Sequences of primers utilized for 5′ and 3′ RACE analyses.

1<p>Published in ref. 18.</p><p>Sequences of primers utilized for 5′ and 3′ RACE analyses.</p

Promoter regions mapped by the clustering of TSSs in the early region of the HPV18 genome into five distinct promoter regions.

<p>The TSSs were identified by sequencing individual clones from 5′ RACE assays conducted on polyA⁺ RNA samples from U2OS cells that were transiently transfected with the HPV18 genome (169 clones from the 22- and 71-hour timepoints, upper diagram) and from U2OS subclone #1.13 cells (173 clones, middle diagram), which stably maintain HPV18 episomal DNA. In the bottom diagram, data from the transient transfection and #1.13 are summarized. Each bar in the diagrams represents a single TSS, and the height of each bar represents the percentage of mRNAs that initiated from that TSS in a particular promoter region. For each promoter region, the nt position of the most prevalent TSS is indicated above the highest bar. At the bottom of the figure, the locations of the defined promoter regions (P₁₀₂, P₅₂₀, P₈₁₁, P₁₁₉₃ and P₃₃₈₅, indicated with square brackets) in the HPV18 genome are indicated.</p

Mapping of the HPV18 transcription polyadenylation cleavage sites (CSs) by 3′ RACE.

<p>The assays were conducted in U2OS cells that were transiently transfected with the HPV18 genome (timepoints are indicated above each gel) and in HPV18-positive U2OS subclone #1.13 cells that were cultured in subconfluent or confluent conditions. (A) 3′ RACE analysis of the HPV18 early region CS with the HPV18-specific primer Pr3976. The 3′ RACE product (indicated by an arrow) represents the CS at 4270. (B) 3′ RACE analysis of the HPV 18 late region CS with the HPV18-specific primer Pr7020. Three 3′ RACE products (marked as 1, 2 and 3 and indicated by arrows) represent the CSs at nt 448, nt 7693 and nt 7298, respectively. (C) 3′ RACE analysis with the HPV18-specific primer Pr2500 for analysis of the CSs of E1-encoding transcripts. Three 3′ RACE products (marked as 1, 2 and 3 and indicated by arrows) represent the CSs at nt 4270, nt 3339 and nt 2741, respectively.</p

Summary of the HPV18 transcripts that were mapped in transiently transfected U2OS cells and in subclone #1.13 cells by 5′ RACE analyses with primers Pr1397 (RNA A) and Pr3517-1 (RNAs B-U).

<p>Top, a linear depiction of the HPV18 genome with the ORFs, LCR, E1^∧E4 and E2^∧E8 coding sequences spanned over two exons (dashed line), along with the promoter regions P₁₀₂, P₅₂₀, P₈₁₁, P₁₁₉₃ and P₃₃₈₅ and the major polyadenylation cleavage sites CS 4270 and CS 7298. All transcripts depicted herein (designated with letters A-U, shown at left) are shown with exons (solid boxes) and introns (lines) and with the splicing donor and acceptor sites (nt numbers). The coding potential is described to the right of each transcript. The RNA species previously described in HPV18-infected raft cultures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116151#pone.0116151-Wang1" target="_blank">[18]</a> are indicated by asterisks. In addition, the splicing pattern -3165/3434- (as for RNA H herein) was reported previously in both raft culture and clinical samples (41).</p

Transcriptional analyses of HPV18 E2 variants A: Schematic representations of two reporter plasmids.

<p>One reporter consists of full-length native HPV18 URR region acting as a promoter that drives the Firefly luciferase gene. The other reporter uses HPV18 E2-binding sites 3 and 4 as promoter. B: U2OS cells were transfected with the 18URR-Luc+ reporter plasmid and the non-specific pRL-TK reporter and together with different concentrations (0.1 ng, 10 ng, 100 ng and 500 ng) of the HPV18 E2C-1, E2C-2 and E8ˇE2 expression plasmids; 0.1 ng and 100 ng of the HPV18 E2 plasmid were added as controls. Twenty-four hours after the transfection, luciferase activities were measured and normalized to the Renilla values obtained from the pRL-TK reporter. These data indicate the effects of E2C-1, E2C-2, E8ˇE2 and E2 on the URR promoter activity relative to the reporter alone. Average values of two independent experiments are given. C: U2OS cells were transfected with the 18 E2BS 3&4-Luc+ reporter plasmid and non-specific pRL-TK reporter and together with different concentrations (0.1 ng, 0.5 ng, 1 ng, 3 ng, 5 ng or 10 ng) of HPV18 E2C-2 or 0.1 ng, 1 ng, 10 ng or 100 ng of the BPV1 E2C expression plasmids. Twenty-four hours after the transfection, luciferase activities were measured and normalized to the Renilla values obtained from the pRL-TK reporter. These data indicate the effects of HPV18 E2C-2 and BPV1 E2C on the E2BS 3&4 promoter activity relative to the reporter alone. Average values of three independent experiments are given. D: U2OS cells were transfected with the 18 E2BS 3&4-Luc+ reporter plasmid and non-specific pRL-TK reporter and together with different concentrations (1 ng, 10 ng and 100 ng) of the HPV18 E2C-1, E8ˇE2 and E2 expression plasmids. Twenty-four hours after the transfection, luciferase activities were measured and normalized to the Renilla values obtained from the pRL-TK reporter. These data indicate the effects of HPV18 E2C-1, E8ˇE2 and E2 on the E2BS 3&4 promoter activity relative to the reporter alone. Average values of two independent experiments are given. E and F: 5′ RACE analysis of HPV18 transcripts that were produced in U2OS cells during transient replication of <i>wt</i> or different mutant (E2C-2, E2C-1 and 2-E2C) genomes. HPV18-specific primers, Pr904, which binds to a site in the E7 ORF (panel E), and Pr3157 (panel F), which binds to a site in the E4 ORF, were used. Products representing the RNAs that are initiated from promoters P₁₀₂, P₅₂₀ and P₈₁₁ are shown.</p

Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385.

<p><b>A:</b> Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 <i>wt</i>, E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included <b>B:</b> U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C_N). The value obtained from the HPV18 <i>wt</i> 3-day time point was set to 1, and the C_N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. <b>C:</b> U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8^∧E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. <b>D and E:</b> U2OS cells were transfected with 2 µg of HPV18 <i>wt</i> minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C_N) was performed. The value obtained from the HPV18 <i>wt</i> 3-day time point was set to 1, and the C_N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 <i>wt</i> replication, whereas panel E shows the effect of E2C-2.</p

Mapping of the HPV18 splice junctions in U2OS cells by 5′ RACE.

<p>The assays were conducted with U2OS cells that had been transiently transfected with the HPV18 genome (timepoints indicated above each gel) and with HPV18-positive U2OS subclone #1.13 cells that were cultured in subconfluent or confluent conditions. (A) 5′ RACE analysis of HPV18 transcripts that were produced in U2OS cells during transient replication. An HPV18-specific primer (Pr3517-1) that binds to a site in the E4 ORF was used. All distinct products (regions marked as 1, 2, 3 and 4) were purified, cloned and sequenced. The natures of the represented transcripts are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116151#s3" target="_blank">results</a> and are shown in Fig. 6. (B) 5′ RACE analysis of RNAs in U2OS cells with an HPV18-specific primer (Pr904-1) that binds to a site in the E7 ORF. The dominant RACE product (size ∼700 bp) represents the spliced transcripts as -233/416- (RNAs A, C and K in Fig. 6). Products representing the RNAs that initiated from promoters P₁₀₂, P₅₂₀ and P₈₁₁ are indicated. (C) 5′ RACE analysis of E1-encoding mRNAs with an HPV18-specific primer (Pr1397-1) that binds to a site in the E1 ORF. The indicated products (1–5) were cloned and sequenced. Products representing the RNAs that initiated from promoters P₁₀₂, P₅₂₀, P₈₁₁ and P₁₁₉₃ are indicated. (D) Primer extension assay of HPV18 genome transiently replicating in U2OS cells. Signals are obtained with HPV18-specific primer Pr881. Lane 1 serves as marker (ΦX174 DNA/HinfI; Promega), lane 2 is HPV18 genome and lane 3 is mock sample. Bands representing transcripts arisen from HPV18 promoters are indicated. (E) Primer extension assay quantitation of transiently replicating HPV18 in U2OS cells. Values indicate proportion of overall promotoral activity.</p

Frequency of human consensus transcription start site motifs in promoter regions.

<p>Frequency of human consensus transcription start site motifs in promoter regions.</p

Analysis of E8ˇE1 overexpression on HPV18 genome replication and transcription.

<p><b>A:</b> U2OS cells were transfected with 2 µg of the HPV18 E8 mutant minicircle alone or with different concentrations (10 ng, 50 ng and 250 ng) of the E8ˇE1 expression plasmid. The E8ˇE2 expression plasmid (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, and a qPCR-based analysis of the viral genome relative copy number (C_N) was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116151#s2" target="_blank">Materials and Methods</a> section. The value obtained from the HPV18 E8 mutant 3-day timepoint was set to 1, and the CN values of the other samples are expressed relative to this point. The averages of the results of two experiments are presented with standard deviations. <b>B:</b> U2OS cells were transfected with different concentrations (0.1 ng, 1 ng, 10 ng, 100 ng and 500 ng) of E8ˇE1 or 250 ng of the E8ˇE2 expression plasmid together with the reporter plasmid URR-Luc containing the native HPV18 URR and a non-specific reporter pRL-TK. Twenty-four hours after the transfection, luciferase activities were measured and normalized to those for Renilla luciferase expression from the thymidine kinase promoter of the co-transfected plasmid pRL-TK. These data indicate the effect of E8ˇE1 on promoter activity relative to the reporter alone. The averages of the results of two experiments are presented with standard deviations.</p

(A) Schematic maps of the parental plasmid pMC-HPV18 and the HPV18 miniplasmid produced from pMC-HPV18 in <i>E. coli</i> ZYCY10P3S2T according to the method described in ref. 22.

<p>(B) Schematic diagram of the HPV18 genome indicating the LCR and ORFs. The first nucleotides of the start codons for each ORF are indicated. The binding positions of the primers used for the 5′ and 3′ RACE assays in U2OS cells are shown above as dashed arrows. The numbers in the primer names indicate the binding positions (5′ end) on the HPV18 genome. Primer Pr3976 was described previously (ref. 44). The viral promoters P₅₅, P₁₀₂ and P₈₁₁ and the transcription polyadenylation cleavage sites (CS₄₂₇₀ and CS₇₂₉₉) that were mapped during productive HPV18 infection are indicated (44).</p