Genomic structure and transcription of episomal and integrated HPV16 DNA.

<p>(A) The circular HPV16 genome becomes linearized and inserted into the host cell genome upon integration. The breakpoints of integrated HPV16 DNA can be located anywhere in the L2-L1 region (5′ breakpoint) and E1-E2-E5 region (3′ breakpoint), respectively. The breakpoint regions are indicated by dotted underlines. The circular HPV16 genome is reproduced with slight modifications with permission, from Doorbar, (2006), (Clinical Science), (110), (525–541). © the Biochemical Society. (B) In case of episomal HPV16 DNA, early transcription is initiated at the early promoter P97 and terminated at the early poly-A signal (PAE at pos. 4215). The early transcript shown contains two exons (e) with ORFs for E6, E7, E1?E4 and E5. An intron (i) of 2477 nucleotides is removed by splicing at the indicated donor and acceptor positions. Amplification of HPV16 oncogene transcripts by the APOT assay gives rise to a constant-size RT-PCR amplicon of ∼1 kb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone.0066693-Klaes1" target="_blank">[21]</a>. The APOT primers are indicated by the arrows. (C) Early transcription from integrated HPV16 DNA will lead to HPV16-cellular fusion transcripts, because the viral PAE signal is missing and instead a cellular poly-A signal (PA) is adopted. If the 3′ breakpoint is located upstream of the splice acceptor at position 3358, an alternative cellular splice acceptor (Ac) will be used, and the HPV16-cellular DNA junction sequence (red bar) will be spliced out as part of a viral-cellular intron (upper part). If the 3′ breakpoint is located downstream of the splice acceptor 3358, the HPV16-cellular DNA junction sequence (blue bar) will remain as part of a viral-cellular exon, and the DNA and RNA junction sequences are colinear (lower part). The HPV16-cellular fusion transcripts are amplified in APOT assays as RT-PCR products that are shorter or longer than the ∼1-kb amplicon derived from episomal HPV16 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone.0066693-Klaes1" target="_blank">[21]</a>.