Disc large 1 expression is altered by human papillomavirus E6/E7 proteins in organotypic cultures of human keratinocytes

Abstract

Loss of cell polarity is a fundamental process in cell transformation. Among polarity proteins, we focused on human disc large (DLG1), which is localized mainly at adherens junctions and contributes to the control of cell proliferation. We previously demonstrated that its expression is altered in HPV-associated cervical neoplastic lesions, but the mechanisms beyond this remain unknown. In this study, we analysed the contribution of HPV proteins to the changes in DLG1 expression in the squamous epithelium. We observed tissue and intracellular misdistribution of DLG1 when high-risk HPV-18 E7 or E6/E7 proteins were expressed in organotypic raft cultures. The viral oncoproteins induce the loss of DLG1 from the cell borders and an increase in the level of DLG1 protein, reflecting the pattern observed in cervical lesions. These findings were corroborated in cultures bearing the entire HPV-18 genome. Interestingly, changes in tissue distribution and abundance of DLG1 were also detected in organotypic cultures expressing the low-risk HPV-11 E7 or E6/E7 proteins, suggesting a conserved function among different HPV types. However, for low-risk HPVs, the subcellular localization of DLG1 at cell-to-cell contacts was predominantly maintained. This report offers new evidence, we believe, of the involvement of HPV proteins in DLG1 expression pattern and our data support previous observations regarding DLG1 expression in cervical lesions.

HPV E6 and E7 oncoproteins cooperatively alter the expression of Disc Large 1 polarity protein in epithelial cells

“…Notably, in organotypic epithelial raft cultures generated from human keratinocytes expressing HPV-18 E6 and E7 oncoproteins, we detected an increased and mislocalized expression of DLG1, in agreement with the observations in SILs biopsies [18]. This finding may indicate the participation of these viral proteins in DLG1 deregulation, and ultimately during this pathological process.…”

“…Having corroborated the effects of E718, we next analysed DLG1 expression in the presence of both HPV oncoproteins, as performed for our previous analysis in raft cultures [18] and as naturally occurs in histological HPV-infected cervical samples [16,18]. For this, we carried out triple transfection assays in HEK293 cells with vectors expressing E618, E718 and DLG1.…”

“…Interestingly, our previous studies also suggest a role for HPV E7 in the differential expression of DLG1, as the single presence of this viral protein promotes an increase in DLG1 abundance in raft tissues and in monolayer epithelial cell cultures [18]. HPV E7 deregulates signal transduction pathways involved in cell proliferation and transcriptional regulation in order to favour viral replication.…”

“…As mentioned before, DLG1 deregulation has been observed in histological samples and organotypic raft cultures, which expressed not only E6 but also the HPV-E7 oncoprotein [16][17][18]. Therefore, we wanted to investigate in more detail the specific contribution of E7 to the observed changes in DLG1 expression.…”

“…pseyfp2-E618 construction was used as template for obtaining the seyfp2-E618ΔPBM mutant version in which the E6 PBM (E T Q V, C-terminal aminoacids) was deleted by PCR mutagenesis. The HPV-18 E7 (E718) coding sequence was amplified by PCR from pcDNA3-His-E718 [18] and cloned into the pLPC-mCherry and pegfp-c1 vector, resulting in pLPC-mCherry-E718 and pegfp-E718. The mTurq2-DLG1 and pcDNA3-HA-DLG1 (DLG1 protein fused to the HA, human influenza hemagglutinin epitope) vectors were previously described [5,26].…”