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

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 results 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.