Reporter assays for HPV16 E1/E1C and E2 mRNA splicing using nanoluciferase and secreted luciferase

We have generated two reporter plasmids that either monitor expression of human papillomavirus type 16 (HPV16) E2 mRNAs using splice site SA2709 or HPV16 E1C mRNAs utilizing splice site SA2582. In plasmid pE2sLuc, the entire E2 open reading frame was replaced by the secreted luciferase (sLuc) reporter gene and in the pE1nLuc plasmid the E1/E1C open reading frame was fused in frame with the nanoluciferase (nLuc) reporter gene. We demonstrate that these reporter plasmids can be used to investigate cis-acting RNA elements and trans-acting factor that regulate splicing to the E2 splice site SA2709 and the alternative E1C- splice site SA2582. Furthermore, we generated a stable C33A cell line carrying HPV16 reporter plasmid pE1nLuc. Using this cell line, we showed that splicing to the HPV16 E2 3'-splice site SA2709 could be perturbed by over expression of SR-proteins or by incubation with a pan-Akt kinase inhibitor. These proteins and substances shifted splicing from E2 splice site SA2709 to the upstream HPV16 3'-splice site SA2582 and enhanced nLuc production, thereby demonstrating the utility of the pE1nLuc cell line as a bioassay for HPV16 E2 mRNA splicing.