Ethics Statement

Human cervical exfoliated cells were collected from patients at NTT Medical Center Tokyo with written informed consent. The study adhered to the declaration of Helsinki and was approved by the Ethics Committee at NTT Medical Center Tokyo.

Cell culture

W12 cell clones 20850 (5 to 50 copies of HPV16 episomes per cell) and 20863 (500 to 1,000 copies of HPV16 episomes per cell) were cultured in the presence of mitomycin C-treated 3T3m feeder cells as described previously [17,18]. CaSki and human embryonic kidney 293 (HEK293) cells were cultured at 37°C with 5% CO₂ in DMEM supplemented with 10% fetal bovine serum, 50 U/mL of penicillin, and 50 µg/mL of streptomycin.

Extraction of episomal and total DNA

To extract episomal HPV DNA from W12 cells, a modified Hirt method was used [19]. Briefly, subconfluent cells on a 6-cm dish were digested with 500 μL of lysis buffer consisting of 25 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.6% SDS, and 50 μg/mL of RNase A at room temperature for 5 min. After the addition of 350 μL of precipitation buffer consisting of 3 M CsCl, 1 M potassium acetate, and 0.67 M acetic acid, the solution was incubated on ice for 15 min, and precipitated by centrifugation (14,000 x g, 15 min, 4°C). Cleared supernatant was applied onto a Miniprep column (Favorgen, Ping-Tung, Taiwan) and passed through the column by centrifugation (14,000 x g, 1 min). The column was then washed with 750 μL of wash buffer consisting of 10 mM Tris-HCl (pH 7.5), 80 mM potassium acetate, 40 μM EDTA, and 60% ethanol. DNA was eluted with 60 μL of elution buffer consisting of 10 mM Tris-HCl (pH 7.5) and 0.1 mM EDTA. The resultant DNA fraction mainly contains extrachromosomal DNAs.

Total DNA was isolated from cervical exfoliated cells using the QIAamp DNA blood kit (QIAGEN, Hilden, Germany). The DNA samples were genotyped by PGMY-reverse blot hybridization assay [20,21], and HPV16-positive samples without other detectable HPVs from LSIL and ICC specimens were used for subsequent PCR. Total DNA was extracted from CaSki cells as described above for clinical samples. The concentrations of extracted DNAs were about 80 ng/μL from the cultured cells and 2 to 4 ng/μL from clinical samples.

Full-circle PCR

PCR was performed in 10 μL consisting of 1× PrimeSTAR^® GXL Buffer (Takara, Ohtsu, Japan), 200 μM dNTP mixture, 0.25 μM forward primer, 0.25 μM reverse primer, 0.25 U of PrimeSTAR^® GXL DNA polymerase (Takara), and 2 to 4 ng of template DNA. Primers used are as follows: 1742F, 5’-tgc tgt cta aac tat tat gtg tgt ctc-3’; 1873R, 5’-gcg tgt ctc cat aca ctt ca-3’; 1869F, 5’-cac gcc aga atg gat aca aa-3’; 1746R, 5’-cag caa ttt ttc aat tgt ttc tc-3’. Positions of 5’-end nucleotide of primers in the reference HPV16 genome (HPV16REF in Papillomavirus Episteme, http://pave.niaid.nih.gov) are used as primer names with either forward (F) or reverse (R) orientation. The plasmid containing the full-length HPV16 genome, HPV16/pUC19 [22], was used as a template for positive PCR. The PCR protocol was 98°C for 30 s, followed by 30 cycles of 98°C for 10 s, 60°C for 15 s, 68°C for 2 min. The PCR products were separated on a 0.7% agarose gel in 1 x TAE buffer and visualized with ethidium bromide.

Deep sequencing

Approximately 8-kb amplicons generated by full-circle PCR with the primer-pair 1742F/1873R were separated on a 0.7% agarose gel and purified using the Wizard gel purification kit (Promega, Madison, WI). To prepare DNA libraries compatible with Illumina NGS, the purified DNA (16 ng) was fragmented and adaptor-ligated using the Nextra DNA sample prep kit (Epicentre, Madison, WI), followed by DNA purification with the QIAquick PCR purification kit (QIAGEN). The resultant DNA libraries were amplified using a minimal PCR amplification step with Phusion High-Fidelity DNA polymerase (New England Biolab, Ipswich, MA) as follows: 72°C for 3 min, 95°C for 30 s, followed by 13 cycles of 95°C for 10 s, 62°C for 30 s, 72°C for 3 min. Adaptor-ligated DNAs in the range of 500 to 700 bp were then size-selected by 1% agarose gel electrophoresis and purified using the Wizard kit. Five to 8 libraries from different clinical samples were multiplexed with adaptors containing different index sequences and simultaneously subjected to deep sequencing. Cluster generation and sequencing were performed for 151 cycles on a MiSeq sequencer (Illumina, San Diego, CA) with the MiSeq Reagent kit (300 cycle) (Illumina) according to the manufacturer’s instructions. Fluorescent images were analyzed using the Illumina MCS1.1/RTA1.13.56 base-calling pipeline to obtain FASTQ-formatted sequence data. To construct contiguous DNA sequences (contigs), total sequences of paired-end 151-mer reads were assembled using ABySS-pe v1.3.3 [23] with the following parameters: k80, n1200, c1000, t10, e10, q20, and s160; or k80, n120, c100, t10, e10, q20, and s160. The assembly generated 2 to 20 contigs, followed by further assembly of these sequences into the complete circular sequence by Phrap [24].

The fidelity of de novo assembly was examined by alignment of paired-end reads to the assembled sequence using Burrows-Wheeler Aligner (BWA) v0.6.1 [25]. Prior to read mapping, read sequences of Phred quality score >20 were selected by PoPoolation v1.2.2 [26], and trimmed for 20 bases at 5’-terminus and 51 bases at 3’-terminus because 5’ and 3’-terminus of reads generate relatively low fidelity sequences. The processed 80-base sequences were mapped to the assembled sequence as a paired-end mode by BWA. The mapping results were visualized by Integrative Genomics Viewer (IGV) v2.0.34 [27] or Tablet v1.12.12.05 [28]. The numbers of paired-end reads obtained from each sample and used for de novo assembly are described in Table 1, and the average depth of the HPV16 genome ranged from x 7,624 for ICC sample 6 to x 48,221 for W12 sample. Nucleotide mismatches compared to the assembled reference genome and positions of SNPs in each sample were identified by using SAMtools v0.1.18 [29] with in-house Perl scripts (available upon request) or VarScan v2.3.2 [30]. Based on a quality score confidence threshold of Phred quality score > 30 (error probability < 0.001) that was used to extract variation positions in the read sequences, we defined a position as homogeneous if the mutation frequency is < 0.5% and a position to be heterogeneous if the mutation frequency is > 0.5%. The presence of nucleotide substitutions and insertions/deletions was finally verified by visual inspection of mismatched read sequences using IGV and Tablet.

Transient HPV16 replication assay

An HPV16 origin-positive plasmid was constructed by insertion of a PstI fragment of the HPV16 genome (from 7,005 to 7,906 and 1 to 880) into pGL4.50 (Promega). Expression plasmids for N-terminal FLAG-tagged HPV16 E1 and N-terminal FLAG-tagged HPV16 E2 were constructed by cloning of the codon-optimized cDNA of E1 and E2 into pCMV-β (Clontech, Palo Alto, CA). Expression plasmids for E1 mutants were generated by using the QuickChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies, La Jolla, CA). Transient replication assays were performed as described by Fradet-Turcotte et al. [31] with some modifications. Briefly, HEK293 cells were plated 20 h before transfection in a 24-well plate at a density of 32,000 cells/well, and transfected with 2 ng of the origin-positive plasmid and 2 ng of pGL4.75 (Promega), together with 20 ng of the E2 expression plasmid and the indicated amounts of E1 expression plasmid using the FuGENE6 reagent (Promega). The total quantity of transfected plasmid DNA was adjusted to 100 ng with the empty plasmid p3xFLAG-CMV10 (Sigma-Aldrich, St. Louis, MO) as carrier DNA. At 72 h after transfection, firefly and Renilla luciferase activities were measured using the Dual-Glo Luciferase assay system (Promega) on an ARVO MX luminescence counter (PerkinElmer, Waltham, MA), and the level of replication was quantified as the ratio of the two luciferase activities.

Nucleotide sequence accession number

All assembled HPV genome sequences have been deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers AB818687 to AB818693 for HPV16, AB819272 to AB819274 for HPV52, and AB819275 to AB819279 for HPV58. The sequence reads of the HPV genomes are available from the DDBJ Sequence Read Archive under accession number DRA001083.

Full-circle PCR amplifying full-length HPV16 genome sequences

Using PrimeSTAR^® GXL polymerase, which is specifically designed for the fast and accurate amplification of long DNA sequences, and the HPV16-specific primers 1742F and 1873R, we generated two amplicons from HPV16/pUC19 plasmid DNA: a short fragment of ~130 bp and a large fragment consisting of the entire HPV16/pUC19 sequence of approximately 11 kb (with ~130-bp overlap) (Figure 1A, lanes 2 to 7, and see Figure 1B). This PCR reaction is slightly less efficient at creating the large amplicon than the same reaction using the non-overlapping primers 1869F and 1746R, which create only one amplicon (Figure 1A, lanes 9 to 14). The reactions with PrimeSTAR^® GXL polymerase were both specific (only 1 or 2 PCR amplicons, depending on the primers used) and sensitive (we successfully amplified as little as 0.01 pg of input template DNA, which corresponds to about 900 copies of the plasmid). The introduction of cellular DNA did not inhibit this reaction, even at the sub-picogram level of template DNA (Figure S1).

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Figure 1. Amplification of full-length HPV16 genomes by full-circle PCR.

(A) PCR was performed with PrimeSTAR^® GXL DNA polymerase and HPV16-specific primer-pairs as indicated. The amounts of HPV16/pUC19 used for the PCR template are also indicated above. The PCR products were analyzed by agarose gel electrophoresis. M, DNA size markers. (B) Scheme for full-circle PCR. PrimeSTAR^® GXL DNA polymerase generates short and long DNA products with primer-pair 1742F/1873R. (C) Full-circle PCR with DNA extracted from W12 cells, clone 20863 (high-copy HPV16 episomes) (lane 2) and clone 20850 (low-copy HPV16 episomes) (lane 3). M, DNA size marker (lanes 1) (D) Full-circle PCR using DNA isolated from 7 clinical specimens: 5 LSIL (lanes 2 to 6), and 2 ICC (lanes 7 and 8). M, DNA size marker (lanes 1).

https://doi.org/10.1371/journal.pone.0080583.g001

Using the primer-pair 1742F/1873R and PrimeSTAR^® GXL polymerase, we also amplified two DNA fragments from extrachromosomal HPV16 DNA isolated from W12 cells that were originally isolated from an LSIL biopsy (Figure 1C). The short and long amplicons matched the predicted sizes of 132 bp and ~8 kb, respectively (Figure 1B), and the long amplicon covered the complete HPV16 genome, as confirmed by Sanger sequencing (results not shown). Hence we refer to this overlapping PCR hereafter as full-circle PCR. To further test the utility of full-circle PCR with PrimeSTAR^® GXL polymerase, DNA prepared from CaSki cells, known to contain 60 to 600 copies of integrated tandem repeated HPV16 genomes [32,33], was used as the PCR template. As expected, a mixture of 8 and 6.5-kb amplicons were obtained (Figure S2).

Importantly, full-circle PCR was able to generate approximately 8-kb amplicons using DNA isolated from HPV16-positive cervical exfoliated cells in patients with either LSIL (Figure 1D, lanes 2 to 6) or ICC (lanes 7 and 8). This observation prompted us to use these long PCR products for genetic variation analyses of full-length HPV16 genomes derived from clinical samples.

De novo assembly of full-length HPV16 genome sequences by deep sequencing

In order to comprehensively analyze HPV genetic variation through deep sequencing, we sought a methodology capable of determining the full-length reference sequence of the HPV16 genome from high-throughput short-read sequence data. First, to develop the system, the 8-kb DNA amplified from W12 DNA by full-circle PCR was fragmented and adaptor-ligated to yield a DNA library compatible with Illumina next generation sequencers. Deep sequencing of the resultant library generated sequence data from more than a million paired-end reads. De novo assembly of those paired-end reads by ABySS-pe and Phrap generated one contiguous sequence of 7,904 bp length, which was a perfect match for the full-length W12 HPV16 genome sequence in GenBank (accession No. AF125673). Next, the long PCR products from CaSki DNA were subjected to deep sequencing analysis. The de novo assembly procedure once again resulted in construction of one contiguous sequence of 7,905-bp length, identical to the full-length CaSki HPV16 genome sequence in GenBank (accession No. U89348). Mapping of the total read sequences to the CaSki reference sequence demonstrated the presence of two large deletions; from nucleotide positions 1 to 470 and 6,907 to 7,905 (Figure S3), which perfectly matched a deleted region previously reported in CaSki cells [33]. Thus, it is likely that the 6.5-kb DNA products obtained from CaSki DNA contain this 1,469-bp deletion. Overall, these results demonstrate the accuracy of long PCR, deep sequencing and bioinformatics to obtain full-length HPV16 genome sequences.

To determine the complete genome sequence of HPV16 contained in clinical specimens, we analyzed the 8-kb full-circle PCR products from 7 clinical samples by deep sequencing and assembled the complete viral genome sequence as described above. Table 1 presents the results of this analysis and demonstrates that de novo assembly yielded one contiguous sequence for each sample. The length of each sequence ranged from 7,903 to 7,907 bp due to the differences in the length of the non-coding region between E5 and L2 (Figure S4).

The HPV16 genome is classified into 4 major variant-lineages based on a unique combination of single nucleotide polymorphisms (SNPs): European-Asian, including the sublineages European (EUR) and Asian (As), African 1 (AFR1), African 2 (AFR2), and North-American/Asian-American (NA/AA) [34]. As presented in Table 1, SNP analysis of the assembled HPV16 genomes assigned the following variants to each clinical sample: 2 LSIL samples, EUR; 1 LSIL sample, NA; 1 LSIL sample, AFR2; and 1 LSIL and 2 ICC samples, As. Furthermore, phylogenetic tree analysis with the assembled complete genome sequences of HPV16 confirmed the assignments in variant-lineages (Figure S5). These results indicate that one HPV16 variant predominates in each clinical specimen.

Analysis of nucleotide variations in HPV16 genomes

Before analyzing the sequence variation present in clinical samples, we first validated the efficacy and error rates of deep sequencing on our platform. For this purpose, we used a linearized full-length HPV16 DNA that was excised from HPV16/pUC19 by BamHI digestion. The read sequences obtained were aligned to the prototype HPV16 sequence (GenBank accession No. K02718), and the number of reads containing nucleotide mismatches was determined at each position of the viral genome. This analysis enabled us to calculate mutation frequencies at each nucleotide position (the average depth of reads count per nucleotide position was 7,035). As shown in Figure 2, an overall profile of the mutation/error frequency demonstrated an error rate below 0.3% distributed throughout the whole sequence, which reflects intrinsic errors generated in the deep sequencing analysis. The average error rate per nucleotide position throughout the whole sequence was determined to be 0.0070%. Visualization and manual inspection of aligned read sequences revealed that the two minor peaks observed at nt positions 6,242 and 6,434 (see Figure 2) were derived from mis-mapping of reads to ends of the linear HPV16 DNA (BamHI site, at nt 6,154).

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Figure 2. Mutation frequency profile of full-length HPV16 genomes prepared from a plasmid.

The read sequences obtained with full-length HPV16 genomes prepared from a cloned plasmid were aligned to the reference HPV16 sequence, and mutation/error frequencies at each nucleotide position relative to the total coverage (number of reads that encompass each nucleotide, up to the maximum x 8,000) are presented in the landscape of the full-length HPV16 genome. A threshold line for a reliable mutation frequency (0.5%) is indicated with the red dotted line. The genome organization of HPV16 is indicated above: p97, the early promoter; p670, the late promoter; polyA(early) and polyA(late), the early and late polyadenylation signals, respectively.

https://doi.org/10.1371/journal.pone.0080583.g002

Our deep sequencing analysis of HPV16 DNA amplified by PrimeSTAR^® GXL polymerase demonstrated that the average error rate was 0.0076% (results not shown), which is comparable to the error rate reported by the manufacturer (0.0062%), indicating high fidelity DNA amplification by this polymerase. Based upon this low error rate and our analytical method’s ability to pick up nucleotide variation with a minimum confidence score threshold of Phred quality score 30 (error probability < 0.001), we decided to use 0.5% as a threshold value of mutation frequency to distinguish real mutations from putative errors.

We applied this variation analysis method to HPV16 genome sequences obtained from W12 cells. As shown in Figure 3, alignment of the paired-end reads to the W12 reference sequence revealed no major nucleotide substitutions with a frequency >0.5% in the read coverage , suggesting that HPV16 genome sequences are highly homogeneous in W12 cells.

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Figure 3. Mutation frequency profile of full-length HPV16 genomes in W12 cells.

The read sequences obtained with full-length HPV16 genomes prepared from W12 cells were aligned to the reference HPV16 sequence (AF125673), and mutation/error frequencies at each nucleotide position are presented in the landscape of the full-length HPV16 genome. A threshold line for a reliable mutation frequency (0.5%) is indicated with the red dotted line. The genome organization of HPV16 is indicated above.

https://doi.org/10.1371/journal.pone.0080583.g003

Next we employed this established analysis procedure to examine HPV genomic variation in clinical samples. We report two types of mutation profiles found in 7 clinical samples: a highly homogeneous profile as observed with W12 cells, and a profile with 1 to 3 specific positions of nucleotide substitution. Four samples (#1, #4, #5, and #7) showed an almost homogeneous profile without specific nucleotide substitution (two representative profiles are presented in Figure S6) and 3 samples (#2, #3, and #6) demonstrated a mutation profile with several positions of nucleotide substitution (Figure 4). The nucleotide substitutions were located in the early region of the HPV16 genome, E6 and E1, and the non-coding region between E5 and L2, with frequencies from 0.60 to 5.42% (Table 2). Intriguingly, the nucleotide substitutions observed in the E1 gene of sample 6 cause amino-acid (aa) changes in the E1 protein: methionine at aa position 326 to isoleucine, and glutamine at aa position 381 to glutamic acid. The nucleotide substitution in the E6 gene detected in sample 6 also leads to an aa change in the E6 protein, glutamic acid to aspartic acid at aa position 25.

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Figure 4. Mutation frequency profile of full-length HPV16 genomes in clinical specimens.

The read sequences obtained with full-length HPV16 genomes prepared from clinical specimens (2 LSIL samples, #2 and #3; 1 ICC sample, #6) were aligned to their de novo assembled complete genome sequences, and mutation/error frequencies at each nucleotide position are presented in the landscape of the full-length HPV16 genome. A threshold line for a reliable mutation frequency (0.5%) is indicated with the red dotted line. Peaks above 0.5% are indicated with red asterisks. The genome organization of HPV16 is indicated above.

https://doi.org/10.1371/journal.pone.0080583.g004

We were also able to employ our full-circle PCR and deep sequencing approach to analyze clinical samples that contained HPV52 and HPV58 DNA (Figure S7 and Table S1), suggesting that this method is not specific to the analysis of HPV16 and that it is also applicable to the oncogenic HPV types prevalent in East Asian countries including China and Japan [35-37].

Effect of E1 amino-acid changes on its replication function

The de novo assembled HPV16 genomes from clinical specimens revealed aa polymorphisms of the E1 protein in its DNA-binding domain at aa positions 294 and 326: a replacement of leucine (prototype) at position 294 with methionine (L294M) in 2 LSIL specimens (#3 and #4) and that of isoleucine (prototype) at position 326 with methionine (I326M) in all the specimens (Figure 5A). Further, nucleotide variation analyses showed a novel aa variation at position 381 from glutamine (prototype) to glutamic acid (Q381E) in sample 6. To explore the biological significance of these E1 variations, we mutated the corresponding aa residues in the European prototype E1 protein and tested the mutated E1’s ability to support HPV16 origin-dependent DNA replication. The E1 mutant Y379F (in which tyrosine at 379 is replaced with phenylalanine), which is expected to lose replication activity as reported previously [38], was also included as a representative of a replication-deficient E1 protein.

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Figure 5. Characterization of E1 variants by transient HPV16 replication assay.

(A) Left panel: schematic representation of the HPV16 E1 protein. The prototype E1 protein has leucine at position 294 and isoleucine at position 326, both of which are located in the DNA-binding domain, and glutamine at position 381 located in the oligomerization domain. Right panel: summary of aa variations found in the E1 protein from the clinical samples (#1 to #7). Proto, the E1 protein of European prototype. (B) E1-dependent replication of HPV16 origin-positive plasmid. The HPV16 origin-positive plasmid expressing Firefly luciferase (Fluc) and the origin-deficient plasmid expressing Renilla luciferase (Rluc) were transfected into HEK293 cells with increasing amounts of the prototype E1 expression plasmid together with the E2 expression plasmid. At 72 h after transfection, the ratio of the two luciferase activities (Fluc/Rluc) was measured as levels of the origin-dependent replication. The origin-deficient plasmid expressing Firefly luciferase (pGL4.50) was used as a negative control for HPV replication. Statistically significant differences (Welch’s t-test, p<0.01) are indicated with *. (C) Replication activity of E1 variants. Increasing amounts of expression plasmids for FLAG-tagged prototype or variant E1 proteins were transfected into HEK293 cells, and the levels of replication were measured at 72 h after transfection. Error bars represent the standard deviation of triplicate transfections. Statistically significant differences (Welch’s t-test, p<0.01) are indicated with *. The data are representative of three independent experiments. (D) Western blot analysis of E1 variants. FLAG-tagged prototype or variant E1 proteins expressed in HEK293 cells were detected with anti-FLAG antibody (Sigma-Aldrich) and the ECL prime Western blotting detection reagent (GE Healthcare, Buckinghamshire, England). Tubulin was visualized with anti-α/β-tubulin antibody (Cell Signaling, Danvers, MA) as loading control.

https://doi.org/10.1371/journal.pone.0080583.g005

In the transient HPV16 replication assay, two reporter plasmids were transfected into HEK293 cells together with expression vectors for FLAG-tagged E1 and E2. Firefly luciferase (Fluc) is expressed from the HPV16 origin-positive plasmid, whereas Renilla luciferase (Rluc) is expressed from the origin-deficient plasmid, so the ratio of the two luciferase activities (Fluc/Rluc) in the presence of the E1/E2 expression vectors represents levels of HPV origin-dependent replication. As previously reported [31], the Fluc/Rluc ratio obtained with the origin-positive plasmid increased in a manner dependent on the amount of the E1 expression plasmid (Figure 5B). Under these assay conditions, two E1 variants, L294M and I326M, showed replication activities almost comparable to the parental E1 (Figure 5C). In contrast, Q381E exhibited a reduced activity for supporting HPV16 replication, and Y379F completely lost the replication activity as expected. Western blot analyses revealed that the expression levels of I326M, Q381E, and Y379F were comparable to that of the prototype E1 (Figure 5D), indicating that the reduced replication activity of Q381E is not due to reduced protein level.

Detection of deletions in HPV16 genomes

Because our deep sequencing analysis was able to precisely define the deletion in the HPV16 genome of CaSki cells, we examined HPV16 genome deletions in the 7 clinical samples by read mapping to the assembled reference sequence. Of the 5 LSIL samples, 4 LSIL samples did not show any deletions and 1 LSIL sample (#2) demonstrated the discontinuous distribution of read depth indicative of genome deletions (Figure S3, arrows). Further detailed visualization of the mapped read sequences revealed that 27 bases were deleted from nucleotide positions 3,476 to 3,502 in 78.9% of total read sequences covering this region (Figure 6A). Independent full-circle PCR for sample 2 and subsequent deep sequencing analysis yielded a comparable deletion frequency (86.6%, results not shown), demonstrating the reproducibility of the analysis overall. PCR amplification of the E2 region (Figure S8) and subsequent Sanger sequencing confirmed the presence of the deletion (results not shown). In addition, read sequences lacking 1 to 3 bases in the region between E5 and L2 were also detected in sample 2 in 89.0% of the total read coverage of that region (Figure 6B). Although the read depth in the E5 region was low (Figure S3), no deletion was found in this region. These data suggest that deleted viral genomes are present as a mixed state with intact full-length HPV16 genomes in this clinical specimen. In contrast to HPV16 genomes in CaSki cells, no deletion was detected in HPV16 genome sequences obtained from the 2 ICC samples.

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Figure 6. HPV16 genome deletions in a clinical specimen revealed by read mapping.

Mapping of paired-end read sequences to de novo assembled full-length HPV16 genome sequence for LSIL sample 2 is visualized by Tablet. Deletions in the E2/E4 region (A) and in the region between E5 and L2 (B) are shown. (C) The genome organization of HPV16. The deleted regions in E2/E4 and between E5 and L2 are indicated with red and blue dotted-line boxes, respectively.

https://doi.org/10.1371/journal.pone.0080583.g006