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Multiple ASF/SF2 Sites in the Human Papillomavirus Type 16 (HPV-16) E4-Coding Region Promote Splicing to the Most Commonly Used 3'-Splice Site on the HPV-16 Genome
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2010 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, ISSN 20519389, Vol. 84, no 16, 8219-8230 p.Article in journal (Refereed) Published
Abstract [en]

Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3 '-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3 '-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3 '-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3 '-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.

Place, publisher, year, edition, pages
2010. Vol. 84, no 16, 8219-8230 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-135655DOI: 10.1128/JVI.00462-10ISI: 000279983200029OAI: oai:DiVA.org:uu-135655DiVA: diva2:375631
Available from: 2010-12-08 Created: 2010-12-07 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Cellular and Viral Factors that Control Human Papillomavirus Type 16 Late Gene Expression
Open this publication in new window or tab >>Cellular and Viral Factors that Control Human Papillomavirus Type 16 Late Gene Expression
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human papillomavirus type 16 (HPV-16) is the major cause of cervical cancer. We speculate that inhibition of HPV-16 late gene expression is a prerequisite for establishment of persistence and progression to cervical cancer. This is based on the findings that the late proteins are found only in the nuclei of terminally differentiated epithelium, and are never detected in human papillomavirus infected cervical cancer cells. It is therefore of great importance to understand how HPV-16 controls the onset of the immunogenic proteins L1 and L2 in an infected cancer cell. HPV-16 late gene expression is tightly regulated by differentiation-dependent transcription as well as by post-transcriptional mechanisms.

The long-term goal of these studies was to understand how HPV late gene expression is regulated. The specific aim of this thesis was to identify cellular and viral factors that force the virus to switch on the late genes, and to determine the mechanism of action of these factors. This will help us to understand under which circumstances HPV establish persistent infections that could progress to cancer.

We found three cellular factors; PTB, ASF/SF2 and SRp30c, and one viral factor; AdE4orf4, that in four distinctive ways were involved in the regulation of HPV-16 late gene expression. Interestingly, over-expression of PTB, AdE4orf4 or SRp30c produced different types of spliced late mRNAs. PTB induced the unspliced L2/L1 mRNA, while AdE4orf4 and SRp30c induced the spliced L1 and L1i mRNA, respectively. The three proteins had different mechanisms of action and different target sites within the HPV-16 genome, which revealed the many and complex pathways in HPV-16 gene regulation. These findings have contributed to a broader understanding of how the expression of HPV-16 late genes is controlled.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 63 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 670
National Category
Microbiology in the medical area
Research subject
Medical Virology
Identifiers
urn:nbn:se:uu:diva-150706 (URN)978-91-554-8069-1 (ISBN)
Public defence
2011-05-31, BMC, C10:305, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2011-05-10 Created: 2011-04-04 Last updated: 2011-07-01Bibliographically approved

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