Journal of Medical Virology 87:1754–1760 (2015)

Hypermutation in the E2 Gene of Human Papillomavirus Type 16 in Cervical Intraepithelial Neoplasia Iwao Kukimoto,1* Seiichiro Mori,1 Satoru Aoyama,2 Kousho Wakae,2 Masamichi Muramatsu,2 and Kazunari Kondo3 1

Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan Department of Molecular Genetics, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan 3 Department of Obstetrics and Gynecology, NTT Medical Center Tokyo, Tokyo, Japan 2

Persistent infection with oncogenic human papillomavirus (HPV) causes cervical cancer. However, viral genetic changes during cervical carcinogenesis are not fully understood. Recent studies have revealed the presence of adenine/thymine-clustered hypermutation in the long control region of the HPV16 genome in cervical intraepithelial neoplasia (CIN) lesions, and suggested that apolipoprotein B mRNA editing enzyme, catalytic polypeptidelike (APOBEC) proteins, which play a key role in innate immunity against retroviral infection, potentially introduce such hypermutation. This study reports for the first time the detection of adenine/thymine-clustered hypermutation in the E2 gene of HPV16 isolated from clinical specimens with low- and high-grade CIN lesions (CIN1/3). Differential DNA denaturation PCR, which utilizes lower denaturation temperatures to selectively amplify adenine/thyminerich DNA, identified clusters of adenine/thymine mutations in the E2 gene in 4 of 11 CIN1 (36.4%), and 6 of 27 CIN3 (22.2%) samples. Interestingly, the number of mutations per sample was higher in CIN3 than in CIN1. Although the relevance of E2 hypermutation in cervical carcinogenesis remains unclear, the observed hypermutation patterns strongly imply involvement of APOBEC3 proteins in editing the HPV16 genome during natural viral infection. J. Med. Virol. 87:1754–1760, 2015. # 2015 Wiley Periodicals, Inc. KEY WORDS:

human papillomavirus; hypermutation; cervical intraepithelial neoplasia; APOBEC protein

INTRODUCTION Persistent infection with oncogenic human papillomavirus (HPV) is a primary cause of cervical cancer [zur Hausen, 2002], and HPV type 16 (HPV16) is the most prevalent genotype in cervical cancer cases worldwide [de Sanjose et al., 2010]. HPV infection alone, however, is not sufficient to drive full carcinogenesis. The development of invasive cervical cancer requires accumulation of multiple mutations in the host genome, leading to activation of cellular oncogenes, or inactivation of tumor suppressor genes. Recent cancer genomics studies revealed that the cervical cancer genome accumulates mutation patterns typical of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) proteins, which play a critical role in innate cellular immunity against retroviral infection by converting cytosine (C) to uracil (U) in DNA/RNA [Conticello, 2008], and that among at least 11 APOBEC proteins including activation-induced cytidine deaminase, APOBEC1, -2, -3A, -3B, -3C, -3DE, -3F, -3G, -3H, and -4, APOBEC3B (A3B) is the most likely candidate involved in cervical carcinogenesis [Alexandrov et al., 2013; Burns et al., 2013; Roberts et al., 2013]. Furthermore, A3B has recently been implicated in the development of HPV-positive head-and-neck cancer [Henderson et al., 2014], suggesting a general role for A3B in HPV-associated carcinogenesis. The mutational signatures characteristic of the APOBEC proteins have also been detected in the long Grant sponsor: Japanese Ministry of Health, Labor and Welfare for the Re-emerging Infectious Diseases (grant-in-aid)  Correspondence to: Iwao Kukimoto, Ph.D, Pathogen Genomics Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-murayama, Tokyo 208-0011, Japan. E-mail: [email protected] Accepted 19 March 2015 DOI 10.1002/jmv.24215 Published online 24 April 2015 in Wiley Online Library (wileyonlinelibrary.com).

C 2015 WILEY PERIODICALS, INC. 

HPV16 E2 Hypermutation in CIN

control region of the HPV16 genome in cervical intraepithelial neoplasia (CIN) lesions [Vartanian et al., 2008]. Moreover, the A3 subfamily proteins have been shown to possess an ability to introduce adenine (A) or thymine (T) clustered hypermutation in the E2 gene of the HPV16 genome maintained in cultured cervical keratinocyte cells [Wang et al., 2014]. Whether such E2 hypermutation takes place in natural HPV infection, however, has not been investigated. Here, using differential DNA denaturation PCR (3D-PCR), a method that can detect A/T hypermutation with high sensitivity, this study explored HPV16 E2 hypermutation in clinical specimens. MATERIALS AND METHODS

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followed by staining with ethidium bromide to identify the 314-bp E2 DNA. Subsequently, 3D-PCR was performed to amplify a nested region of the first amplicon, from nucleotide positions 3376 to 3588, with 0.5 ml of the first PCR product in a total 10-ml reaction volume as follows: 86.6–80.6˚C (at 0.6˚C intervals) for 5 min, followed by 35 cycles, in 6 separate reactions each at 86.6–83.6˚C (at 0.6˚C intervals) or 83.6–80.6˚C (at 0.6˚C intervals) for 45 s, 50˚C for 30 s, and 65˚C for 38 s, and a final elongation step at 65˚C for 10 min, using inner primers, 50 -TCC TGA AAT TAT TAG GCA GCA CTT-30 and 50 -CGT CCT TTG TGT GAG CTG TTA AAT-30 , and rTaq on a Veriti. The final PCR products were run on 1.5% agarose gels, followed by staining with ethidium bromide to identify the 213-bp E2 DNA.

Clinical Specimens In a cohort of Japanese women who had visited the NTT Medical Center Tokyo as outpatients (n ¼ 1088) [Kondo et al., 2012], cervical exfoliated cells were1 collected from each patient 1using a Cervex-brush combi and stored in Thinprep collection media. Total DNA was extracted from a 200-ml aliquot of the suspended cell samples using the QIAamp DNA Blood Mini kit (Qiagen, Valencia, CA), resulting in a final elution volume of 100-ml Tris-EDTA buffer, followed by PGMY-PCR, and reverse blot hybridization-based HPV genotyping [Kondo et al., 2012]. The Pap smear test identified 326 abnormal cytology cases in our cohort, among which 69 cases (21.2%) were HPV16-positive. Histological diagnosis was made for the patients having abnormal cytological results as a routine clinical procedure using hematoxylin-eosin-stained sections obtained by punch biopsy according to the World Health Organization classification. Forty-two HPV16-positive DNA samples, which included 11 CIN grade 1 (CIN1) and 31 CIN grade 3 (CIN3) cases, were randomly selected and subjected to E2 PCR. The institutional research ethics committee approved the collection of samples for analyses of HPV DNA, and written informed consent was obtained from each patient. Differential DNA Denaturation PCR 3D-PCR for the E2 gene was performed as previously described [Wang et al., 2014]. Firstly, a region of the E2 gene, from nucleotide positions 3304 to 3617, was amplified from a 2-ml aliquot of the purified DNA in a total 20-ml reaction volume by conventional PCR as follows: 94˚C for 4 min, followed by 35 cycles, each at 94˚C for 16 s, 55˚C for 20 s, and 68˚C for 50 s, and a final elongation step at 65˚C for 10 min, using primers, 50 -ATG GGA AGT TCA TGC GGG TGG TCA-30 and 50 -TGG GTG TAG TGT TAC TAT TAC AGT TAA T-30 , and rTaq (Takara, Ohtsu, Japan) on a Veriti thermal cycler (Applied Biosystems, Life Technologies, Carlsbad, CA). The first round PCR products were run on 1.5% agarose gels,

Nucleotide Sequencing The 3D-PCR products generated at the lowest denaturation temperature for each sample were gelexcised and ligated into the pGEM-T vector (Promega, Madison, WI) using the Mighty Mix DNA ligation kit (Takara), followed by transformation into Escherichia coli DH5a. Four to six bacterial clones were randomly selected from each sample, and subjected to plasmid preparation by the QIAprep Spin Miniprep kit (Qiagen), followed by sequencing with the SP6 primer on a 3730xl DNA analyzer (Applied Biosystems). To analyze nucleotide substitutions in the 3D-PCR products, the first PCR products were purified using the Wizard gel purification kit (Promega) and sequenced in bulk without cloning using the first PCR primers on a 3730xl sequencer, followed by comparison of the sequence of each 3D-PCR clone with that of the corresponding first PCR product. Statistical Analysis Statistical analysis was performed using R version 2.11.1. Welch’s t-test was used to examine differences in mutation number between CIN1 and CIN3 samples and differences in age distribution among samples with or without hypermutation. Fisher’s exact test was used to examine differences in the prevalence rate for hypermutation between CIN1 and CIN3. Two-sided P-values were calculated and considered to be significant at less than 0.05. RESULTS 3D-PCR for the HPV16 E2 Gene Using a 3D-PCR technique, we examined the presence of A/T hypermutation in the E2 gene of the HPV16 genome in low- and high-grade CIN lesions (CIN1 and CIN3). 3D-PCR utilizes lower melting temperatures than that used in conventional PCR to denature target DNA, which favors selective amplification of A/T-rich DNA in the final PCR product. First, a region of the E2 gene was amplified by J. Med. Virol. DOI 10.1002/jmv

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conventional PCR. All 11 CIN1 samples and 27 of 31 CIN3 samples generated the expected 314-bp PCR product, whereas three CIN3 samples failed to generate any PCR products and one CIN3 sample yielded a shorter product (data not shown). These four CIN3 samples were excluded from subsequent 3D-PCR analysis because these samples contained disrupted or deleted E2 gene. The first round PCR products were then subjected to 3D-PCR, which targets a nested region of the first PCR amplicon, under conditions of different denaturation temperatures. As shown in Figure 1, four CIN1 and six CIN3 samples yielded the expected PCR product at a lower denaturation temperature compared to the reference HPV16 plasmid (GenBank accession No. K02718), which suggests an enrichment of A/T-rich DNA in these PCR products. Interestingly, whilst the four CIN1 samples showed a low denaturation temperature range, from 84.2 to 84.8˚C, the six CIN3 samples demonstrated an even lower temperature range, from 81.2 to 83.0˚C (Fig. 1 and Table I), suggesting the existence of more extensive A/T hypermutation in the CIN3 samples.

hypermutations, the CIN3 samples exhibited a strong preference for C to T hypermutation in the codingstrand of the E2 gene. Direct sequencing of the first round PCR products did not demonstrate the presence of such hypermutation in the 3D-PCR amplicon (data not shown), suggesting that E2 hypermutation constitutes only a minor fraction of the total viral population in individual clinical specimens. Dinucleotide context analysis of C to T substitutions revealed a preference for C that is 50 -flanked by T or C as a target site for hypermutation, both in CIN1 and CIN3 (Fig. 3). Amongst the samples tested by 3D-PCR, the prevalence rate for hypermutation in CIN1, 36.4% (4/11), was higher than that in CIN3, 22.2% (6/27); however, the difference was not statistically significant (P ¼ 0.43, Fisher’s exact test). On the other hand, mutation numbers in the CIN3 samples, 17–29 mutations per clone, were significantly higher than those measured in the CIN1 samples, 8–19 mutations per clone (P ¼ 0.009, Welch’s t-test) (Fig. 4A). In contrast, no difference in age distribution was observed among samples with or without hypermutation (P ¼ 0.55, Welch’s t-test) (Fig. 4B).

Hypermutation in the HPV16 E2 Gene in Clinical Specimens

DISCUSSION

To further substantiate the presence of A/T hypermutation, 3D-PCR products generated at the lowest denaturation temperature were excised from gels and inserted into a plasmid vector followed by cloning and sequence determination. As expected, sequence analysis of individual clones revealed the presence of extensive hypermutation that was extremely biased for substitution of either C to T or guanine (G) to A, when compared to the sequences of the first round PCR products (Fig. 2). Interestingly, whilst the CIN1 samples revealed patterns of both C to T and G to A

This study has demonstrated the presence of A/T hypermutation in CIN1/3 specimens by performing 3D-PCR with subsequent sequence analysis. The patterns of mutation, which are clusters of either C to T or G to A substitutions, are typical of the APOBEC protein family [Conticello, 2008]. A previous study using cultured cervical keratinocytes as a model system to analyze persistent HPV infection proposed a potential role for A3s in introducing hypermutation into the HPV16 genome [Wang et al., 2014]. Interferon-b treatment of cervical keratinocyte W12 cells, which are derived from HPV16-positive

Fig. 1. Differential DNA denaturation PCR analysis of the HPV16 E2 gene in CIN1/3 specimens. Gel electrophoresis analysis of 3D-PCR products from CIN1 (#1, #2, #5, #6, and #7) and CIN3 (#12, #15, #18, #33, #40, and #42) samples. Plasmid indicates the full-length HPV16 genome cloned into the pUC19 vector. Arrow and asterisk indicate the position of the E2 amplicon (213 bp) and the first PCR product (314 bp), respectively. In some samples, smaller products were generated by mis-annealing of the primers to the first PCR amplicon. Denaturation temperatures for each PCR reaction are indicated above.

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TABLE I. Profile of CIN1/3 Specimens for Differential DNA Denaturation PCR #

Histology

HPV type

Age

Tm* (˚C)

Average mutations per clone

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

CIN1 CIN1 CIN1 CIN1 CIN1 CIN1 CIN1 CIN1 CIN1 CIN1 CIN1 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3 CIN3

16 16, 16, 16 16 16 16, 16, 16, 16, 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16

35 36 46 28 29 35 31 37 41 49 32 37 29 40 35 46 30 32 40 35 41 51 34 31 46 38 28 34 33 34 33 27 67 26 35 47 27 33 38 57 37 32

84.2 86.0 86.0 86.0 84.8 84.2 84.2 86.0 86.0 86.0 86.0 81.2 86.0 86.0 81.2 86.0 86.0 82.4 ND** ND** 85.4 86.0 86.0 86.0 86.0 86.0 86.0 ND*** 86.0 ND** 85.4 86.0 83.0 86.0 86.0 86.0 86.0 86.0 86.0 81.2 86.6 83.0

11

52, 55 52

52, 58 18, 42, 84 53 52, 53

8.75 14.25 18.25

26

29

26

17

25.33 17

*

The lowest denaturation temperature to generate the 213-bp E2 DNA as a robust band. E2 DNA not amplified. Short E2 DNA amplified.

**

***

CIN1 lesions, and maintain the HPV16 genome as episomes, leads to induction of several A3 family members, together with concomitant introduction of C to T hypermutation into the E2 gene of HPV16 episomes. Consistent with that in vitro study, the results in this study indicate that the hypermutation

in the E2 gene is a truly physiological phenomenon occurring in natural HPV infection. The dinucleotide preference of C to T substitutions in the E2 gene for TpC and CpC (Fig. 3) strongly suggests the involvement of A3A, 3B, 3C, and 3G in introducing E2 hypermutation [Stenglein et al., 2010; J. Med. Virol. DOI 10.1002/jmv

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CIN1

CIN3

mutation

mutation

#1

11 (4)

#12

26 (4)

#5

9 (2)

#15

29 (4)

9 (1)

#18

26 (4)

8 (1)

#33

17 (4)

12 (1)

#40

25 (4)

#6

17 (1)

26 (1)

15 (1)

26 (1)

#42

13 (1)

#7

17 (6)

18 (2)

50 nucleotides

18 (1)

G to A

C to T

other

19 (1)

Fig. 2. Landscape of nucleotide substitutions in individual clones generated at the lowest denaturation temperature for each sample. substitutions in the coding-strand of the E2 gene are indicated with indicates the number of nucleotide substitutions in each clone. The indicates the number of independent clones sequenced.

Burns et al., 2013; Wang et al., 2014]. Recently, it has been reported that the levels of A3A and 3B mRNA are upregulated in low- and high-grade cervical lesions compared to normal tissue, and that HPV16 oncoproteins E6/E7 are responsible for this upregulation [Warren et al., 2015]. Furthermore, A3B mRNA expression and enzymatic activity are upregulated in normal immortalized keratinocytes harboring a full-length HPV16 or HPV18 genome,

from 3D-PCR products Positions of nucleotide vertical lines. Mutation number in parenthesis

and this effect is attributable to the E6 gene [Vieira et al., 2014]. Because the expression levels of E6/E7 are increased during progression into CIN3 [zur Hausen, 2002], the higher number of mutations observed in CIN3 samples may reflect higher levels and activity of E6/E7, leading to the elevated expression of A3B that would enhance its ability to introduce hypermutation. On the other hand, the detection rate of E2 hypermutation was not

Proportion (%)

70 60

CIN1

50

CIN3

40

expected

30 20 10 0

TpC

GpC

CpC

ApC

Fig. 3. Dinucleotide context analysis of C to T substitutions in 3D-PCR products from CIN1 (#1, #5, and #6) and CIN3 (#12, #15, #18, #33, #40, and #42) samples. Dotted lines indicate the expected proportions of each dinucleotide pair in the E2 coding sequences of the 3D-PCR amplicon in the first PCR product.

J. Med. Virol. DOI 10.1002/jmv

HPV16 E2 Hypermutation in CIN

B

10

50 30

40

Age

Mutation number 15 20 25

60

A

1759

CIN1

CIN3

-

+

Hypermutation

Fig. 4. Profile of E2 hypermutation. (A) Boxplot showing the difference of average mutation number per sample between CIN1 and CIN3 samples. (B) Boxplot showing age distribution among samples with or without hypermutation.

significantly different between CIN1 and CIN3, although the distribution of low-level mutations that cannot be assessed by 3D-PCR remains to be examined. Since the deaminase activity of A3s requires singlestranded DNA as their substrate, viral replication, or transcription provides a window of opportunity for A3s to gain access to the HPV genome and introduce C to U conversions. It is presumed that a G to A conversion occurs in the coding-strand of the E2 gene when A3s target the transcription template-strand for a C to U conversion and then incorporates A versus U in the complementary coding-strand during DNA replication. Although it is not clear why CIN3 samples showed a preference for hypermutation of C to T rather than G to A in the coding-strand of the E2 gene (Fig. 2), it may be related to different modes of viral replication and transcription between CIN1 and CIN3. While CIN1 lesions supports vegetative replication of the HPV genome together with transcriptional activation of the E2 gene in the upper layers of the infected epithelium, viral replication is generally limited, and transcription of the E2 gene relative to E6/E7 is reduced in CIN3 lesions [Xue et al., 2012]. Thus, changes in the balance between viral replication and transcription may have some effect on A3s strand availability. Furthermore, epigenetic changes, such as DNA methylation, and histone modification, which are likely to occur in the HPV genome during persistent infection [Johannsen and Lambert, 2013], may affect viral replication and transcription, and thus also affect the availability of the single-stranded DNA targeted by A3s. The clinical significance of E2 hypermutation was retrospectively evaluated by examining follow-up cytological diagnoses of the CIN1 cases. Among the four CIN1 cases that showed E2 hypermutation, three cases (#1, #5, and #6) exhibited regression to normal cytology, while one case (#7) had no follow-up examination.

Similarly, in the hypermutation-negative CIN1 cases, 5 of 7 cases showed regression, while one case had no follow-up examination, and one case kept low-grade squamous intraepithelial lesions. These results do not suggest a clear association of E2 hypermutation with CIN progression or regression, although further investigation with larger sample sizes is needed. Moreover, all the CIN3 cases had cervical conization except one case that underwent a hysterectomy, which precludes an assessment of clinical relevance of E2 hypermutation in CIN progression into cervical cancer. Lastly, limitations of this study include relatively small sample sizes, making it difficult to understand clinical implications of HPV16 hypermutation for cervical carcinogenesis. Another limitation is the dependence on 3D-PCR for detection of hypermutation, which can only detect heavily A/T-rich DNA compared to the reference. Thus, non-biased methods to determine a full range of HPV genome sequences in clinical specimens, such as deep sequencing, with a large set of samples will be required to comprehensively assess the biological significance of hypermutation and its clinical impact on cervical cancer development in future studies. ACKNOWLEDGMENTS We are grateful to Drs. Tadahito Kanda, Yoshiyuki Ishii, and Takamasa Takeuchi for critical reading of the manuscript. REFERENCES Alexandrov LB,Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, Bignell GR, Bolli N, Borg A, Borresen-Dale AL, Boyault S, Burkhardt B, Butler AP, Caldas C, Davies HR, Desmedt C, Eils R, Eyfjord JE, Foekens JA, Greaves M, Hosoda F, Hutter B, Ilicic T, Imbeaud S, Imielinski M, Jager N, Jones DT, Jones D, Knappskog S, Kool M, Lakhani SR, Lopez-Otin C, Martin S, Munshi NC, Nakamura H, Northcott PA, Pajic M, Papaemmanuil E, Paradiso A, Pearson JV, Puente XS, Raine K, Ramakrishna M, Richardson AL, Richter J, Rosenstiel P, Schlesner M, Schumacher TN, Span PN, Teague JW, Totoki Y, Tutt AN, Valdes-Mas R, van Buuren MM, van ’t Veer L, Vincent-Salomon A, Waddell N, Yates LR, Zucman-Rossi J, Futreal PA, McDermott U, Lichter P, Meyerson M, Grimmond SM, Siebert R, Campo E, Shibata T, Pfister SM, Campbell PJ, Stratton MR. 2013. Signatures of mutational processes in human cancer. Nature 500:415–421. Burns MB, Temiz NA, Harris RS. 2013. Evidence for APOBEC3B mutagenesis in multiple human cancers. Nat Genet 45:977–983. Conticello SG 2008. The AID/APOBEC family of nucleic acid mutators. Genome Biol 9:229. de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B, Tous S, Felix A, Bravo LE, Shin HR, Vallejos CS, de Ruiz PA, Lima MA, Guimera N, Clavero O, Alejo M, Llombart-Bosch A, Cheng-Yang C, Tatti SA, Kasamatsu E, Iljazovic E, Odida M, Prado R, Seoud M, Grce M, Usubutun A, Jain A, Suarez GA, Lombardi LE, Banjo A, Menendez C, Domingo EJ, Velasco J, Nessa A, Chichareon SC, Qiao YL, Lerma E, Garland SM, Sasagawa T, Ferrera A, Hammouda D, Mariani L, Pelayo A, Steiner I, Oliva E, Meijer CJ, Al-Jassar WF, Cruz E, Wright TC, Puras A, Llave CL, Tzardi M, Agorastos T, Garcia-Barriola V, Clavel C, Ordi J, Andujar M, Castellsague X, Sanchez GI, Nowakowski AM, Bornstein J, Munoz N, Bosch FX. 2010. Human papillomavirus genotype attribution in invasive cervical cancer: A retrospective crosssectional worldwide study. Lancet Oncol 11:1048–1056. Henderson S, Chakravarthy A, Su X, Boshoff C, Fenton TR. 2014. APOBEC-mediated cytosine deamination links PIK3CA helical

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Hypermutation in the E2 gene of human papillomavirus type 16 in cervical intraepithelial neoplasia.

Persistent infection with oncogenic human papillomavirus (HPV) causes cervical cancer. However, viral genetic changes during cervical carcinogenesis a...
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