Ribeiro, A. L. | Search bioRxiv

bioRxiv2024

Virus-host interaction: investigating novel transcription factors involved in coupling HPV life cycle and epithelial differentiation.

High-risk human papillomavirus (HPV) causes almost all cervical cancer, and HPV-18 is the second most prevalent type. The HPV life cycle is intimately associated with the epithelial differentiation program, and the repertoire of cellular transcription factors (TFs) has a crucial role in coordinating viral gene expression across epithelial layers. We aimed to identify host TFs involved in the virus differentiation-dependent life cycle. We initially compared the DNA binding activity of 345 TFs in nuclear extracts of undifferentiated and high calcium-induced differentiated HaCaT cells. Next, we searched in silico for putative binding sites within the HPV-18 long control region (LCR) for the TFs affected by differentiation. Chromatin immunoprecipitation assays (ChIP) were used to assess direct binding of selected TFs, protein levels were evaluated by immunohistochemistry in rafts obtained from human foreskin keratinocytes (HFK) and HPV-18 E6/E7 immortalized HFK, and the impact upon LCR activity was measured in reporter assays. The binding activity of 40 TFs was influenced by differentiation among which 16 putative binding sites within the LCR were identified. We next focused our analysis in PAX6, HMGB1, FOXI1, and NFE2, and all bound to the LCR except FOXI1. Whether FOXI1 activates HPV-18 early promoter activity, PAX6, HMGB1, and NFE2 inhibit viral transcription in undifferentiated normal HFK in which these proteins are mostly expressed. Finally, HPV-18 E6/E7 impaired the normal expression of these TFs throughout epithelial layers. In conclusion, we describe the involvement of PAX6, HMGB1, NFE2, and FOXIl in coupling HPV-18 transcriptional regulation with the epithelia differentiation program, a key event for the productive HPV life cycle. Furthermore, these TFs may also be relevant for HPV-driven carcinogenesis. The identification of proteins involved in the differentiation-dependent HPV life cycle and viral induced transformation may contribute to unveiling novel therapeutic targets for HPV-related malignancies. Author SummaryHPV-16 and HPV-18 respond together for over 70% of cervical cancers. HPV infection is established in basal layers cells of the stratified epithelia and the viral life cycle is highly dependent on the epithelial differentiation program. In this study, we focused on identifying host transcription factors (TFs) involved in the finely tuned regulation of the HPV-18 differentiation-dependent life cycle. We initially screened 345 cellular TFs and found 40 with differential binding activity after differentiation, among which we chose to focus on PAX6, HMGB1, FOXI1, and NFE2. Besides investigating TFs binding to HPV-18 LCR in silico and with immunoprecipitation assays, we evaluated protein levels throughout epithelial layers and demonstrated that the impact of these TFs upon HPV-18 early promoter activity depends on the differentiation status of cells. Finally, we show that HPV-18 oncoproteins lead to abnormal expression of these TFs. Our results contribute to a clearer understanding of pivotal mechanisms governing spatiotemporal regulation of viral early transcription and open avenues for innovative therapeutic strategies for HPV-associated diseases.

molecular biology↗

bioRxiv2019

OPTN translocates to an ATG9A-positive compartment to regulate innate immune signalling and cytokine secretion

Optineurin (OPTN) is a multifunctional protein involved in autophagy, secretion as well as NF-{kappa}B and IRF3 signalling and mutations are associated with several human diseases including primary open-angle glaucoma (POAG), amyotrophic lateral sclerosis (ALS), Pagets disease of bone (PDB) and Crohns disease (CD). Here we show that, in response to viral RNA, OPTN translocates to foci in the perinuclear region, where it negatively regulates NF-{kappa}B and IRF3 signalling pathways and downstream pro-inflammatory cytokine secretion. These OPTN foci consist of a tight cluster of small membrane vesicles, which are positive for marker proteins of the trans-Golgi network/recycling compartment - most notably ATG9A. Disease mutations linked to POAG cause aberrant formation of this compartment in the absence of stimuli, which correlates with the ability of OPTN to inhibit signalling. Using proximity labelling proteomics (BioID), we identify the linear ubiquitin assembly complex (LUBAC), CYLD and TBK1 as part of the OPTN interactome and show that these proteins, along with NEMO, are recruited to this OPTN-positive perinuclear compartment. Together, we propose OPTN might be responsible for dampening the NF-{kappa}B and IRF3 signalling responses through the sequestration of LUBAC and other positive regulators of these pathways in this dsRNA-induced compartment leading to altered pro-inflammatory cytokine secretion.\n\nSummaryDisease associated OPTN mutations impact on the formation of the perinuclear compartment and result in hypo- or hyper-activation of the immune response, which could drive the development of human diseases such as POAG, ALS and also Pagets disease of bone.

cell biology↗