HPV16 E5 is produced from an HPV16 early mRNA spliced from SD226 to SA3358

The HPV16 E5 open reading frame (ORF) is present on the majority of all alternatively spliced HPV16 mRNAs, but it is currently unknown how well it is translated into E5 protein. To identify HPV16 mRNAs that are efficiently translated into E5, we have generated cDNA plasmids expressing individual, alternatively spliced HPV16 mRNAs with the potential to produce E5. By replacing the E5 ORF with sLuc, we could quantitate sLuc and determine how well each cDNA was translated. Our results showed that the upstream E1 and E7 AUGs inhibited translation of the E5 ORF and revealed that only one HPV16 mRNA produced high levels of E5. This was an HPV16 early mRNA spliced from SD226 to SA3358. These results were confirmed in the context of the entire HPV16 genome. Taken together, our results indicate that E5 is expressed early in the HPV16 replication cycle since it is translated efficiently only by one early mRNA.