The Regulation of Human Papillomavirus Type16 Early Gene Expression
(2022) In Lund University, Faculty of Medicine Doctoral Dissertation Series- Abstract
- Abstract
Human papillomaviruses (HPVs) are small non enveloped viruses that contain a double-stranded circular DNA genome of approximately 8kb in size. It has been estimated that approximately 5% of all cancers are caused by HPV infection. Cervical cancer represents the majority of HPV-associated anogenital cancers worldwide. HPV type16 (HPV16) accounts for 65% of cervical cancer and approximately 90% of the other HPV-associated cancers, such as head-and-neck-squamous cell carcinomas (HNSCC). Persistent infection is the critical risk factor for HPV16-associated cancer progression. Dysregulation of HPV16 gene expression, especially oncogenes E6/E7, viral replication and transcription factors E1/E2, and the highly pathogenic major... (More) - Abstract
Human papillomaviruses (HPVs) are small non enveloped viruses that contain a double-stranded circular DNA genome of approximately 8kb in size. It has been estimated that approximately 5% of all cancers are caused by HPV infection. Cervical cancer represents the majority of HPV-associated anogenital cancers worldwide. HPV type16 (HPV16) accounts for 65% of cervical cancer and approximately 90% of the other HPV-associated cancers, such as head-and-neck-squamous cell carcinomas (HNSCC). Persistent infection is the critical risk factor for HPV16-associated cancer progression. Dysregulation of HPV16 gene expression, especially oncogenes E6/E7, viral replication and transcription factors E1/E2, and the highly pathogenic major structural protein L1, may contribute to establishment of persistence that may, in the end, result in cancer. In this study, we demonstrated that cellular splicing factor hnRNP D and m6A modification of HPV16 mRNA play significant roles in the regulation of HPV16 gene expression. In addition, the long control region (LCR) of the HPV16 genome appears to control E6/E7 mRNA splicing, indicating that the LCR DNA may contain splicing regulatory elements.
Cellular RNA binding protein (RBP) hnRNP D acts as a splicing inhibitor of HPV16 E1/E2 and E6/E7 mRNAs, generating intron-containing E1 and E6 mRNAs. N- and C-termini of hnRNP D contributed to HPV16 mRNA splicing control differently. The N- terminus of hnRNP D played the core inhibitory function. Furthermore, the RGG domain of the C- terminus contributed to splicing inhibition by interacting with the splicing machinery. Also, hnRNP D intensively interacted with HPV16 mRNAs in an RRM1 dependent manner. As a result, the cytoplasmic levels of intron-retained HPV16 mRNAs were increased in the presence of hnRNP D. In addition, we detected direct binding of hnRNP D to HPV16 mRNAs in an HPV16-driven tonsillar cancer cell line and in HPV16-immortalized human keratinocytes. Furthermore, knockdown of hnRNP D in HPV16-driven cervical cancer cells enhanced the production of the HPV16 E7 oncoprotein. Our results suggest that hnRNP D plays significant roles in the regulation of HPV gene expression and HPV-associated cancer development.
HPV16 mRNA m6A modification also affected alternative splicing. Overexpression of m6A demethylase ALKBH5 promoted E6 mRNA production and altered L1 splicing by regulating exon skipping. Overexpression of methyltransferase METTL3 induced E1 mRNA production and altered L1 oppositely to ALKBH5. YTHDC1 worked as an m6A reader that could induce E6 mRNA production. Our results suggest that m6A-modifications of HPV16 mRNAs contribute to the control of HPV16 gene expression.
In summary, in this thesis we report that the cellular hnRNP D protein plays a major role in the control of production of the HPV16 E1 mRNA as well as in the production of the E7 oncogene mRNA. We also found yet unidentified splicing regulatory elements in the HPV16 E6 coding region that affected E6/E7 mRNA splicing. In addition, non-transcribed sequences in the LCR may contribute to E6/E7 splicing regulation by altering the association of RNA polymerase with RNA binding proteins required for HPV16 mRNA splicing during a co-transcriptional splicing process. Finally, we found that m6A modification of HPV16 mRNAs contribute to control of HPV16 gene expression at the level of RNA processing. Taken together, these results enhance our understanding of the carcinogenic human papillomavirus HPV16.
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Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/f64fea6c-54dd-4497-951d-91862b275916
- author
- Cui, Xiaoxu LU
- supervisor
-
- Stefan Schwartz LU
- Naoko Kajitani LU
- Ola Forslund LU
- opponent
-
- Docent Klingström, Jonas, Karolinska Institutet
- organization
- publishing date
- 2022
- type
- Thesis
- publication status
- published
- subject
- keywords
- Human papillomavirus, splicing, hnRNP D, m6A, LCR
- in
- Lund University, Faculty of Medicine Doctoral Dissertation Series
- issue
- 2022:79
- pages
- 90 pages
- publisher
- Lund University, Faculty of Medicine
- defense location
- Segerfalksalen, BMC A10, Sölvegatan 17 i Lund
- defense date
- 2022-06-01 09:00:00
- ISSN
- 1652-8220
- ISBN
- 978-91-8021-240-3
- language
- English
- LU publication?
- yes
- id
- f64fea6c-54dd-4497-951d-91862b275916
- date added to LUP
- 2022-05-08 11:07:38
- date last changed
- 2022-05-12 12:07:55
@phdthesis{f64fea6c-54dd-4497-951d-91862b275916,
abstract = {{Abstract<br/>Human papillomaviruses (HPVs) are small non enveloped viruses that contain a double-stranded circular DNA genome of approximately 8kb in size. It has been estimated that approximately 5% of all cancers are caused by HPV infection. Cervical cancer represents the majority of HPV-associated anogenital cancers worldwide. HPV type16 (HPV16) accounts for 65% of cervical cancer and approximately 90% of the other HPV-associated cancers, such as head-and-neck-squamous cell carcinomas (HNSCC). Persistent infection is the critical risk factor for HPV16-associated cancer progression. Dysregulation of HPV16 gene expression, especially oncogenes E6/E7, viral replication and transcription factors E1/E2, and the highly pathogenic major structural protein L1, may contribute to establishment of persistence that may, in the end, result in cancer. In this study, we demonstrated that cellular splicing factor hnRNP D and m6A modification of HPV16 mRNA play significant roles in the regulation of HPV16 gene expression. In addition, the long control region (LCR) of the HPV16 genome appears to control E6/E7 mRNA splicing, indicating that the LCR DNA may contain splicing regulatory elements. <br/>Cellular RNA binding protein (RBP) hnRNP D acts as a splicing inhibitor of HPV16 E1/E2 and E6/E7 mRNAs, generating intron-containing E1 and E6 mRNAs. N- and C-termini of hnRNP D contributed to HPV16 mRNA splicing control differently. The N- terminus of hnRNP D played the core inhibitory function. Furthermore, the RGG domain of the C- terminus contributed to splicing inhibition by interacting with the splicing machinery. Also, hnRNP D intensively interacted with HPV16 mRNAs in an RRM1 dependent manner. As a result, the cytoplasmic levels of intron-retained HPV16 mRNAs were increased in the presence of hnRNP D. In addition, we detected direct binding of hnRNP D to HPV16 mRNAs in an HPV16-driven tonsillar cancer cell line and in HPV16-immortalized human keratinocytes. Furthermore, knockdown of hnRNP D in HPV16-driven cervical cancer cells enhanced the production of the HPV16 E7 oncoprotein. Our results suggest that hnRNP D plays significant roles in the regulation of HPV gene expression and HPV-associated cancer development.<br/>HPV16 mRNA m6A modification also affected alternative splicing. Overexpression of m6A demethylase ALKBH5 promoted E6 mRNA production and altered L1 splicing by regulating exon skipping. Overexpression of methyltransferase METTL3 induced E1 mRNA production and altered L1 oppositely to ALKBH5. YTHDC1 worked as an m6A reader that could induce E6 mRNA production. Our results suggest that m6A-modifications of HPV16 mRNAs contribute to the control of HPV16 gene expression.<br/>In summary, in this thesis we report that the cellular hnRNP D protein plays a major role in the control of production of the HPV16 E1 mRNA as well as in the production of the E7 oncogene mRNA. We also found yet unidentified splicing regulatory elements in the HPV16 E6 coding region that affected E6/E7 mRNA splicing. In addition, non-transcribed sequences in the LCR may contribute to E6/E7 splicing regulation by altering the association of RNA polymerase with RNA binding proteins required for HPV16 mRNA splicing during a co-transcriptional splicing process. Finally, we found that m6A modification of HPV16 mRNAs contribute to control of HPV16 gene expression at the level of RNA processing. Taken together, these results enhance our understanding of the carcinogenic human papillomavirus HPV16.<br/><br/>}},
author = {{Cui, Xiaoxu}},
isbn = {{978-91-8021-240-3}},
issn = {{1652-8220}},
keywords = {{Human papillomavirus, splicing, hnRNP D, m6A, LCR}},
language = {{eng}},
number = {{2022:79}},
publisher = {{Lund University, Faculty of Medicine}},
school = {{Lund University}},
series = {{Lund University, Faculty of Medicine Doctoral Dissertation Series}},
title = {{The Regulation of Human Papillomavirus Type16 Early Gene Expression}},
url = {{https://lup.lub.lu.se/search/files/118016486/Thesis_Cui.pdf}},
year = {{2022}},
}