Biological Activity Characterization of the Diagnostically Relevant Human Papillomavirus 16 E1C RNA

Christy Susan Varghese,Daniela Höfler,Bernd Hessling,Claudia Tessmer,Ilse Hofmann,Michael Pawlita,Rainer Will

doi:10.3390/microbiolres12030038

Christy Susan Varghese, Daniela Höfler + Show 5 more

Open Access

https://doi.org/10.3390/microbiolres12030038

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Abstract

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.

Highlights

Persistent infection by high-risk HPV types, predominantly human papillomavirus 16 (HPV16) [1], can cause anogenital and oropharyngeal cancers
Spliced HPV16 E1C RNA was found to be almost exclusively associated with highgrade CxCa precursor lesions and possess the potential as a biomarker to distinguish from low-grade lesions [8,9]
Despite the low endogenous levels of E1C RNA, Höfler et al have shown the prevalence of E1C transcript peaks in CIN3 lesions (54% of 158 cervical cell samples) [9]

Summary

Introduction

Persistent infection by high-risk HPV types, predominantly HPV16 [1], can cause anogenital and oropharyngeal cancers. Regulation of HPV16 transcription with at least two promoters and multiple alternatively used splice donor and acceptor sites is complex and is tightly linked to the differentiation stage of the infected keratinocyte [5]. Expression of the early genes E6 and E7 enhance proliferation and lateral expansion of the infected basal and parabasal cells, while E1 and E2 are essential for the initial amplification and maintenance of viral DNA. In further differentiated upper epidermal cell layers, viral DNA is replicated and the late genes L1 and L2, encoding the capsid proteins, are expressed together with

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