VIROLOGY'~~~, lOl-107(1991)

Nucleotide and Amino Acid Sequence Variation in the Ll and E7 Open Frames of Human Papillomavirus Type 6 and Type 16 JOSEPH P. ICENOGLE,*a’ PUSHPA SATHYA,1- DONNA L. MILLER,* RUTH ANN TUCKER,* AND WILLIAM E. RAWLSt~2 *Viral Exanthems and Herpesvirus Branch, Division of Viral and Rickettsial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333, and tDepartment of Pathology, McMaster University, Hamilton, Ontario, Canada L8N 325 Received February 8, 199 1; accepted May 15, 199 1 Human papilbmavirus (HPV) type 6 and type 16 DNA sequence variants were found b and E7 open reading frames, using tempbtes generated with the polymeraere chein found in patients from widely separated locations, such as the United States, the sequence variants of tiPV 16 were found in women with invasive cervical carcinoma di. Variation in the predicted amino acid sequences of the WV 16 Ll and nucleotide change at position 6433 wes found in about 60% of the HPV 16 DNAs, amino acid sequence from threonine to alanine at the equivalent positii in the changes were found in the HPV 16 E7 proteins at amino acid positiis2S, 29, and 47. Variatiibn at affect known properties of the E7 protein, including binding to the retinoblastoma protein. ~31s~ ~csdemicRWJ+inc.

INTRODUCTION

1989; Firzlaff et al., 1989; Edmonda and Vousden, 1989). Mutations in the late ORFs, Ll and L2, which code for structural proteins, could a t host immune responses (Strang et al., 1990; Steek md Galiimore, 1990; Dillner et a/., 1990). Mutdions in the upstream regulatory region (URR), the major tr~~~~~j~~i control region, have been observed with XPV 6 DNAs isolated from vulvar carcinomas (Rando et al., 1986; Kasher and Roman, 1988). Thus far, work conelating sequence information with bidogicd properties has been done primarily with laboratory-derived sequence variants. The extent of natural intragertofypic variation in HPV is largely unknown. In the present study, natural intra~~o~pic variation in HPVs was measured by sequenolng about 250 bases of the Ll ORF and most of the E7 ORF. Sequences of HPV DNAs from women living in gewraphitally separated locations and from women with highand low-grade cervical disease were d&errninsd since these DNAs were expected to give the maximum sequence variation. The nt sequence and predicted amino acid sequence changes found in such samples are reported here.

Human papillomaviruses (HPVs) are a diverse group of viruses with about 60 recognized genotypes, roughly a third of which have been isolated from human genital mucosa (de Villiers, 1989). Subsets of the HPVs found in human genital mucosa have been associated with specific diseases. For example, HPV 6 and HPV 11 are usually found in benign exophytic and cervical lesions, while HPV 16 and HPV 18 are commonly found in severe dysplasias and cervical carcinomas (Reeves et al., 1989b). However, most cervical HPV 16 and HPV 18 infections do not lead to the development of preinvasive or invasive lesions. Data are accumulating which suggest that HPV nucleotide (nt) sequence variation may in part determine whether a HPV-associated lesion progresses or regresses. Mutations in the early open reading frames (ORFs), E2, E6, and E7, may affect the likelihood that the virus will be associated with malignant progression (Werness et al., 1990; Lambert eta/., 1990; Storey et a/., 1990; Chesters et al., 1990). Specific amino acid residues in one early protein, E7, have been shown to be important for E-/-induced transformation, transactivation, and immortalization; for E7 binding to retinoblastoma protein; and for phosphorylation of E7 by casein kinase II (Storey et a/., 1990; Chesters et a/., 1990; Gage et al., 1990; Munger et a/., ’ To whom ’ Deceased

requests for reprints November 1990.

should

MATERlAtS Sources and preparation

AND METHODS of WV DNA

Samples of HPV DNA were obtained from cervical scrapes, cervicovaginal lavages, cervic&l bbp~&, and biopsies of exophytic condyiomas. Sampies were from

be addressed.

101

0042-6822191

$3.00

Copyright 43 i 991 by Academic press, Inc. All rights of reproducWm in any form reserved

I

1

ICENOGLE ETAL.

102 TABLE

1

was suspended in water at concentrations from 0.01 to 1 PgIpI and stored at -70”.

SEQUENCEVARIANTSIN HPV 6 Ll ORF Nucleotide

6598

6625

6661

Reference HPV 6b” Georgia-65 India-D4, D5, D7, D9 Philippines-A4 Alaska-C36 Georgia-B4, B6, G6 Georgia-Bl, G2, G3, G5, Gl Philippines-A6 Georgia-G4, G7

A T T T T T T T T

C -b A A A

G A A A A -

a The reference sequence has been previously verified using the PCR-generated template amplified from cloned HPV 6b (Icenogle et al., 1990; Schwarz et al., 1983). b A reference nucleotide was obtained at this position.

patients residing in diverse geographic locations, including Alaska, Georgia, New Delhi, the Republic of Panama, and Manila. Total DNA was prepared by proteinase K treatment, phenol-chloroform extraction, and ethanol precipitation (Ausubel et a/., 1989). The DNA

Preparation

of single-stranded

ranging

DNA templates

Templates were prepared by using the asymmetric polymerase chain reaction (PCR) amplification as described previously (Gyllensten and Erlich, 1989; Icenogle et al., 1990). Primers defining a 413-bp region of the Ll ORFfrom nt 6383 to nt 6795 of HPV 6b and from nt 6244 to nt 6656 of HPV 16 or primers defining the entire coding region of the E7 ORF from nt 562 to nt 855 of HPV 16 were used. The Ll ORF primers for HPV 6 were 5’-ACAGGCllTGGTGCTATGAAlllT-3’ and 5’-GTACTGCGTGTGGTATCTACCACAG-3’, and the Ll ORF primers for HPV 16 were 5’-ACTGGCllTGGTGCTATGGACllT-3’ and 5’GTACTGCGTGTAGTATCAACAACAG-3’. The E7 ORF primers for HPV 16 were 5’ GAATXATGCATGGAGATACACCTAC-3’ and 5’-CAGGATCCTGGTITCTGAGAACAGATGG-3’. The thermal cycling program was: (i) raise to 95” as rapidly as possible, (ii) hold at 95” for 15 set, (iii) cool to 50” over 2 min, (iv) hold at 50” for 15 set, (v) raise to 72” over 1.5 min, and (vi) hold at 72” for 1 min. A 7-min

TABLE 2 SEQUENCEVARIANTSOBTAINEDIN THE Ll ORF AND E7 ORF OF HPV 16 DNAs Ll ORF Nucleotidee Referenceb Alaska-C51

Michigan-B8

Panama-l 56 CaSkid Alaska-C32 Panama-Al 3 Panama-F30 SiHad Panama-C20 Alabama-B24 Panama-349 Missouri-B1 1 Panama-200 Panama-Cl 0” Panama-B20 Georgia-B9 Alabama-B23

6433

6531

A -c _ G G G G G G G G G G

T _ _ _ -------c--wc

Not done

G G G

-

6558 C _ ___-

E7 ORF 6567

6582

645

647

666

678

701

732

789

795

T _ -

A _ -

A -

A -----_----___

G -

T -

C -

T -

T _ -

T -

-

-

-

-

-

-

-

-

c

-

A------

-

-

-

_

_

-

-

-

-

T

-

-

-

G

_

-

_

C C C

C C C C

G G G G G

___---__ --------___ T--------C T T T

------------c

T-------T

-

-

-

a An A at 6433 results in a codon for thr. G at 6433 results in a codon for ala. An A at 645 results in a codon for leu. C at 645 results in a codon for phe. An A at 647 results in a codon for asn. G at 647 results in a codon for ser. C at 701 results in a codon for pro. Tat 701 results in a codon for leu. b Seedorf et a/. (1985). c A reference nucleotide was obtained at this position. d CaSki and SiHa cell lines were originally obtained from cervical carcinoma (Pattillo et al., 1977; Fried1 et al., 1970). ’ Sequencing templates from the Ll ORF did not produce a readable sequence.

HPV SEQUENCE VARIATION TABLE

3

SEQUENCESIN THE E7 ORF FROMWOMEN WITHINVASIVECERVICAL CARCINOMAAND FROMASYMPTOMATICWOMEN E7 ORF Nucleotide Reference Invasive Al2 Al3 84 B20 Cl0 c20 D6 F15 F30 F38 F43

Asymptomatic 136 148 156 200 206 349

645

647

666

678

701

732

789

795

AAGTCTTT -a -

-G----CG

-

-

_ C

C

G

-

-

c---_ -

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

C -

C C

G -

A-----

-

G

@A reference nucleotide was obtained at this position.

incubation at 72” was done after the final cycle. Twenty-two cycles were typically done for doublestranded products. Double-stranded PCR products were separated from any residual primers and deoxynucleoside triphosphates with Centricon 30 microconcentrators (Amicon Division, W. R. Grace & Co.). Double-stranded products were then used as templates in a second PCR amplification for 15 cycles of the abovedescribed program, using only one of the primers. This procedure produced significant amounts of singlestranded DNA products for use as sequencing templates (Icenogle et a/., 1990). Sequencing Sequences were determined by using dideoxynucleotide chain termination during elongation of the synthetic DNA primers with Sequenase (United States Biochemical). Inclusion of ar-36S dATP into the reactions allowed termination products to be visualized by autoradiography. Polymerase-induced errors are a significant concern when sequencing PCR-derived templates. A DNA from a typical 30-cycle PCR amplification is expected to differ once in every 400 to 4000 bp because of errors introduced by the Taq polymerase (Saiki era/., 1988). If the PCR product had been cloned and a single clone

103

had been sequenced, then naturally occurring sequence variation and Taq polymerase-derivedsequence errors in that particular clone could not be differentiated. However, since the errors introduced by the polymerase should be distributed throughout the product, sequencing the bulk product averages the polymerase-derived errors and should give the correct sequence of the original HPV DNA. Even though this method of template production should theoretically give the correct sequences, the accuracy of the method was verified by fi) determining the sequence of PCR templates produced from cloned HPV 6b DNA (Icenogle ef al., 1990), (ii) repeating the PCR template production and the sequence determination using a second aliquot of some of the HPV DNAs, and (iii) determining the sequence of both strands of some PCR products. Nucleotides 6392 through 6768 of the cloned reference HPV 6b DNA were sequenced using this method, and a sequence identical to that determined by other methods was obtained (Schwarz et al., 1983). Three hundred fifty bases of the nt sequence in the Ll ORF of 4 HPV 16 DNAs were determined (samples B24, C32, C51, and 200), and the reference HPV 16 sequence was obtained at all positions except as listed in Table 2. Repeating the sequencing of these four samples starting with a second aiiquot of DNA extracted from each patient’s sample gave identical results. Sequences of both strands of 19 doublestranded products were determined without evidence of polymerase-induced errors. For the Ll ORF 413 base templates, sequence information from about 250 bases was collected. For the TABLE

4

SEQUENCESIN THE Ll ORF FROMWOMEN WITH I~I~MIVE GEMCAL CARCINOMAAND FROMASYMPTOM~~TIC WQMEN Ll ORf= Nucleotide Reference Invasive Al3 84 820 c20 D6 F30 F38

6433

6531

6558

A

T

c

G G

-* -

--

G G

-

T _. .--

6567

6582

T

A -.--

.-

-_

G

Asymptomatic 156 200 206 349

G G

-

-

-

T

--.

-

--

-. -

-

a A reference nucleotide was obtained at this portion.

T

104

ICENOGLE ET AL.

Ll Class Class

1 2

HPV HPV HPV HPV HPV HPV HPV

16 16 31 33 11 6B 18

REFERENCE REFERENCE REFERENCE REFERENCE REFERENCE REFERENCE

ORF COMPARISON

. ..RHLFNRAG . ..-------. ..--F--+. ..--F----,..--F----. ..--F----. ..--FW----

T A E a 1

VGENVPDDLYIKGSGSTANLASSNYFPTPSGS... --------------------------------... ---S--T-----------T--N-T--------.,. L--A-----------T--sIQ--AF-------...

---P-----LV--GNNRSSV---I-VH ---P---T-I-----NRTSVG--I-~

-----. -----.

.. ..

M-DT--Qs-----T-MR-spG-~-S-S----...

E7 ORF COMPARISON HPV 16 REFERENCE SiHa Cells PANAMA-Cl0 QG-U Cells MISSOURI-B11 HPV 31 REFERENCE HPV 33 REFERENCE HPV 18 REFERENCE

I . ..LQPETTDLYC%QLNDS.:%DEIDGPAGQAEPDR... ***_-__-_------F---------------------... l **--------------

s--------------------...

m*s-________-_.m-s--------------------... ,..--------------------------------L--... . ..----A---H-----P---D---V--s--------T... . ..-Y--P---------S---D-DEGL-R-D---Q-AT... . ..QNEIPV--L-H---S--E--N-----VNH-HL-A-...

FIG. 1. Comparison of predicted amino acids in HPV 16 variants with amino acid sequences predicted from published sequences of other HPV genotypes. The vertical box highlights the amino acid position which differentiates between Class 1 and Class 2 HPV 16. The first leucine indicated in the E7 ORF comparison is amino acid 15 of the E7 protein. The retinoblastoma protein (Rb) binding site determined by synthetic peptide competition experiments is indicated, and amino acids required for maximal antagonist activity of the peptide are underlined (Jones et a/., 1990). The casein kinase II recognition site is indicated (CKII). QG-U is a cell line with an altered E7 sequence (Shirasawa era/., 1989). A major immunoreactive region of E7 is DLYCYEQLNDSSEE (Jenison et a/., 1991).

E7 ORF templates, sequence to nt 801 was determined.

information

from nt 582

Typing of HPV DNAs A comparison of the amplified regions of the known Ll ORF sequences of cervical HPVs shows that the ends of this region are highly conserved whereas the central portion is highly variable (Schwa= et al., 1983; Dartmann et a/., 1986; Seedorf et al., 1985; Cole and Danos, 1987; Goldsborough et al., 1989; Cole and Streek, 1986). There are a number of locations in the region where a given nt exists in only one sequenced genotype. Since between 18 and 37 such unique bases exist when HPV 6b, HPV 11, HPV 16, HPV 18, HPV 31, and HPV 33 are compared, unambiguous identification of HPV type results from sequence information on this region alone. Typing of samples analyzed here was done by this sequence comparison method. RESULTS Variation

in the HPV 6 Ll ORF

The Ll ORF of 18 HPV 6 DNAs from condylomas were sequenced from nt 6507 to nt 6661. Variant nts relative to the reference HPV 6b sequence were found at only three positions: 6598, 6625, and 6661 (Table 1). Sixteen of the 18 samples also had sequence information collected in the larger interval from nt 6486 to nt

6739 (all samples except B5 and C36). Only those changes listed in Table 1 were found in these 16 samples, even in the larger region. The observed nt changes give no changes in the predicted amino acid sequence of the HPV 6 Ll protein. Variation

in HPV 16 Ll and E7 ORFs

Thirty-two HPV 16 Ll ORF sequences were determined from nt 6347 to nt 6614. Representative sequence variants from various locations are shown in Table 2. The Ll ORF sequence variants could be easily separated into two classes. One class was identical to the reference virus in the sequenced region, and the other class had a G at nt 6433, which results in a change from a codon for threonine to one for alanine (Seedorf et al., 1985). Of the 32 HPV 16 DNAs sequenced in the Ll ORF, 14 were identical to the reference sequence and 18 had a G at 6433. The 14 HPV DNAs that were identical to the reference HPV 16 DNA were obtained from patients in Alaska, Michigan, Panama, Georgia, and Alabama. Thirteen HPV DNAs with a G at 6433 are shown in Table 2. The remaining 5 had the same Ll ORF sequence and were from the same locations as DNAs which are included in Table 2. Sequences of the HPV 16 E7 ORFs extending from nt 582 through nt 801 were also done (Table 2). Three samples had nucleotide changes that resulted in changes in the predicted amino acid sequence (SiHa cells, Missouri-B 11, and Panama-C 10).

HPVSEQUENCE VARIATION

v8hfa4it7p%~~~withinvasive andin a~ptorn~~cpatients

TheHPV16E7protein hasbeen associated with transhming ~ctity

(Storey et al., 1990: Chesters et

a/., 1990; G er a/., 1990; Munger et al., 1989). Thus, E7 ORF sequences from nt 582 to nt 801 of a number of HPV 16 DNAs from women with invasive cervical carcinoma and from asymptomatic women were compared (Reeves eZ al,, 1989a). Although variants were observed in HPVs from both groups of women, there was no simple correlation between invasive disease and the HPV 16 E7 ORF sequence variant present (Table 3). The Ll ORF sequences from nt 6319 to nt 6602 were also compared. Again there was no simple correlation between the HPV variant present and invasive disease (Table 4). The HPV Ll ORF and E7 ORF sequence variants found in these samples were the same as those found in samples from a wide geographic distribution (Table 2). DtSCUSSlON The section of the Ll ORF which was sequenced contained a highly variable section bounded by relatively conserved regions at both the nt and the amino acid level. However, no HPV 6 Ll ORF variant had changes in the predicted amino acid sequence. In addition, none of the variant HPV 6 DNAs analyzed had a sequence identical to reference HPV 6b DNA, indicating that reference HPV 6b is relatively uncommon, at least in condylomas. The 32 HPV 16 DNAs sequenced in the Ll ORF yielded six Ll ORF sequences which could be easily grouped into two classes. Class 1 Ll ORF was identical to the reference HPV 16, and Class 2 Ll ORF had a G at nt 6433, resulting in a change in the predicted amino acid from threonine to alanine (Seedorf et a/., 1995). Fourteen of 32 HPV 16 DNAs were Class 1 and 18 were Cla$s 2. Many Class 2 viruses had additional nt changes in the sequenced region of the Ll ORF, but none resuited in additional changes in the predicted amino acid sequence. The A to G change at nucleotide 6433 occurred in all HPV 16 DNAs differing from reference DNA. No other change in the Ll or E7 ORFs occurred in all variants. In other words, nucleotide position 6433 is a stronger marker for sequence variation than any other position analyzed here. Only 12 HPV 16 sequence variants (counting reference DNA as a variant) were found, while 48 total variants (6 [Ll ORF variants] X 8 [E7 ORF variants]) would be expected if recombination between variants was frenuent. Recombination between different genotypes

isapparently a rarewentduring~~~~~~~~US infixtions. Thesamesequencevariantsof bdhHP~~~ndHPV 16 were observed in specimens &% parts of the world, suggesting that distributed (Table 2). Thus, tracking of HPV variants between many geographic locations such ae has been done with poliovirus will not be possible (Rico-He&~ et al., 1987). Transmission studies of HPV 16 from individual to individual are difficult to design since the prevaience of HPV 16 is high and the presenting dieeaee can teke years to develop (Reeves et a/., 1989b; Sytjanen, 1989). The possibility of tracking variants of HPV 16 from person to person should be fee&We since any subsequent infection of an indiidual by other sequence variants could be detected. Stud&a could be designed using methods to quickiy i DNAs differing by as little as a single nt, using PCR techniques (Kwok et al., 1990). The naturally occurring amino acid variation described here in the Ll ORF was compared with the variation occurring between sequenc& s of HPVs isolated from genital specime~+e (F hreonine-to-alanine difference between Cl&es 1 and Class 2 HPV 16 Ll ORFs occurs at a loca%on where the reference DNA for most genital HPV types has a threonine; HPV 6b, which causes ben@n exophytic lesions, is the only exception thus far identifted. None of the 18 HPV 6s isolated from condyloma have altered amino acids at this position. The threonine-to-alanine change occurs 16 amino acids upstream from the most variable region in the sequenced portion of Ll . The possibility exists that the threonine-to-alenine change affects the tertiary f&ding of t such that regions of Ll which might be r important properties (e.g., receptor binding) would be altered. The natural variation in E7 proteins is 213~0summarized in Fig. 1. The changes in the variant E7 ORFs are located at amino acid positions 28, 29, and 47. Positions 28 and 29 are immediate&y downstream from cysteine 24 and glutamic acid 26, both of which are important for various functions of E7 such as traneformation of the NIH 3T3 cell line and binding to retinobleatoma protein (Chesters et al., 1990; Jones et a/., 1990). A casein kinase II phosphorylation site occurs at amino acid 30 to 37 of the E7 protein with eerinee 31 and 32 being phosphorylated (Fir&& et a/., 1&IQ o8a et al., 1990). The degree of ion by casein kinase II has been s e 8 feotor in the oncogenic potential of HPVs (Firzla!I et &, 1QSS). The __

ICENOGLE ETAL.

’ phosphotylation ofE7.Thechangeat position29 in Panama sample number C 10 has been previously observed in a cervical carcinoma cell line (Shirsawa eta/., 1989). This observation further reinforces the argument that this change in E7 might alter the transforming properties of the E7 protein. The amino acid substitution at position 47 occurs at a proline which is conserved among HPV E7 proteins in HPV 16, HPV 31, HPV 33, and HPV 18. Substitution of leucine for this proline may alter the structure and function of the resulting protein. Finally, amino acids 2 1 to 34 of the HPV 16 E7 protein form the immunoreactive region of this protein (Jenison et al., 1991). Amino acid changes at positions 28 and 29 might affect antibody binding. Thus, it is reasonable to hypothesize that the naturally occurring amino acid changes in the E7 protein observed in this study might alter its biological properties. The lack of a simple correlation between the HPV 16 variant present and invasive disease does not eliminate the possibility of different oncogenic potentials among the HPV 16 variants. By doing functional analysis of naturally occurring HPV 16 variants, for example, determining the ability of E7 amino acid variants to immortalize keratinocytes, differences might still be detected. Such differences might be difficult to detect when comparing patients with invasive disease and asymptomatic patients since factors such as the patient’s immune response might modulate the development of cervical disease.

ACKNOWLEDGMENTS We gratefully acknowledge Alan Parkinson and Michael Davidson, CDC. Arctic Investigations Laboratory, Anchorage; Pradeep Seth, All India Institute of Medical Sciences, New Delhi; Michael Campion. Saint Joseph’s Hospital, Atlanta; Curtis Hayes, U.S. Naval Medical Research Unit, Manila; and William C. Reeves, Gorgas Memorial Laboratory, Republic of Panama for samples analyzed in this study. We thank William C. Reeves and Suzanne Vernon for careful review of the manuscript.

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frame E7 immortalizingfunction in rat embryo fibroblast cells. 1. Gen. Viral. 71, 449-453. COLE, S. T., and DANOS, 0. (1987). Nucleotide sequence and comparative analysis of the human papillomavirus type 18 genome. 1, Mol. Biol. 193, 599-608. COLE, S. T., and STREEK,R. E. (1986). Genome organization and nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer. J. Viral. 58, 991-995. DARTMANN, K., SCHWARZ, E.. GISSMANN, L., and ZUR HAUSEN, H. (1986). The nucleotide sequence and genome organization of human papilloma virus type 1 1. Virology 151, 124-l 30. DEVILLIERS.E. M. (1989). Heterogeneity of the human papillomavirus group. J. Viral. 63, 4898-4903. DILLNER,J., DILLNER,L., UITER, G., EKLUND,C., ROTOLA,A., COSTA, S., and DILUCA, D. (1990). Mapping of linear epitotes of human papillomavirus type 16: The Ll and L2 open reading frames. lnf. J. Cancer 45, 529-535. EDMONDS,C., and VOUSDEN,K. H. (1989). A point mutational analysis of human papillomavirus type 16 E7 protein. J. Viral. 63, 26502656. FIRZLAFF,J. M., GALLOWAY, D. A., EISENMAN,R. N., and LUSCHER,B. (1989). The E7 protein of human papillomavirus type 16 is phosphorylated by casein kinase Il. New Biologisf 1, 44-53. FRIEDL,F., KIMURA,I., OSATO, T., and ITO, Y. (1970). Studies on a new human cell line (SiHa) derived from carcinoma of uterus. I. Its establishment and morphology. Proc. Sot. Exp. Biol. Med. 135, 543-545. GAGE, J. R., MEYERS,C., and WEITSTEIN,F. 0. (1990). The E7 proteins of the nononcogenic human papillomavirus type 6b (HPV-6b) and of the oncogenic HPV-16 differ in retinoblastoma protein binding and other properties. J. Viral. 64, 723-730. GOLDSBOROUGH,M. D., DISILVESTRE,D.. TEMPLE, G. F., and LORINCZ, A. T. (1989). Nucleotide sequence of human papillomavirus type 3 1: A cervical neoplasia-associated virus. virology 171,306-31 1. GYLLENSTEN,U. B., and ERLICH,H. A. (1989). Generation of singlestranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc. A&/. Acad. Sci. USA 85,7652-7656. ICENOGLE,J., MILLER, D. L., TUCKER,R. A., and HOLLOWAY,B. (1990). Rapid sequence analysis of the capsid region of human papillomaviruses using single-stranded DNA generated by the polymerase chain reaction. In “Papillomaviruses: Molecular and Cellular Biology” (P. M. Howley and T. R. Broker, Eds.), Vol. 124, pp. 89-97. A. R. Liss. New York. JENISON,S. A., Yu, X., VALENTINE,J. M., and GALLOWAY,D. A. (1991). Characterization of human antibody-reactive epitopes encoded by human papillomavirus type 16 and 18. J. Viral. 65, 1208-l 2 18. JONES,R. E., WEGRZYN.R. J., PATRICK,D. R., BALISHIN,N. L., VUOCOLO, G. A., RIEMEN,M. W., DEFEO-JONES,D., GARSKY,V. M., HEIMBROOK, D. C., and OLIFF, A. (1990). Identification of HPV-16 E7 peptides that are potent antagonists of E7 binding to the retinoblastoma suppressor protein. J. Biol. Chem. 265, 12,782-l 2,785. KASHER, M. S., and ROMAN, A. (1988). Characterization of human papillomavirus type 6b DNA isolated from an invasive squamous carcinoma of the vulva. Virology 165, 225-233. KRUBKE,J., KRAUS,J., DELIUS,H., CHOW, L., BROKER,T., IFTNER,T.. and PFISTER, H. (1987). Genetic relationship among human papillomaviruses associated with benign and malignant tumours of patients with epidermodysplasia verruciformis. J. Gen. Viral. 66, 30913103. KWOK, S., KELLOGG, D. E., MCKINNEY, N., SPASIC, D., GODA, L., LEVENSON, C., and SNINSKY, J. J. (1990). Effects of primer-template misml+,+Pc nn +ha ~,-J\,mnrsoc, A.,.:- .s.....+:--. I d..--- :--. -- - J-1’

HPV SEQUENCEVARIATION

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