORFs, and the 5' and 3' parts of the E2 ORF, available for 7 variants, clustered HPVS variants into three groups (Fig. 3). This result allows discussion of the nomenclature of HPVs and provides some clues as to the mechanisms of evolution of HPVS. According to a recent definition of HPV genotypes, two types share less than 90% identical nucleotides in their Li, E6, and E7 nucleotide sequences (iSa). However, the distinction between subtypes and variants remains to be defined, and phylogenetic trees could provide a basis for such a classification (42). The three HPVS phylogenetic groups differ by 3.2 to 4.7% of nucleotides in their LCR, E6, E7, and E2 sequences (1,854 nt) and could correspond to subtypes designated a, b, and c (Fig. 3B). Isolates within each of the phylogenetic groups show less than 2% nucleotide divergence and could constitute variants (Fig. 3B). Interestingly, the four HPVSb variants encode the same E6 protein (Fig. 1), and at least three of them encode the same E7 protein (Table 3). Similarly, two variants of subtype a (5a3 and 5a4) encode the same E6 protein, and two others (Sal and 5a2) encode the same E7 protein. A phylogenetic study of HPV16 identified Eurasian and
2925
African lineages (3). In contrast, the phylogenetic analysis of HPVS isolates originating from different areas did not reveal any clear relationship between the grouping and the geographical origin (Fig. 3). Variants belonging to different groups (SaS and 5bl) were found in two sisters (Table 1), and two highly divergent variants (Sa5 and 5a6) were isolated from the same patient (7) (Table 1). Variants belonging to group b were found on four continents. It must be stressed that, while HPV16-associated disease is prevalent in the general population (36), only patients with a rare genetic
predisposition
are
susceptible
to
lifelong EV
disease and produce high levels of HPV5 virions (27). HPV5 has been found only seldomly in immunosuppressed transplant recipients (2, 22, 26, 41), and the existence of latent and subclinical HPV5 infections in the general population, although conceivable, has not been proven yet (16). The persistent infection, usually disseminated to the whole body, and the high level of viral replication observed in EV (27) should favor the occurrence of point mutations. However, it is likely that the cellular "EV gene(s)" somehow exerts such a strong selection pressure on the emergence of new infectious HPV5 variants that the same variants arise independently of the geographical origin of the patients. As for the functional significance of the genetic heterogeneity of HPVS, it does not affect the enhancer activity of the LCR or the transactivating properties of the E2 protein, as analyzed in a transient expression system. In contrast to the HPV18 LCR (40), the HPV5 LCR showed no constitutive enhancer activity in SW13 cells. It is likely that such an activity might require keratinocytes from EV patients (30). In conclusion, the comparison of 17 HPVS isolates from 12 EV patients demonstrates both the genetic heterogeneity of this HPV type and the stability of HPVS variants. Whether this genetic variability affects the oncogenic potential of HPVS through distinct biological activities or specific antigenic properties remains to be elucidated. ACKNOWLEDGMENTS We acknowledge the contributions of M.-F. Avril, C. BlanchetBardon, M. Lutzner, and P. C. Van Voorst Vader, who provided some patient specimens. We thank F. Thierry for the gift of SW13 cells, the pTKM vector, and the BPV1 E2 expression vector, F. Tekaia for helpful advice in phylogenetic studies, and F. Breitburd for critical reading of the manuscript. REFERENCES 1. Barbosa, M. S., D. R. Lowy, and J. T. Schiller. 1989. Papillomavirus polypeptides E6 and E7 are zinc-binding proteins. J. Virol. 63:1404-1407. 2. Barr, B. B. B., E. C. Benton, K. McLaren, M. H. Bunney, I. W. Smith, and K. Blessing. 1989. Human papillomavirus infection and skin cancer in renal allograft recipients. Lancet i:124-129. 3. Chan, S. Y., L. Ho, C. K. Ong, V. Chow, B. Drescher, M. Durst, J. ter Meulen, L. VBia, J. Luande, H. N. Mgaya, and H. U. Bernard. 1992. Molecular variants of human papillomavirus type 16 from four continents suggest ancient pandemic spread of the virus and its coevolution with humankind. J. Virol. 66:2057-2066. 4. Chen, C., and H. Okayama. 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745-2752. 5. Coggin, J. R., and H. zur Hausen. 1979. Workshop on papillomaviruses and cancer. Cancer Res. 39:545-546. 6. Cole, S. T., and 0. Danos. 1987. Nucleotide sequence and comparative analysis of the human papillomavirus type 18 genome: phylogeny of papillomaviruses and repeated structure of the E6 and E7 gene products. J. Mol. Biol. 193:599-608. 7. Deau, M.-C., M. Favre, and G. Orth. 1991. Genetic heterogeneity among human papillomaviruses (HPV) associated with
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