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

Jiang, Zixiao ; Zhao, Yani ; Zheng, Yunji LU ; Hao, Chengyu LU ; Cui, Xiaoxu LU ; Jönsson, Johanna LU orcid ; Wu, Chengjun ; Kajitani, Naoko LU and Schwartz, Stefan LU (2026) In Journal of Virological Methods 342.
Abstract

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... (More)

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.

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author
; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Gene regulation, HnRNP, HPV, Papillomavirus, Splicing, SR proteins
in
Journal of Virological Methods
volume
342
article number
115358
publisher
Elsevier
external identifiers
  • pmid:41620032
  • scopus:105029271517
ISSN
0166-0934
DOI
10.1016/j.jviromet.2026.115358
language
English
LU publication?
yes
id
804948e6-2974-400a-be5b-ef44f656f619
date added to LUP
2026-02-18 09:09:04
date last changed
2026-02-18 09:09:19
@article{804948e6-2974-400a-be5b-ef44f656f619,
  abstract     = {{<p>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.</p>}},
  author       = {{Jiang, Zixiao and Zhao, Yani and Zheng, Yunji and Hao, Chengyu and Cui, Xiaoxu and Jönsson, Johanna and Wu, Chengjun and Kajitani, Naoko and Schwartz, Stefan}},
  issn         = {{0166-0934}},
  keywords     = {{Gene regulation; HnRNP; HPV; Papillomavirus; Splicing; SR proteins}},
  language     = {{eng}},
  publisher    = {{Elsevier}},
  series       = {{Journal of Virological Methods}},
  title        = {{Reporter assays for HPV16 E1/E1C and E2 mRNA splicing using nanoluciferase and secreted luciferase}},
  url          = {{http://dx.doi.org/10.1016/j.jviromet.2026.115358}},
  doi          = {{10.1016/j.jviromet.2026.115358}},
  volume       = {{342}},
  year         = {{2026}},
}