Differences in Stability of Viral and Viral-Cellular Fusion Transcripts in HPV-Induced Cervical Cancers

Abstract

HPV-DNA integration results in dysregulation of viral oncogene expression. Because viral-cellular fusion transcripts inherently lack the viral AU-rich elements of the 3’UTR, they are considered to be more stable than episome-derived transcripts. The aim of this study is to provide formal proof for this assumption by comparing the stability of viral early transcripts derived from episomal and integrated HPV16 DNA, respectively. Full-length cDNA of three fusion transcripts comprising viral and cellular sequences in sense orientation were amplified and cloned into the adeno-viral-vector pAd/CMV/V5-DEST. The most abundant HPV16 oncogene transcript E6*I-E7-E1vE4-E5 with and without 3’UTR, served as reference and control, respectively. Human primary keratinocytes were transduced using high titer virus stocks. qRT-PCR was performed to determine mRNA stability in relation to GAPDH in the presence of actinomycin-D. In four independent transduction experiments, all three viral-cellular fusion transcripts were significantly more stable compared to the episome-derived reference. Among the three viral-cellular fusion transcripts the most stable transcript was devoid of the instability core motif “AUUUA”. Unexpectedly, there was no significant difference in the stability between the episome-derived transcripts either with or without 3’UTR, indicating that the AU-rich elements of the 3’UTR are not contributing to RNA stability. Instead, the three “AUUUA” motifs located in the untranslated region between the viral E4 and E5 genes may be responsible for the instability. This is the first report showing that authentic viral-cellular fusion transcripts are more stable than episome-derived transcripts. The longer half-life of the fusion transcripts may result in increased levels of viral oncoproteins and thereby drive the carcinogenic process.

Long-read sequencing unveils high-resolution HPV integration and its oncogenic progression in cervical cancer

“…This is considered an important molecular event in cervical carcinogenesis 12 – 14 . Upon integration, deregulated expression of viral oncogenes E6 and E7 is frequently observed, which may result from the disruption of their negative regulators E1 and/or E2 15 , and the increased stability of E6 and E7 mRNAs expressed from the viral-host fusion transcripts 16 , 17 . Of note, a study also showed that there was no significant association between the expression levels of viral oncogenes and the physical state of the viral genome based on cervical biopsy materials, implying that the constitutive expression rather than the expression level of viral oncogenes seems to play a decisive role in the transformation and the maintenance of malignant phenotype 18 .…”

MicroRNA-Based Fingerprinting of Cervical Lesions and Cancer

“…The majority of integration sites are transcriptionally active, however, different patterns of DNA and RNA integration breakpoints have been noticed [ 56 ]. Importantly, expression of viral-cellular transcripts increases stability of HPV oncogenes [ 57 , 58 ]. HPV integrations exhibit features of enhancers or activators of flanking genes that can act on their target genes over long distances [ 59 , 60 ].…”