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Cellular and Viral Factors that Control Human Papillomavirus Type 16 Late Gene Expression
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Stefan Schwartz)
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: urn:nbn:se:uu:diva-150706ISBN: 978-91-554-8069-1OAI: oai:DiVA.org:uu-150706DiVA: diva2:410043
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
List of papers
1. Polypyrimidine tract binding protein induces human papillomavirus type 16 late gene expression by interfering with splicing inhibitory elements at the major late 5' splice site, SD3632
Open this publication in new window or tab >>Polypyrimidine tract binding protein induces human papillomavirus type 16 late gene expression by interfering with splicing inhibitory elements at the major late 5' splice site, SD3632
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2008 (English)In: Journal of Virology, ISSN 0022-538X, Vol. 82, no 7, 3665-3678 p.Article in journal (Refereed) Published
Abstract [en]

We have initiated a screen for cellular factors that can induce human papillomavirus type 16 (HPV-16) late gene expression in human cancer cells. We report that the overexpression of polypyrimidine tract binding protein (PTB), also known as heterologous nuclear ribonucleoprotein I (hnRNP I), induces HPV-16 late gene expression in cells transfected with subgenomic HPV-16 plasmids or with full-length HPV-16 genomes and in persistently HPV-16-infected cells. In contrast, other hnRNPs such as hnRNP B1/A2, hnRNP F, and hnRNP Q do not induce HPV-16 late gene expression. PTB activates SD3632, the only 5' splice site on the HPV-16 genome that is used exclusively by late mRNAs. PTB interferes with splicing inhibitory sequences located immediately upstream and downstream of SD3632, thereby activating late gene expression. One AU-rich PTB-responsive element was mapped to a 198-nucleotide sequence located downstream of SD3632. The deletion of this element induced HPV-16 late gene expression in the absence of PTB. Our results suggest that the overexpression of PTB interferes with cellular factors that interact with the inhibitory sequences. One may speculate that an increase in PTB levels or a reduction in the concentration of a PTB antagonist is required for the activation of HPV-16 late gene expression during the viral life cycle.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-99656 (URN)10.1128/JVI.02140-07 (DOI)000254139800045 ()18216120 (PubMedID)
Available from: 2009-03-18 Created: 2009-03-18 Last updated: 2011-07-01Bibliographically approved
2. Adenovirus E4orf4 induces HPV-16 late L1 mRNA production
Open this publication in new window or tab >>Adenovirus E4orf4 induces HPV-16 late L1 mRNA production
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2009 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 383, no 2, 279-290 p.Article in journal (Refereed) Published
Abstract [en]

The adenovirus E4orf4 protein regulates the switch from early to late gene expression during the adenoviral replication cycle. Here we report that overexpression of adenovirus E4orf4 induces human papillomavirus type 16 (HPV-16) late gene expression from subgenomic expression plasmids. E4orf4 specifically overcomes the negative effects of two splicing silencers at the two late HPV-16 splice sites SD3632 and SA5639. This results in the production of HPV-16 spliced L1 mRNAs. We show that the interaction of E4orf4 with protein phosphatase 2A (PP2A) is necessary for induction of HPV-16 late gene expression. Also an E4orf4 mutant that fails to bind the cellular splicing factor ASF/SF2 fails to induce L1 mRNA production. Collectively, these results suggest that dephosphorylation of SR proteins by E4orf4 activates HPV-16 late gene expression. Indeed, a mutant ASF/SF2 protein in which the RS-domain had been deleted could itself induce HPV-16 late gene expression, whereas wild type ASF/SF2 could not.

Keyword
HPV, Splicing, Adenovirus, E4orf4, ASF/SF2
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-119726 (URN)10.1016/j.virol.2008.09.041 (DOI)000262605700013 ()19026433 (PubMedID)
Available from: 2010-03-01 Created: 2010-03-01 Last updated: 2011-07-01Bibliographically approved
3. 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
Open this publication in new window or tab >>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
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-135655 (URN)10.1128/JVI.00462-10 (DOI)000279983200029 ()
Available from: 2010-12-08 Created: 2010-12-07 Last updated: 2011-07-01Bibliographically approved
4. Serine/arginine-rich protein 30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism
Open this publication in new window or tab >>Serine/arginine-rich protein 30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism
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2011 (English)In: Journal of General Virology, ISSN 0022-1317, E-ISSN 1465-2099, Vol. 92, no 10, 2411-2421 p.Article in journal (Refereed) Published
Abstract [en]

Two splice sites on the human papillomavirus type 16 (HPV-16) genome are used exclusively by the late capsid protein L1 mRNAs: SD3632 and SA5639. These splice sites are suppressed in mitotic cells. This study showed that serine/arginine-rich protein 30c (SRp30c), also named SFRS9, activated both SD3632 and SA5639 and induced production of L1 mRNA. Activation of HPV-16 L1 mRNA splicing by SRp30c required an intact arginine/serine-repeat (RS) domain of SRp30c. In addition to this effect, SRp30c could enhance L1 mRNA production indirectly by inhibiting the early 3′-splice site SA3358, which competed with the late 3′-splice site SA5639. SRp30c bound directly to sequences downstream of SA3358, suggesting that SRp30c inhibited the enhancer at SA3358 and caused a redirection of splicing to the late 3′-splice site SA5639. This inhibitory effect of SRp30c was independent of its RS domain. These results suggest that SRp30c can activate HPV-16 L1 mRNA expression via a bimodal mechanism: directly by stimulating splicing to late splice sites and indirectly by inhibiting competing early splice sites.

Keyword
HPV-16, splicing, SR-proteins, SRp30c, polyadenylation
National Category
Microbiology in the medical area
Research subject
Medical Virology
Identifiers
urn:nbn:se:uu:diva-150707 (URN)10.1099/vir.0.033183-0 (DOI)000295856000022 ()
Available from: 2011-04-05 Created: 2011-04-04 Last updated: 2016-10-10Bibliographically approved

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