Eight nucleotide substitutions inhibit splicing to HPV-16 3'-splice site SA3358 and reduce the efficiency by which HPV-16 increases the life span of primary human keratinocytes

PLoS One. 2013 Sep 9;8(9):e72776. doi: 10.1371/journal.pone.0072776. eCollection 2013.


The most commonly used 3'-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing*
  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Cell Line
  • Conserved Sequence
  • DNA-Binding Proteins / genetics
  • Enhancer Elements, Genetic
  • Gene Expression
  • Gene Expression Regulation, Viral
  • Gene Order
  • Genes, Reporter
  • Genome, Viral
  • Human papillomavirus 16 / genetics*
  • Humans
  • Keratinocytes / metabolism*
  • Keratinocytes / virology*
  • Mutation
  • Nuclear Proteins / metabolism
  • Oncogene Proteins, Viral / genetics
  • Open Reading Frames
  • Plasmids / genetics
  • Protein Binding
  • RNA Splice Sites*
  • RNA, Viral / genetics*
  • RNA-Binding Proteins / metabolism
  • Serine-Arginine Splicing Factors


  • DNA-Binding Proteins
  • E2 protein, Human papillomavirus type 16
  • Nuclear Proteins
  • Oncogene Proteins, Viral
  • RNA Splice Sites
  • RNA, Viral
  • RNA-Binding Proteins
  • oncogene protein E4, Human papillomavirus type 16
  • Serine-Arginine Splicing Factors

Grants and funding

Research funded by the Swedish Research Council and the Swedish Cancer Society (Cancerfonden). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.