Biological Activity Characterization of the Diagnostically Relevant Human Papillomavirus 16 E1C RNA
The spliced human papillomavirus 16 (HPV16) E1C RNA is associated with high-grade precursor lesions and cervical cancer. This qualifies E1C as a biomarker for high-grade lesions in HPV-based cervical cancer precursor screening. Here, we aimed to characterize the biological activity of HPV16 E1C RNA....
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| Format: | Article |
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MDPI AG
2021-06-01
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| Series: | Microbiology Research |
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| Online Access: | https://www.mdpi.com/2036-7481/12/3/38 |
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| author | Christy Susan Varghese Rainer Will Claudia Tessmer Ilse Hofmann Bernd Hessling Michael Pawlita Daniela Höfler |
| author_facet | Christy Susan Varghese Rainer Will Claudia Tessmer Ilse Hofmann Bernd Hessling Michael Pawlita Daniela Höfler |
| author_sort | Christy Susan Varghese |
| collection | DOAJ |
| description | The spliced human papillomavirus 16 (HPV16) E1C RNA is associated with high-grade precursor lesions and cervical cancer. This qualifies E1C as a biomarker for high-grade lesions in HPV-based cervical cancer precursor screening. Here, we aimed to characterize the biological activity of HPV16 E1C RNA. In HEK-293T cells overexpressing HPV16 E1C RNA, we detected 9 kDa E1C protein in the cytoplasm using immunological assays with a newly generated E1C-specific monoclonal antibody or in mass spectrometry only after proteasome inhibition with MG132, indicating instability of the E1C protein. In HPV16-transformed cervical cancer cell lines in which the level of endogenous E1C RNA is much lower, E1C protein was not detected even after proteasome inhibition. Transient E1C overexpression in HEK-293T cells, co-transfected with a firefly luciferase reporter gene under the control of the HPV16 upstream regulatory region (URR), activated the HPV16 URR by 38%. This activation was also present when E1C translation was abolished by mutation. However, a construct expressing a random RNA sequence with similar GC content and 45% homology to the E1C RNA sequence also stimulated URR activity, indicating that special E1C RNA motifs might be responsible for the activation. In HPV16-transformed cell lines W12-episomal (W12-epi), W12-integrated HPV (W12-int), CaSki and SiHa stably overexpressing E1C RNA from lentiviral transduction, levels of endogenous HPV16 RNAs E6*I and E7 remained unchanged, while E1^E4 levels were significantly reduced by 20–30% in W12-epi, W12-int and CaSki cells. Overall, our study shows that E1C RNA is active and might contribute to transformation independent of the E6*I or E7 pathways. However, E1C overexpression resulted in only subtle changes in HPV16 RNA expression and very low copies of endogenous E1C RNA were detected in cervical cancer cell lines. This could weigh towards a less prominent role of E1C RNA in natural HPV transformation. |
| first_indexed | 2024-03-10T10:03:49Z |
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| institution | Directory Open Access Journal |
| issn | 2036-7481 |
| language | English |
| last_indexed | 2024-03-10T10:03:49Z |
| publishDate | 2021-06-01 |
| publisher | MDPI AG |
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| series | Microbiology Research |
| spelling | doaj.art-a8ae2030b6034f8caa5527bcd7543deb2023-11-22T01:44:41ZengMDPI AGMicrobiology Research2036-74812021-06-0112353955210.3390/microbiolres12030038Biological Activity Characterization of the Diagnostically Relevant Human Papillomavirus 16 E1C RNAChristy Susan Varghese0Rainer Will1Claudia Tessmer2Ilse Hofmann3Bernd Hessling4Michael Pawlita5Daniela Höfler6Molecular Diagnostics of Oncogenic Infections, Department Infection, Inflammation and Cancer, German Cancer Research Center (DKFZ), 69120 Heidelberg, GermanyCellular Tools/Vector & Clone Repository, Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), 69120 Heidelberg, GermanyAntibodies, Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), 69120 Heidelberg, GermanyAntibodies, Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), 69120 Heidelberg, GermanyMass Spectrometry Based Protein Analysis, Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), 69120 Heidelberg, GermanyMolecular Diagnostics of Oncogenic Infections, Department Infection, Inflammation and Cancer, German Cancer Research Center (DKFZ), 69120 Heidelberg, GermanyMolecular Diagnostics of Oncogenic Infections, Department Infection, Inflammation and Cancer, German Cancer Research Center (DKFZ), 69120 Heidelberg, GermanyThe spliced human papillomavirus 16 (HPV16) E1C RNA is associated with high-grade precursor lesions and cervical cancer. This qualifies E1C as a biomarker for high-grade lesions in HPV-based cervical cancer precursor screening. Here, we aimed to characterize the biological activity of HPV16 E1C RNA. In HEK-293T cells overexpressing HPV16 E1C RNA, we detected 9 kDa E1C protein in the cytoplasm using immunological assays with a newly generated E1C-specific monoclonal antibody or in mass spectrometry only after proteasome inhibition with MG132, indicating instability of the E1C protein. In HPV16-transformed cervical cancer cell lines in which the level of endogenous E1C RNA is much lower, E1C protein was not detected even after proteasome inhibition. Transient E1C overexpression in HEK-293T cells, co-transfected with a firefly luciferase reporter gene under the control of the HPV16 upstream regulatory region (URR), activated the HPV16 URR by 38%. This activation was also present when E1C translation was abolished by mutation. However, a construct expressing a random RNA sequence with similar GC content and 45% homology to the E1C RNA sequence also stimulated URR activity, indicating that special E1C RNA motifs might be responsible for the activation. In HPV16-transformed cell lines W12-episomal (W12-epi), W12-integrated HPV (W12-int), CaSki and SiHa stably overexpressing E1C RNA from lentiviral transduction, levels of endogenous HPV16 RNAs E6*I and E7 remained unchanged, while E1^E4 levels were significantly reduced by 20–30% in W12-epi, W12-int and CaSki cells. Overall, our study shows that E1C RNA is active and might contribute to transformation independent of the E6*I or E7 pathways. However, E1C overexpression resulted in only subtle changes in HPV16 RNA expression and very low copies of endogenous E1C RNA were detected in cervical cancer cell lines. This could weigh towards a less prominent role of E1C RNA in natural HPV transformation.https://www.mdpi.com/2036-7481/12/3/38human papillomavirus 16cervical cancerE1C RNAE1C proteinURR |
| spellingShingle | Christy Susan Varghese Rainer Will Claudia Tessmer Ilse Hofmann Bernd Hessling Michael Pawlita Daniela Höfler Biological Activity Characterization of the Diagnostically Relevant Human Papillomavirus 16 E1C RNA Microbiology Research human papillomavirus 16 cervical cancer E1C RNA E1C protein URR |
| title | Biological Activity Characterization of the Diagnostically Relevant Human Papillomavirus 16 E1C RNA |
| title_full | Biological Activity Characterization of the Diagnostically Relevant Human Papillomavirus 16 E1C RNA |
| title_fullStr | Biological Activity Characterization of the Diagnostically Relevant Human Papillomavirus 16 E1C RNA |
| title_full_unstemmed | Biological Activity Characterization of the Diagnostically Relevant Human Papillomavirus 16 E1C RNA |
| title_short | Biological Activity Characterization of the Diagnostically Relevant Human Papillomavirus 16 E1C RNA |
| title_sort | biological activity characterization of the diagnostically relevant human papillomavirus 16 e1c rna |
| topic | human papillomavirus 16 cervical cancer E1C RNA E1C protein URR |
| url | https://www.mdpi.com/2036-7481/12/3/38 |
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