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The Role of Polyadenylation in Human Papillomavirus Type 16 Late Gene Expression
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

High-risk type human papillomaviruses (HPVs) are associated with cancer. HPVs are strictly epitheliotropic and infect basal cell layers, establishing a life cycle strongly linked to the differentiation stage of the infected cells. The viral capsid late genes, L2 and L1, are only expressed in terminally differentiated epithelium. Late gene expression involves regulation of most gene processing events including transcription, splicing, polyadenylation, mRNA stability and translation.

Both L2 and L1 have elements present in the open reading frames (ORFs) negatively affecting mRNA levels and translation. The negative elements in L1 were mapped to the first 514 nucleotides, with the strongest inhibitory effect located in the first 129 nucleotides. The negative elements in the L2 sequence were concentrated in two locations on the gene. Both genes were mutated by changing the nucleotide sequence while retaining the amino acid sequence. Mutating the first 514 nucleotides in L1 deactivated the negative elements while the entire L2 gene had to be mutated to achieve the same result. The L2 protein was found to localise the L1 protein into a punctuated pattern in the nucleus.

In the HPV-16 genome the negative elements reside in regions important for regulation of polyadenylation and splicing, critical for late gene expression. By exchanging parts of the L2 gene in subgenomic constructs with the corresponding mutant sequence we show that certain features of the L2 elements direct splicing to the L1 splice acceptor, and also regulate the efficiency of the early polyadenylation site. Cumulative binding of hnRNP H to the L2 mRNA gradually increased polyadenylation efficiency. Most interestingly, hnRNP H levels were downregulated in more differentiated epithelial cells.

Elucidation of how expression of the immunogenic late proteins is regulated would be greatly beneficial in prevention and treatment of HPV infection and thereby cancer.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2005. , p. 82
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 4
Keywords [en]
Molecular biology, HPV-16, polyadenylation, mRNA stability, codon usage, hnRNP H, DSE, splicing, negative element, RNA processing
Keywords [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-4774ISBN: 91-554-6141-7 (print)OAI: oai:DiVA.org:uu-4774DiVA, id: diva2:165709
Public defence
2005-02-25, B42, Biomedicinska centret, BMC, Husargatan 3, Uppsala, 09:15
Opponent
Supervisors
Available from: 2005-02-04 Created: 2005-02-04Bibliographically approved
List of papers
1. Specific inactivation of inhibitory sequences in the 5' end of the human papillomavirus type 16 L1 open reading frame results in production of high levels of L1 protein in human epithelial cells
Open this publication in new window or tab >>Specific inactivation of inhibitory sequences in the 5' end of the human papillomavirus type 16 L1 open reading frame results in production of high levels of L1 protein in human epithelial cells
2002 In: Journal of Virology, ISSN 11861841, Vol. Mars, no 76, p. 2739-52Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-92572 (URN)
Available from: 2005-02-04 Created: 2005-02-04Bibliographically approved
2. Mutational inactivation of two distinct negative RNA elements in the human papillomavirus type 16 L2 coding region induces production of high levels of L2 in human cells
Open this publication in new window or tab >>Mutational inactivation of two distinct negative RNA elements in the human papillomavirus type 16 L2 coding region induces production of high levels of L2 in human cells
2003 In: Journal of Virology, ISSN 14557653, Vol. November, no 77, p. 11674-84Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-92573 (URN)
Available from: 2005-02-04 Created: 2005-02-04Bibliographically approved
3. A Downstream Polyadenylation Element in Human Papillomavirus Type 16 L2 Encodes Multiple GGG Motifs and Interacts with hnRNP H
Open this publication in new window or tab >>A Downstream Polyadenylation Element in Human Papillomavirus Type 16 L2 Encodes Multiple GGG Motifs and Interacts with hnRNP H
2005 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 79, no 14, p. 9254-9269Article in journal (Refereed) Published
Abstract [en]

Production of human papillomavirus type 16 (HPV-16) virus particles is totally dependent on the differentiation-dependent induction of viral L1 and L2 late gene expression. The early polyadenylation signal in HPV-16 plays a major role in the switch from the early to the late, productive stage of the viral life cycle. Here, we show that the L2 coding region of HPV-16 contains RNA elements that are necessary for polyadenylation at the early polyadenylation signal. Consecutive mutations in six GGG motifs located 174 nucleotides downstream of the polyadenylation signal resulted in a gradual decrease in polyadenylation at the early polyadenylation signal. This caused read-through into the late region, followed by production of the late mRNAs encoding L1 and L2. Binding of hnRNP H to the various triple-G mutants correlated with functional activity of the HPV-16 early polyadenylation signal. In addition, the polyadenylation factor CStF-64 was also found to interact specifically with the region in L2 located 174 nucleotides downstream of the early polyadenylation signal. Staining of cervix epithelium with anti-hnRNP H-specific antiserum revealed high expression levels of hnRNP H in the lower layers of cervical epithelium and a loss of hnRNP H production in the superficial layers, supporting a model in which a differentiation-dependent down regulation of hnRNP H causes a decrease in HPV-16 early polyadenylation and an induction of late gene expression.

Keywords
Base Sequence, Capsid Proteins/*genetics, Cell Differentiation, Cervix Uteri/cytology/metabolism, Female, Hela Cells, Heterogeneous-Nuclear Ribonucleoprotein Group F-H/*physiology, Humans, Molecular Sequence Data, Oncogene Proteins; Viral/*genetics, Open Reading Frames, Polyadenylation, RNA/metabolism, RNA Splicing, Research Support; Non-U.S. Gov't
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-92574 (URN)10.1128/JVI.79.14.9254-9269.2005 (DOI)15994820 (PubMedID)
Available from: 2005-02-04 Created: 2005-02-04 Last updated: 2017-12-14Bibliographically approved

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