An innate immune response is crucial to inhibit pathogen spread until adaptive immunity is activated. Human papillomaviruses are able to inhibit various pathways including interferon signalling, chemokine expression and other cytokines. Interleukin-1β, for instance, is a pro-inflammatory cytokine which is produced by cells of the monocyte lineage as well as keratinocytes upon infection. IL-1ß is able to activate immune cells and is involved in the up-regulation of adhesion molecules on endothelial cells. Because of its strong pyrogenic effects the production of IL-1ß is tightly controlled on the transcriptional as well as post-translational level. Here, we investigated the influence of the oncogenes E6 and E7 of HPV16 on the expression, post-translational processing and secretion of IL-1ß.
In our studies we used primary keratinocytes (pK), non-tumorigenic keratinocytes that were retrovirally immortalized by the HPV16 oncogenes E6 or/and E7 (E6, E7 and E6/7 cells) as well as the HPV16-positive cervical carcinoma (CxCa) cell lines CaSki and SiHa for molecular analysis.
Upon adenoviral infection, pK and E7-immortalised cells secrete high amounts of IL-1ß while E6 and E6/7 cells or CxCa cells do not respond. qPCR revealed that in CxCa cells the transcription of the IL-1ß gene is reduced when compared to pK. Conversely, despite similar mRNA levels in immortalised cells, only pK and E7 cells show a constitutive expression of pro-IL-1ß while the protein is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1β levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. Transfection of an E6 expression plasmid into E7 cells lead to a decrease of pro-IL1ß. Treatment of E6 and E6/7 cells with the proteasome inhibitor MG132 was able to restore intracellular pro-IL1ß levels. Mutagenesis analysis of the N-terminal part of pro-IL-1ß led to the identification of several lysine residues responsible for degradation. Currently, a siRNA library screen is conducted to identify factors involved in this process.
In HPV positive cells, IL-1ß is abrogated in two ways: by an E6-mediated post-translational and proteasome-dependent mechanism in immortalised cells and by additional transcriptional silencing in tumor cells. We conclude that the post-translational degradation of IL-1ß upon infection with HPV16 represents an early mechanism of viral immune escape, while the transcriptional silencing of IL-1ß takes place during an additional selection process towards tumour formation.