Regulation of HPV16 Early Gene Expression

Research output: ThesisDoctoral Thesis (compilation)

Abstract

Human papillomavirus type 16 (HPV16) is the most common high-risk type and is
associated with more than 50% cervical cancer cases as well as a number of head
and neck cancer cases. The life cycle of HPV16 is strictly linked to cell
differentiation in the squamous epithelium with expression of the HPV16 early
genes. The oncoproteins E6 and E7 are essential for preventing apoptosis and
inducing proliferation of HPV16 infected cells. As cell differentiation proceeds,
viral protein E2 suppresses early promoter p97 and results in downregulation of
E6/E7 proteins and activation of HPV16 late gene expression. It is not surprising
that the HPV16 E2 gene is inactivated in many HPV16-driven cancer cells.
Therefore, the study of HPV16 early gene regulation especially regulation of E1,
E2, E6 and E7 expression is important to understand HPV16 infection. These
studies may contribute to development of antiviral drugs to HPV16.
The HPV16 E6 protein is translated from unspliced mRNAs, whereas the E7 protein
is translated from the mRNAs spliced from HPV16 5’-splice site SD226 to 3’-splice
site SA409. The balanced expression of unspliced and spliced mRNAs is important
to produce the E6 and E7 proteins that affect the HPV16 life cycle progression and
pathogenesis. We have investigated the regulation of splicing from SD226 to
SA409. We determined that cellular splicing factors hnRNP A1 and hnRNP A2B1
function as inhibitors of HPV16 E7 expression. Both hnRNP A1 and hnRNP A2
inhibited splicing to SA409 through direct and specific interaction with a C-less
RNA element located between HPV16 nucleotide position 594 and 604. Even
though inhibition SA409 exerted by hnRNP A1 and hnRNP A2 had different effects.
Overexpression of hnRNP A1 inhibited SA409 and resulted in enhanced expression
of unspliced E6 mRNAs at the expense of the E7 mRNAs. Overexpression of
hnRNP A2 inhibited SA409 but resulted in alternative splicing to 3’-splice site
SA742. The splice site SA742 is used for the production of the HPV16 E6^E7, E1,
and E4 mRNAs.
Since HPV16 E2 is a transcriptional regulator indirectly downregulates HPV16 E6
and E7 expression via suppressing of HPV16 early promoter p97. HPV16 produces
various E2 mRNAs that initiate at either early promoter p97 or late promoter p670
and several splice sites are used. Our study demonstrated that the most efficiently
translated E2 mRNA initiates at late promoter p670 spliced from 5’-splice site
SD880 to 3’-splice site SA2709. An alternative 3’-splice site named SA2582 and
located upstream of the E2 ATG could also produce E2 mRNAs, but it is less
efficiently to be translated into E2 protein. These results suggest that a splicing
enhancer may be located downstream of SA2709 to regulate E2 mRNA expression.
To further understand how HPV16 regulates 3’-splice site SA2709, we introduced
a number of deletions and mutations to HPV16 subgenomic plasmids. We identified
a 19-nucleotide RNA element consisting of three “AC(A/G)AGG” repeats located
downstream of SA2709.This RNA element acted as a splicing enhancer. This
splicing enhancer sequence interacted with cellular RNA binding protein hnRNP G
to enhance splicing to SA2709 and interacted with hnRNP A1 and A2B1 to inhibit
splicing to SA2709. Deletion of the splicing enhancer reduced mRNA splicing to
SA2709, and redirected splicing to the upstream 3’-splice site SA2582. The later
mRNA has poor ability to produce E2 protein, but it may produce a small protein
called E1C. We also wish to establish a bio-assay that can monitor expression of the
various E1 mRNAs and the alternatively spliced E2 mRNAs using either SA2582
or SA2709. We constructed a number of subgenomic HPV16 plasmids containing
green fluorescent protein, secreted luciferase, neomycin resistance gene, or
thymidine kinase reporter genes. These reporter plasmids produce either E1-fusion
proteins or E2-fusion proteins. We observed that the balance between unspliced E1
mRNAs and spliced E2 mRNAs was affected by overexpression of RNA binding
proteins SRp30c, Tra2b, hnRNP A1, and hnRNP G. However, further study is
required to optimize these reporter plasmids for quantitative analysis of HPV16 E1
and E2 mRNA splicing. This bioassay could be used to enhance our understanding
of HPV16 gene regulation and to identify small molecules that interfere with
HPV16 mRNA splicing. Such substances could potentially be used as antiviral
drugs to HPV16 infections and HPV16-driven cancers.

Details

Authors
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Microbiology in the medical area

Keywords

  • HPV16, splicing, mRNA, hnRNP A1, hnRNP G, E6, E7, E1, E2
Original languageEnglish
QualificationDoctor
Awarding Institution
Supervisors/Assistant supervisor
Award date2021 Mar 25
Place of PublicationLund
Publisher
  • Lund University, Faculty of Medicine
Print ISBNs978-91-8021-033-1
Publication statusPublished - 2021
Publication categoryResearch

Bibliographic note

Defense details Date: 2021-03-25 Time: 09:00 Place: Dora Jacobsohn, BMC D15, Klinikgatan 32 i Lund. Join by Zoom: https://lu-se.zoom.us/j/69985617527 External reviewer (s) Name: Kurg, Reet Title: Director and Professor of Molecular Biomedicine Affiliation: Institute of Technology University of Tartu, Estonia

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