Detection of the human papillomavirus 58 physical state using the amplification of papillomavirus oncogene transcripts assay

Journal of Virological Methods 189 (2013) 290–298

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Detection of the human papillomavirus 58 physical state using the amplification of papillomavirus oncogene transcripts assay Arkom Chaiwongkot a , Chamsai Pientong a,∗ , Tipaya Ekalaksananan a , Svetlana Vinokurova b , Bunkerd Kongyingyoes c , Bandit Chumworathayi d , Natcha Patarapadungkit e , Sumalee Siriaunkgul f , Magnus von Knebel Doeberitz b a

Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany c Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand d Department of Obstetrics and Gynaecology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand e Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand f Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand b

a b s t r a c t Article history: Received 11 June 2012 Received in revised form 17 February 2013 Accepted 21 February 2013 Available online 4 March 2013 Keywords: Human papillomavirus 58 Episome- and integrate-derived transcripts Amplification of papillomavirus oncogene transcripts (APOT)

HPV 58 is detected commonly in cervical cancer in East Asian countries. To evaluate the HPV 58 physical state, the amplification of papillomavirus oncogene transcripts (APOT) and hybridisation assays were established. Episome- and integrate-derived transcripts were confirmed by direct sequencing. Twentynine HPV 58 positive samples from various cervical lesions were used. The results showed that the episome-derived transcripts were recognised as two major specific amplified products (1040 and 714 bp). Two splice donor sites were mapped to the 5 splice site of the E1 gene on SD898 and SD899 and spliced to the 3 acceptor site of the E4 gene on SA3353, SA3356 and SA3365. The episome-derived transcripts were found 100% in normal cervical epithelia and low-grade lesions (9/9 cases) while the integrate-derived transcripts were detected in 13.3% of high-grade lesions (2/15 cases) and in 20% of carcinomas (1/5 cases). HPV 58 integration sites were found on chromosomes 4q21, 12q24 and 18q12. Using the established APOT assay, the results revealed not only novel information on the HPV 58 transcription patterns of episomal transcripts, but also integration site. The APOT assay is a reliable and useful tool for the detection of the HPV 58 physical state and its oncogene expression. © 2013 Elsevier B.V. All rights reserved.

1. Introduction High-risk human papillomaviruses (HR-HPVs) infection is recognised as the direct cause of cervical cancer (Lombard et al., 1998; Walboomers et al., 1999), which remains the second leading cancer in women worldwide. Among the HR-HPV types associated with anogenital cancers, HPV 16 is the most common type (Clifford et al., 2003, 2005; Munoz et al., 2003). HPV 58 is a member of the alpha 9 papillomaviruses, along with HPV 16, 31, 33, 35, 52 and 67, which are also classified as HR-HPV types (de Villiers et al., 2004). Due to its limited geographic distribution, HPV 58 is clinically less prevalent than HPV 16 and 18, with a detection rate of only 3% of cervical cancers worldwide (Clifford et al., 2005). However, the incidence of HPV 58 is enormously high in Asia, Africa and some other areas, where the infection rate has been reported to be up to 13–33%, ranked second to HPV 16 (Chan et al., 1999;

∗ Corresponding author. Tel.: +66 43 348385; fax: +66 43 348385. E-mail address: [email protected] (C. Pientong). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.02.011

De Marco et al., 2006; Gonzalez-Losa Mdel et al., 2004; Paesi et al., 2009). In Asian countries, HPV 58 has been detected frequently not only in cervical cancer but also in normal cervical cells, low-grade squamous intraepithelial lesions (LSILs) and high-grade squamous intraepithelial lesions (HSILs) (Chen et al., 2006; de Sanjose et al., 2007; Ho et al., 2006a; Huang et al., 1997; Hwang, 1999; Inoue et al., 2006; Jeng et al., 2005; Liaw et al., 1997; Lin et al., 1998, 2006; Pham et al., 2003; Shah et al., 2009; Zhao et al., 2009). In Thailand, HPV 58 is the third most common type of cervical cancer (Sriamporn et al., 2006; Suthipintawong et al., 2011). This finding suggests that HPV 58 has a potential to induce malignant transformation. Because persistent HR-HPV infection is the major cause of cervical cancer, HPV DNA testing has been accepted as an adjunctive test for cytological based screening, which has resulted in a substantial reduction in the number of invasive cancer cases. However, the HPV DNA test also identifies many transient infections that are not associated with high-grade cervical intraepithelial neoplasia. Thus, an improvement of its specificity is required to develop a better marker for the prediction of cervical cancer.

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Deregulation of HR-HPV E6 and E7 oncogenes expression in the basal cells of cervical epithelia is a critical event during carcinogenesis, whereby these two oncoproteins disrupt the function of the p53 and retinoblastoma proteins in host cell cycle regulation, which subsequently triggers chromosomal instability and results in malignant transformation (Doorbar, 2006; Duensing et al., 2000; Duensing and Munger, 2003; Munger et al., 2004). The disruption of the E2 gene by viral integration into host chromosome is one example where the transcriptional control of the E2 protein could be abolished. Consequently, integrate-derived transcripts become stabilised because of its co-transcribed cellular sequences (Jeon and Lambert, 1995) and thus, increase viral oncogenes expression level. Viral integration has been studied mostly in cervical samples infected with HPV 16 and 18. Some studies reported that viral integration was found in portions of normal and low-grade lesions when DNA-based techniques detecting the E2 gene or E2/E6 genes ratio were employed, suggesting that it may be an early event during the progression to cancer (Kulmala et al., 2006; Peitsaro et al., 2002). In contrast, recent studies using either RNA-based (APOT) or DNA-based (DIPS-PCR) techniques, detecting viral-cellular flanking sequences revealed that the integration of HPV was detected only in high-grade lesions and invasive cancers (Klaes et al., 1999; Matovina et al., 2009; Vinokurova et al., 2008). Integration frequency of HR-HPVs was type dependent, and a low frequency of integration was found in less aggressive HPV 31 and 33 compared to HPV 16, 18 and 45 (Vinokurova et al., 2008). The latter observations support the theory that integration of HR-HPVs is a late event during cervical carcinogenesis. This finding was supported by laboratory investigations using cervical samples, cell lines and raft cultures. A study using raft cultures showed that E6 and E7 oncoproteins expressed from episomal HPV were sufficient to induce centrosome abnormalities and genomic instability (Duensing et al., 2001). DNA aneuploidy and chromosomal aberrations were detected in cervical cells harbouring episomal HPV (Hopman et al., 2004; Melsheimer et al., 2004). A study using cell lines showed that neoplastic phenotypes can be observed in W12 cells displaying only episome-derived transcripts of HPV 16 after long-term cultures (Gray et al., 2010). The evidence supports that viral integration into host chromosome occurs in cells that already exhibit chromosomal instability, which is driven by the overexpression of the E6 and E7 oncogenes. Various methods have been used to detect the integrated HPV genome including restriction enzyme cleavage, self-ligation, inverse polymerase chain reaction (rli PCR) (Kalantari et al., 2001), ligation-mediated PCR (DIPS-PCR) (Luft et al., 2001), restriction site PCR (Yu et al., 2005), amplification of papillomavirus oncogene transcripts (APOT) (Klaes et al., 1999), random PCR (Choo et al., 1996) and fluorescent in situ hybridisation (Couturier et al., 1991). However, the development of sensitive real-time PCR and E1/E2 PCR techniques have raised some questions about the diagnostic value of those methods because integration of the HPV genome has also been found in some low-grade lesions (Cricca et al., 2009; Kulmala et al., 2006; Peitsaro et al., 2002; Wanram et al., 2009). The APOT assay, that was developed previously for detection of HPV transcripts to determine the physical state of HR-HPV types 16, 18, 31, 33 and 45, has been accepted as the standard technique to detect the HPV physical state (Klaes et al., 1999; Nambaru et al., 2009; Vinokurova et al., 2008). Due to the high prevalence of HPV 58 in cervical cancer in certain regions, its physical state was examined previously by real-time PCR. The integration frequency of two studies on invasive cervical carcinoma showed severely contrasting results, with an incidence rate of 12.5% (1/8) in one study (Ho et al., 2006b) and 93.3% (14/15) in another (Chan et al., 2007). No reliable techniques have been used for evaluating the precise frequency of HPV 58 integration status thus far. Therefore, the aim of this study was to establish the APOT

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assay for the detection of the HPV 58 physical state using 29 samples with different grades of cervical lesions. Transcripts derived from episomal and integrated HPV 58 were expected to be obtained from these pilot samples. Primers and probes were designed and conditions were optimised for the PCR and hybridisation assays. The transcription patterns of episome- and integrate-derived transcripts were analysed by sequencing. 2. Materials and methods 2.1. Clinical samples Cervical biopsy samples were collected from patients referred for colposcopy in the gynaecological outpatient unit of the Srinagarind Hospital, Khon Kaen University, Thailand, and Heidelberg University, Germany. Written informed consent was obtained for all participants, and the study was conducted with the institutional review board approval. The samples were divided into two portions. One part of each sample was embedded in paraffin and retained for histopathological analysis to be reviewed by an experienced pathologist. The second part of each sample was transferred into RNAlater® solution and kept at −80 ◦ C for DNA and RNA extractions. 2.2. DNA and RNA extractions DNA and RNA were extracted from fresh cervical biopsies preserved in RNAlater® solution by using AllPrep DNA/RNA Mini Kit (Gentra Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. To control the integrity of the extracted DNA and RNA, the GAPDH gene was amplified using GAPDH1/GAPDH2 primers (product size 564 bp) (Teeratakulpisarn et al., 2007). 2.3. HPV detection and genotyping The extracted DNA was used for HPV detection using GP5+/6+ primers and genotyping by reverse line blot hybridisation as described previously (van den Brule et al., 2002). Samples from Heidelberg University were used for detection of HPV by hybrid capture. Specific nested RT-PCR was performed for the detection of HPV 58 in samples infected with HR-HPV types (Sotlar et al., 2004). 2.4. Detection of the HPV 58 physical state by APOT assay One ␮g of the extracted RNA was reverse transcribed using an oligo (dT)17 primer coupled to a linker sequence, or (dT)17-P3 (5 GAC TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TT-3 ), and 100 units of an engineered version of M-MLV reverse transcriptase, SuperScriptTM II RT (Invitrogen, Carlsbad, USA) at 42 ◦ C for 1 h in a final volume of 20 ␮l. GAPDH1/GAPDH2 primers were used to control cDNA quality (Teeratakulpisarn et al., 2007). P1-58 and P258-specific forward primers for HPV 58 E7 gene, were designed using the reference sequence accession number EU918765 and GeneFisher2 – Interactive PCR Primer Design. The optimised conditions for the first and second rounds of the APOT assay were as follows. First, PCR was performed using P1-58 (5 -ACT TGT GGC ACC ACG GTT CGT TTG TG-3 ) as a forward primer and P3 (5 -GAC TCG AGT CGA CAT CG-3 ) as a reverse primer. A 50 ␮l amplification reaction contained PCR buffer, 2.0 mmol MgCl2 , 0.2 mmol of each dNTP, 12.5 pmol of each primer and 1.5 U Taq DNA polymerase (Invitrogen, Carlsbad, USA). Amplification was performed using the following parameters: initial denaturation at 94 ◦ C for 3 min; 30 cycles of 94 ◦ C for 40 s, 60 ◦ C for 30 s and 72 ◦ C for 4 min; and final extension at 72 ◦ C for 7 min. Second, 4 ␮l of the first-round amplification product was used as a template in a nested PCR under identical conditions using P2-58 (5 -TTC GTT TGT GTA TCA ACA GTA

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Fig. 1. APOT assay for detection of the HPV 58 physical state. Upper panel is the transcripts derived from episomal and integrated HPV. Adaptor linked oligo (dT) primer is used for reverse transcriptase reaction. P1-58 and P2-58 specific forward primers for HPV 58 E7 are designed and used with the adaptor primers, (P3) and (dT)17-P3, for the first and second round PCRs. Lower panel is the amplification products. H1-58-E7 and H2-58-E4 probes are designed for hybridisation assay. Both E7 and E4 genes can be detected on amplified products derived from either episomal or integrated form (Type B). The integration form (Type A) can be observed as the hybridisation signal corresponding with the E7 specific probe only. Direct sequencing of PCR products is performed to identify the HPV nucleotide sequences and viral-cellular nucleotide sequences.

CAA C-3 ) as a forward primer and (dT)17-P3 as a reverse primer. The amplification cycle was the same to the first-round PCR. To identify the amplified products, two specific probes were designed as follows: HPV 58-E7-specific probe (H1-58-E7, 5 -TGC TTA TGG GCA CAT GTA CCA TTG-3 ) and HPV 58-E4-specific probe (H2-58-E4, 5 -AGT ACA CAG GGG ACA AAG CGA CGA CG-3 ). The probes were labelled by Terminal Deoxynucleotidyl Transferase (TdT) and 5 ␮M Biotin 11-dUTP using the Biotin 3 End DNA labelling kit (Thermo Scientific, Rockfold, USA). The labelling reaction was performed at 37 ◦ C for 30 min; after that, 0.2 M EDTA was added to terminate the reaction. TdT extraction was performed using chloroform:isoamyl alcohol. The top aqueous phase was removed, and the purified probes were used in the hybridisation reaction. PCR products from the nested PCR were subjected to electrophoresis in two parallel 1.2% agarose gels stained with ethidium bromide. The PCR products in each gel were blotted onto a nylon membrane (HybondTM N+, Amersham Life Science, UK). The first membrane was hybridised with the H1-58-E7 probe, and the second parallel membrane was hybridised with the H2-58-E4 probe at 55 ◦ C for 16 h using North2South® Hybridisation Buffer. The probe concentration was 0.1 pmole/ml of hybridisation buffer. After hybridisation, the membranes were washed in North2South® Hybridisation Stringency Wash Buffer at 55 ◦ C three times. After blocking in 0.3% skim milk in PBS at room temperature for 15 min, the membranes were incubated with the stabilised streptavidinhorseradish peroxidase conjugate at 42 ◦ C for 15 min and then washed in wash buffer. Substrate equilibration buffer was added prior to detection using the Chemiluminescent Nucleic Acid Detection Module (Thermo scientific, Rockfold, USA). The physical state of HPV 58 was analysed by visualisation of amplimers hybridised with the specific probes. The amplimer of major episome-derived transcripts was approximately 1000 bp in length and hybridised with both of HPV 58-E7-specific and HPV 58-E4-specific probes. In contrast, the amplimers that displayed different sizes from the major E7-E1Eˆ 4 episomal transcripts

were suspected to be transcripts derived from the integrated HPV genomes. 2.5. Sequence analysis of amplified products The PCR products corresponding to hybridised amplimers were excised from the agarose gel and extracted using the QIAquick Gel Extraction Kit (Qiagen, Hilden Germany). The purified products were sequenced directly using the Big-Dye terminator DNAsequencing Kit (Perkin-Elmer, Boston, USA) and then analysed by an ABI Prism 310 Genetic analyser (Applied Biosystems, Foster City, USA). The sequence results were analysed using BLASTN program provided by the National Center for Biotechnology Information, USA. 3. Results 3.1. Optimisation of APOT assay for HPV58 The principle of the APOT assay, which is an RNA-based method, is shown in Fig. 1. It can be used for both detection of the HPV physical state and oncogenes expression from cervical biopsies and swab samples (Klaes et al., 1999; Vinokurova et al., 2008). An oligo (dT)17 primer coupled to a linker sequence, (dT)17-P3, is used to synthesise the cDNA. A nested PCR with P1-58 and P3 primers in the first round and P2-58 and (dT)17-P3 primers in the second round is performed with synthesised cDNA to serve as a template. The expected size of episomal HPV 58 obtained from the major episomal transcript (E7-E1Eˆ 4) is ∼1000 bp; amplimers which displayed a size different from the major product size of ∼1000 bp may be derived from the integrated HPV genome. The physical state is investigated by southern blot hybridisation, using specific probes for E7 (H1-58-E7) and E4 (H2-58-E4). The episomal form is recognised as the ∼1000 bp amplimer, which hybridised with both E7- and E4-specific probes. The integration form can be observed with the hybridisation signal corresponding with the E7 specific probe in Type A only or can

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appear to be positive for both genes in Type B. The sequencing of the amplified products is necessary to distinguish between episomal and integration form. To optimise the PCR condition of the APOT assay, HPV 58 positive samples diagnosed as normal cervical epithelia and were expected to contain the episomal HPV 58 genome were used. The optimal annealing and hybridisation temperatures for nested PCR and southern blot that yielded the expected episomal transcript amplimer size of ∼1000 bp were 60 ◦ C and 55 ◦ C, respectively. No amplification products were detected in the reaction using cDNA prepared from SiHa and C33A cells, which indicated the high specificity of the primers used (data not shown). 3.2. Analysis of the HPV 58 physical state in clinical specimens To obtain transcripts derived from episomal and integrated HPV 58, an APOT assay with its optimised conditions was performed using clinical samples. Fifty HPV 58 positive patients were included initially in this study and that 29 (58.0%) yielded amplifiable transcripts. The 29 clinical samples included normal cervical epithelia (4 cases), low-grade lesions (5 cases), high-grade lesions (15 cases) and squamous cell carcinoma (5 cases). Fig. 2A shows representative examples of amplified products obtained from 12 samples of various cervical lesions. The amplified product size ∼1000 bp, expected to be the episome-derived transcript was detected in most samples (24/29 cases). The product size of ∼700 bp was detected mostly in the normal cervical epithelia (3 of 4 cases; 75%), followed by high-grade lesions (7 of 20 cases; 35%), but was not found in carcinoma. Three samples displayed only the ∼700 bp product size, without co-amplification with ∼1000 bp. Three samples displayed different product sizes (approximately 300, 500, 400, 600 and 900 bp), one of them displayed co-amplification with ∼1000 bp. Product bands >2000 bp and ∼200 bp were detected in more than half of the samples. Amplimers with sizes different from ∼1000 bp were suspected to be integrate-derived transcripts. Southern blot hybridisation was performed using specific probes for E7 (H1-58-E7) and E4 (H2-58-E4) to discriminate between the amplified products of the episome- and integratederived transcripts. Fig. 2B and C show the amplified products with sizes >2000, ∼1000, and ∼700 bp that hybridised with both the E7 and E4 probes. These results were indicative of the episome-derived transcripts. However, the accompanying >2000 and ∼700 bp amplimers suggested that they may be derived from integrated transcripts type B, which have been shown previously by the detection of amplified products derived from the integrated HPV 16 in Caski cells and the integrated HPV 18 in SW756 cells (Klaes et al., 1999). The product bands, which are recognised by the E7-specific probe only, were expected to indicate integratederived transcripts. A sequencing assay was performed further to distinguish between the physical states of these samples. 3.3. DNA sequencing To confirm the results of the southern blot hybridisation, the amplified products from the 29 samples (45 amplimers) that exhibited strong and intense bands in the agarose gel and were at least 200 bp or greater in length were sequenced directly and analysed. Sequence analysis showed that the amplified products with a size of ∼1000 bp contained only the HPV 58 nucleotide sequences, including the E7, E1 and E4 genes; these sequences were obtained from the episome-derived transcripts with the poly(A) signal AATAAA at nucleotide 4209 (poly(A) signal 4209–4214) and corresponded with the results of a previous study (Kirii et al., 1991) in which the poly(A) sequences were found after nucleotide 4230. The >2000 bp product size is assumed to be a result of mis-annealing of the oligo (dT)17-p3 to an adenosine rich (A-rich) region within

Fig. 2. Amplified products by the APOT assay and southern blot hybridisation results of 12 HPV 58 positive clinical samples. (A) 1.2% agarose gel electrophoresis after APOT-PCR. M is 100 bp DNA Ladder. (B and C) Southern blot hybridisations with HPV 58 E7 and E4 specific probes, respectively. Lanes 1–3: normal cervical epithelia; lanes 4–6: low-grade lesions; lanes 7–11: high-grade lesions; lane 12: squamous cell carcinoma. The product size ∼1000 bp, the expected amplimer of major episomal transcript is hybridised with both the E7 and E4 probes (B and C). Amplimers suspected to be derived from integrated transcripts type A (dash arrows) show positive signals only with the E7 probe (B). Amplimers >2000 bp and ∼700 bp, hybridised with both probes, are suspected to be derived from either episomal transcripts or integrated transcript type B. The sequencing assay is further performed to distinguish between episomal and integration form. Block arrows showed one amplified product which hybridised both to the E7 and E4 probes, but sequencing was not performed due to weak intensity of band in the agarose gel.

the E2 gene (3071–3090) of non-spliced transcripts (Fig. 3A). The results of the hybridisation assays indicate that a >2000 bp product size showed a positive signal for both the E7 and E4 genes; the sequencing results revealed that this amplimer contained the same E7, E1, E4 and poly(A) sequences as the ∼1000 bp product. We observed that the signal of the E4 gene was less intense than that of the E7 gene (Fig. 2B and C). It is possible that this >2000 bp product size contained two amplified products, with one of these products deriving from the mis-annealing of the oligo (dT)17-p3 to an A rich region, as mentioned above, and the other product resulting from the difference in structure of the large volume of ∼1000 bp amplified products, which is detected as a large smear and two different bands in the agarose gel after electrophoresis and in the hybridisation assay (Fig. 2A–C). The ∼700 bp product size contained only the HPV 58 nucleotide sequences, including those of the E7, E1 and E4 genes in which the poly(A) sequences were found after nucleotide 3904. From these results, the accurate amplified sizes of HPV 58 episome-derived transcripts were 1040 bp (770–898 and 3353–4230+ 35 bp of (dT)17-p3 primer) and 714 bp (770–898 and 3353–3904+ 35 bp of (dT)17-p3 primer).

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Fig. 3. (A) HPV 58 sequence map with poly(A) signal and possible binding sites for oligo (dT)17-P3, leading to various amplified products. Amplified product sizes >2000 bp and 460–490 bp are suspected to be generated from mis-annealing of the oligo (dT)17-P3 during cDNA synthesis to an adenosine-rich (A-rich) region within the E2 gene (3071–3090) and the E1 gene (1216–1230 and 1241–1260) of non-spliced transcripts, respectively. (B) Episome-derived transcript types and their expected amplified products after APOT-PCR. Two major amplified products (1040 and 714 bp) are derived from the spliced transcripts, E1 sequence splice donor site was at nucleotide 898 and 899 that were spliced to the E4 sequence at acceptor site at nucleotides 3353, 3356 and 3365. A poly(A) signal is found at nucleotide 866, leading to the amplification of a <200 bp product.

Amplified products ∼200 bp revealed E7 and E1 genes followed by poly(A) sequences which were found after nucleotide 887 and 898. A poly(A) signal was found at nucleotide 866; therefore, the actual size of the amplified products was determined to be approximately 152–163 bp. Fig. 3A shows the HPV 58 sequence map with various product sizes obtained from both non-spliced transcripts and major episomal transcripts. The transcript types and splicing patterns of the episomal HPV 58 genome are shown in Fig. 3B. The common donor

site of nucleotide 898 was found in 26 of the 27 episomal samples (96%). Only one sample was found to have a donor site at nucleotide 899. Three different acceptor sites were located: nucleotides 3353 in 92% (25 of 27), 3356 in 4% (1 of 27), and 3565 in 4% (1 of 27), respectively. One of the 27 samples displayed both episome- and integrate-derived transcripts. Sequence analysis was performed on the amplified products with different sizes that were obtained from three different samples (size range from 300, 400, 500, 600 and 900 bp) and were suspected

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Fig. 4. (A) Integrate-derived transcript types. E1 sequence donor site was at nucleotides 898 and 899 that were spliced to acceptor sites on host sequences at different positions in each chromosome. (B) Fusion transcripts of three integration samples. The top diagram of each sample shows the fusion transcript in the 5 to 3 direction. The bottom shows the integrated HPV 58 DNA and host genomic sequences. The two-head arrows indicate varying distances between splice donor sites of viral genomes and splice acceptor sites of the host genome. Dark grey boxes indicate HPV 58 DNA sequences. Light grey boxes indicate exons, introns and intergenic sequences. Black boxes indicate host DNA sequences. Sample 1 shows a transcript spliced to an intron region located upstream of exon 2 of DYNLL1 gene within chromosome 12. Sample 2 shows a transcript spliced to a region between KC6 and ribosomal RPL17P45 within chromosome 18. Sample 3 shows a transcript spliced to a region between CXCL6 and IL8 within chromosome 4. DYNLL1: dynein, light chain, LC8-type 1; RPL17P45: RPL17P45 ribosomal protein L17 pseudogene 45; IL8: Interleukin 8.

to be the integrate-derived transcripts. These products contained HPV 58 E7 and E1 sequences that were flanked by the cellular sequences. The splicing donor sites found at nucleotides 898 and 899 were in the same pattern as the episome-derived transcripts and were spliced to the acceptor sites on the host sequences. The integrate-derived transcripts were detected in two samples from high-grade lesions and in one sample from a carcinoma, which was of mixed form. Fig. 4A demonstrates that two integrate-derived transcripts showed splicing donor sites at nucleotide 898 and one sample at nucleotide 899 of E1 that spliced to acceptor sites on host sequences, indicating transcript type fusion A. The integration sites of the three samples were found at chromosomes 4q21, 12q24 and 18q12 (Fig. 4B). The results showed that integrate-derived transcripts were not detected in normal cervical epithelia and in low-grade lesion (0 of 9 cases), whereas these transcripts were found in 13.3% (2 of 15 cases) and in 20% (1 of 5 cases) of high-grade lesions and carcinomas, respectively. One carcinoma displayed both episome- and integrate-derived transcripts.

4. Discussion The complete HPV 58 genome was published, and its sequences were found to be closely related to the following HPV types: HPV 16, 31, 33, 35, 52 and 67, which have been classified as group 9 alpha papillomaviruses or carcinogenic types (de Villiers et al., 2004; Kirii et al., 1991). HPV 58 infection was shown to be associated with cervical cancer, especially in Eastern Asian countries such as China and Thailand (Chan et al., 1999; Huang et al., 1997; Sriamporn et al., 2006) and in some countries such as Mexico and Costa Rica (Gonzalez-Losa Mdel et al., 2004; Herrero et al., 2005).

Although the presence of HPV 58 has been shown to correlate strongly with premalignant and cancerous lesions, particularly in Asian countries, the information on the integration status of HPV 58 is limited. Inconsistent results relating to the integration frequency of HPV 58 were reported with the use of conventional and real-time PCR (Chan et al., 2007; Ho et al., 2006b; Wu et al., 2009). The study in Taiwan showed that HPV 58 integration was found only in 12.5% (1/8) of cervical cancers using real-time PCR (Ho et al., 2006b). In contrast, a recent study in China using multiple sets of primers to amplify all regions of the HPV 58 genome showed pure integration form in 78.4% (29/37) of cervical cancers (Wu et al., 2009). Consistent with these results were those obtained in Hong Kong using real-time PCR, where the integration frequency was found to be 93.3% (14/15) in invasive cervical carcinoma (Chan et al., 2007). The discrepancy may be due to the amplification-based methods that detected the E2 gene or the ratio of the E2 and E6E7 genes for which the amplification efficiency of each primer pair and ratio value should be optimised for each run. DNA-based (Choo et al., 1996; Kalantari et al., 2001; Luft et al., 2001; Yu et al., 2005) and RNA-based (Klaes et al., 1999) methods have also been established for the detection of integration status of HR-HPV types by analysis of viral-cellular flanking sequences. The APOT assay, the RNA-based method (Klaes et al., 1999; Vinokurova et al., 2008) is recognised as reliable and easy to perform technique compared to DNA-based methods such as DIPS-PCR (Luft et al., 2001) and rli PCR (Kalantari et al., 2001), which require restriction enzyme digestion and a ligation step. Sequence analysis of the amplified products from the APOT assay can distinguish the physical state of the virus by the identification of episome- or integrate-derived transcripts. The APOT assay can be performed not only on RNA extracted from biopsy specimens but also on RNA extracted from cervical swabs (Klaes et al., 1999; Vinokurova et al., 2008).

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In the present study, an APOT assay was established and tested for its applicability to detect the HPV 58 physical state using 29 HPV 58 positive samples. Episome- and integrate-derived transcripts were expected to be obtained from these samples. Compelling information was found in the samples with the episomal form; two common E7-E1Eˆ 4 transcripts corresponding to amplimer sizes 1040 and 714 bp were detected. The 1040 bp product size, in most cases, could be attributed to the expected amplimer of the episomal form. The second amplimer, the 714 bp product, was an unexpected size found commonly in the precancerous samples. For this amplified product, the T rich region after nucleotide 3904 might be recognised by enzymes that are responsible for adding the poly(A) tail. It is also possible that there might be a poly(A) signal upstream of the T rich region, such as TGTAAA, AATAT and GCTAA (Fig. 3A). Sequencing analysis confirmed the results of the hybridisation assay and provided reliable data to determine the physical state. To identify the episomal form of the HPV 58 physical state, at least one of two common amplified products (1040 and/or 714 bp) must be detected. Obtaining an amplified product with the size of <200 bp without splicing to the E4 gene, is not sufficient to identify the HPV physical state as the episomal form. The product size >2000 bp is suspected to be derived from mis-annealing of oligo (dT)-p3 to an A-rich region within the E2 gene (3071–3090) of non-spliced transcripts (Fig. 3A) and is considered as artefact. To identify the integrated HPV genome, viral-cellular flanking sequences must be detected by sequencing analysis. Although there are advantages of the APOT assay, there are also limitations that should be addressed here. It may be difficult to amplify very long integrate-derived transcripts; however, we have not tested this issue in the APOT-PCR reaction. Samples with the mixed form, which contain large amounts of episome-derived transcripts, may lead to difficulty in amplifying the reduced levels of integrate-derived transcripts. However, the quantification of episomal/integration transcripts ratio for testing the amplification efficiency was not performed in the present study, and it was difficult to quantify the ratio in the samples exhibiting both episomal- and integrate-derived transcripts. The other explanation is that the E2 expressed from the episomal HPV can repress viral oncogene transcription activity of the integrated HPV genome, as shown in previous studies using cell lines (Bechtold et al., 2003; Dowhanick et al., 1995; Hwang et al., 1993). One study using both APOT and DIPS-PCR assays showed that in samples containing both episomal and integration forms of either HPV 16 or 18, as measured by the DIPS-PCR assay, solely episomal-derived transcripts were detected by the APOT assay in some samples with the mixed form (Ziegert et al., 2003); large amounts of episomal transcripts or repression of the E2 protein may be the cause. It has been shown that loss of the episome HPV genomes can lead to the expression of integrate-derived transcripts that were repressed previously by the E2 protein expressed from the episomal HPV (Herdman et al., 2006). Integrated HPV genomes may be silent by epigenetic modifications, such as through the methylation of long control region (LCR) that can be observed in Caski cells in which only one of many tandemly repeated integrated HVP 16 genomes was transcriptionally active (De-Castro Arce et al., 2012; Van Tine et al., 2004). Silencing by cellular sequences can also occur at sites close to the integrated HPV genome (von Knebel Doeberitz et al., 1991). As shown previously, SW756 cells containing the tandem repeats of the integrated HPV 18 without disruption of the E4 gene, could express transcript similar to the episomal transcript resulting in amplimer ∼1000 bp (Klaes et al., 1999). Due to several possible explanations mentioned here, precise integration frequency could be obtained using DNAbased assays to detect viral-cellular flanking sequences. However, it was reported by one study using the DIPS-PCR assay that the integration frequency of HPV 16 was found only in 11.3% of high-grade

lesions (Matovina et al., 2009), which is similar to the integration frequency detected by the APOT assay (Vinokurova et al., 2008). Although the sample size presented in the present study was small, it may be that the low integration frequency of HPV 58 in high-grade lesions and carcinoma cases represents the low oncogenic capacity that is similar to HPV 31 and 33, as shown in a previous study (Vinokurova et al., 2008). HPV 58 E6 and E7 oncoproteins could disrupt the normal function of p53 and retinoblastoma proteins, while inducing less chromosomal instability compared to HPV 16 and 18. The integration of HR-HPV types could occur as a consequence of chromosomal instability. It has been shown that E6 and E7 oncoproteins expressed from episomal HPV were sufficient to induce genomic instability (Duensing et al., 2001). Epigenetic changes at HPV LCR may be involved in deregulating viral oncogene expression from the episomal HPV, as shown in W12 cells (Gray et al., 2010). Episomal transcripts of HPV 58 produced two bands of different sizes (1040 and 714 bp), which is an interesting finding. Accordingly, these results should be studied further to investigate the stability of the transcripts and/or the function of the E6 and E7 oncoproteins that were derived from both transcripts, particularly because the transcript resulting in the 714 bp was found only in precancerous lesions. In conclusion, this study established that the APOT assay, with new designed primers and probes, could be used for the discrimination of the HPV 58 physical state. The results of using the APOT assay also revealed novel information on the HPV 58 transcription patterns. Therefore, the APOT assay can serve as a valuable tool not only to study the physical state of HPV 58 but also to study its carcinogenesis. The precise frequency of HPV 58 integration status should be studied further using a larger sample size. Acknowledgements This work was supported by the Khon Kaen University research grant 2009–2011 and a graduate student scholarship from the Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. The authors are thankful to the Department of Applied Tumor Biology, Institute of Pathology, Heidelberg University for both supporting and providing the facilities to perform this research. References Bechtold, V., Beard, P., Raj, K., 2003. Human papillomavirus type 16 E2 protein has no effect on transcription from episomal viral DNA. J. Virol. 77, 2021–2028. Chan, P.K., Li, W.H., Chan, M.Y., Ma, W.L., Cheung, J.L., Cheng, A.F., 1999. High prevalence of human papillomavirus type 58 in Chinese women with cervical cancer and precancerous lesions. J. Med. Virol. 59, 232–238. Chan, P.K., Cheung, J.L., Cheung, T.H., Lo, K.W., Yim, S.F., Siu, S.S., Tang, J.W., 2007. Profile of viral load, integration, and E2 gene disruption of HPV58 in normal cervix and cervical neoplasia. J. Infect. Dis. 196, 868–875. Chen, C.A., Liu, C.Y., Chou, H.H., Chou, C.Y., Ho, C.M., Twu, N.F., Kan, Y.Y., Chuang, M.H., Chu, T.Y., Hsieh, C.Y., 2006. The distribution and differential risks of human papillomavirus genotypes in cervical preinvasive lesions: a Taiwan Cooperative Oncologic Group Study. Int. J. Gynecol. Cancer 16, 1801–1808. Choo, K.B., Chen, C.M., Han, C.P., Cheng, W.T., Au, L.C., 1996. Molecular analysis of cellular loci disrupted by papillomavirus 16 integration in cervical cancer: frequent viral integration in topologically destabilized and transcriptionally active chromosomal regions. J. Med. Virol. 49, 15–22. Clifford, G.M., Smith, J.S., Plummer, M., Munoz, N., Franceschi, S., 2003. Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. Br. J. Cancer 88, 63–73. Clifford, G.M., Rana, R.K., Franceschi, S., Smith, J.S., Gough, G., Pimenta, J.M., 2005. Human papillomavirus genotype distribution in low-grade cervical lesions: comparison by geographic region and with cervical cancer. Cancer Epidemiol. Biomarkers Prev. 14, 1157–1164. Couturier, J., Sastre-Garau, X., Schneider-Maunoury, S., Labib, A., Orth, G., 1991. Integration of papillomavirus DNA near myc genes in genital carcinomas and its consequences for proto-oncogene expression. J. Virol. 65, 4534–4538. Cricca, M., Venturoli, S., Leo, E., Costa, S., Musiani, M., Zerbini, M., 2009. Disruption of HPV 16 E1 and E2 genes in precancerous cervical lesions. J. Virol. Methods 158, 180–183.

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