Novel effect of the high risk-HPV E7 CKII phospho-acceptor site on polarity protein expression.

BACKGROUND: Oncogenic Human Papillomaviruses (HPVs) base their transforming potential on the action of both E6 and E7 viral oncoproteins, which perform cooperative or antagonistic actions and thus interfere with a variety of relevant cellular targets. Among them, the expression of some PDZ-containing polarity proteins, as DLG1 and hScrib, is altered during the HPV life cycle and the consequent malignant transformation. Together with the well-established interference of E6 with PDZ proteins, we have recently shown that E7 viral oncoprotein is also responsible for the changes in abundance and localization of DLG1 observed in HPV-associated lesions. Given that the mechanisms involved remained only partially understood, we here thoroughly analyse the contribution of a crucial E7 post-translational modification: its CKII-dependent phosphorylation. Moreover, we extended our studies to hScrib, in order to investigate possible conserved regulatory events among diverse PDZ targets of HPV. METHODS: We have acutely analysed the expression of DLG1 and hScrib in restrictive conditions for E7 phosphorylation by CKII in epithelial culture cells by western blot and confocal fluorescence microscopy. We made use of genome-edited HPV-positive cells, specific inhibitors of CKII activity and transient expression of the viral oncoproteins, including a mutant version of E7. RESULTS: We here demonstrate that the functional phosphorylation of E7 oncoprotein by the CKII cellular kinase, a key regulatory event for its activities, is also crucial to counteract the E6-mediated degradation of the PDZ-polarity protein DLG1 and to promote its subcellular redistribution. Moreover, we show that the CKII-dependent phosphorylation of E7 is able to control the expression of another PDZ target of HPV: hScrib. Remarkably, we found this is a shared feature among different oncogenic HPV types, suggesting a common path towards viral pathogenesis. CONCLUSIONS: The present study sheds light into the mechanisms behind the misexpression of PDZ-polarity proteins during HPV infections. Our findings stress the relevance of the CKII-mediated regulation of E7 activities, providing novel insights into the joint action of HPV oncoproteins and further indicating a conserved and most likely crucial mechanism during the viral life cycle and the associated transformation.

Application of quantitative immunofluorescence assays to analyze the expression of cell contact proteins during Zika virus infections.

Zika Virus (ZIKV) is an RNA virus that belongs to the Flavivirus (FV) genus. In the last years, several unique characteristics of ZIKV among FV have been revealed, as the multiple routes of transmission and its ability to reach different human tissues, including the central nervous system. Thus, one of the most intriguing features of ZIKV biology is its ability to cross diverse complex biological barriers. The main aim of this study is to contribute to the understanding of the still unclear mechanisms behind this viral activity. We investigated an African strain and two South American ZIKV isolates belonging to the Asian lineage, in order to characterize possible differences regarding their ability to disturb intercellular junctions. The Asian isolates correspond to an imported (Venezuelan) and an autochthonous (Argentinian) ZIKV strain for which there is still no data available. We focused on occludin and DLG1 expression as markers of tight and adherent junctions, respectively. For this, we applied a quantitative immunofluorescence assay that can ascertain alterations in the cell junction proteins expression in the infected cells. Our findings indicated that the different ZIKV strains were able to reduce the levels of both polarity proteins without altering their overall cell distribution. Moreover, the grade of this effect was strain-dependent, being the DLG1 reduction higher for the African and Asian Venezuelan isolates and, on the contrary, occludin down-regulation was more noticeable for the Argentinian strain. Interestingly, among both junction proteins the viral infection caused a relative larger reduction in DLG1 expression for all viruses, suggesting DLG1 may be of particular relevance for ZIKV infections. Taken together, this study contributes to the knowledge of the biological mechanisms involved in ZIKV cytopathogenesis, with a special focus on regional isolates.

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

BACKGROUND: Persistent infection with high-risk Human Papillomavirus (HPVs) is associated with the development of cervical cancer. The transforming capacity of these viruses relies on the cooperative action of the E6 and E7 viral oncoproteins. Among the oncogenic activities of E6, the interaction and interference with cell polarity PDZ proteins have been well established. One of the most characterized PDZ targets of HPV E6 is human Disc large 1 (DLG1), a scaffolding protein involved in the control of cell polarity and proliferation. Interestingly, in cervical squamous intraepithelial lesions, alterations in DLG1 expression were observed in association to tumour progression. Moreover, the expression of both HPV E6 and E7 proteins may be responsible for the changes in DLG1 abundance and cell localization observed in the HPV-associated lesions. METHODS: Due to the relevance of DLG1 deregulation in tumour development, we have performed an in-depth investigation of the expression of DLG1 in the presence of the HPV oncoproteins in epithelial cultured cells. The effects of HPV E6 and E7 proteins on DLG1 abundance and subcellular localization were assessed by western blot and confocal fluorescence microscopy, respectively. RESULTS: We demonstrated that the relative abundance of HPV-18 E6 and DLG1 is a key factor that contributes to defining the expression abundance of both proteins. We also show here that a high expression level of DLG1 may negatively affect HPV-18 E6 nuclear expression. Moreover, the co-expression of HPV-18 E6 and E7 produces a striking effect on DLG1 subcellular localization and a co-distribution in the cytoplasmic region. Interestingly, HPV-18 E7 is also able to increase DLG1 levels, likely by rescuing it from the E6-mediated proteasomal degradation. CONCLUSIONS: In general, the data suggest that HPV-18 E6 and E7 may have opposing activities in regards to the regulation of DLG1 levels and may cooperatively contribute to its subcellular redistribution in the HPV context. These findings constitute a step forward in understanding the differential expression of DLG1 during tumour progression in an HPV-associated model.

Scrib and Dlg1 polarity proteins regulate Ag presentation in human dendritic cells.

We recently reported, for the first time, the expression and regulation of the PDZ polarity proteins Scrib and Dlg1 in human APCs, and also described the viral targeting of these proteins by NS1 of influenza A virus in human dendritic cells (DCs). Scrib plays an important role in reactive oxygen species (ROS) production in Mϕs and uropod formation and migration in T cells, while Dlg1 is important for T cell downstream activation after Ag recognition. Nevertheless, the functions of these proteins in human DCs remain unknown. Here, we knocked-down the expression of both Scrib and Dlg1 in human DCs and then evaluated the expression of co-stimulatory molecules and cytokine production during maturation. We demonstrated that Scrib is necessary for adequate CD86 expression, while Dlg1 is important for CD83 up-regulation and IL-6 production upon maturation, suggesting that Scrib and Dlg1 participate in separate pathways in DCs. Additionally, both proteins are required for adequate IL-12 production after maturation. Furthermore, we showed that the inefficient maturation of DCs induced by Scrib or Dlg1 depletion leads to impaired T cell activation. Our results revealed the previously unknown contribution of Scrib and Dlg1 in human DCs pivotal functions, which may be able to impact innate and adaptive immune response.

Differential expression of DLG1 as a common trait in different human diseases: an encouraging issue in molecular pathology.

Human disc large (DLG1) is a scaffolding protein that through the interaction with diverse cell partners participates in the control of key cellular processes such as polarity, proliferation and migration. Experimental data have mainly identified DLG1 as a tumor suppressor. An outstanding point for DLG1 protein is that altered DLG1 expression and DLG1 gene mutations were observed in different pathologies, including cancer and neurological and immunological disorders. Evident changes in DLG1 abundance and/or cell localization were identified in a number of studies suggesting its participation in molecular mechanisms responsible for the development of such illnesses. In this review, we focus on some of the latest findings regarding DLG1 alterations in different diseases as well as its potential use as a biomarker for pathological progression. We further address the current knowledge on the molecular mechanisms regulating DLG1 expression and the posttranslational modifications that may affect DLG1 cell localization and functions. Despite the advances in this field, there are still open questions about the precise molecular link between alterations in DLG1 expression and the development of each specific pathology. The complete understanding of this concern will give us new scenarios for the design of promising diagnosis and therapeutic tools.

PDZ proteins are expressed and regulated in antigen-presenting cells and are targets of influenza A virus.

In this work, we identified the expression, regulation, and viral targeting of Scribble and Dlg1 in antigen-presenting cells. Scribble and Dlg1 belong to the family of PDZ (postsynaptic density (PSD95), disc large (Dlg), and zonula occludens (ZO-1)) proteins involved in cell polarity. The relevance of PDZ proteins in cellular functions is reinforced by the fact that many viruses interfere with host PDZ-dependent interactions affecting cellular mechanisms thus favoring viral replication. The functions of Scribble and Dlg have been widely studied in polarized cells such as epithelial and neuron cells. However, within the cells of the immune system, their functions have been described only in T and B lymphocytes. Here we demonstrated that Scribble and Dlg1 are differentially expressed during antigen-presenting cell differentiation and dendritic cell maturation. While both Scribble and Dlg1 seem to participate in distinct dendritic cell functions, both are targeted by the viral protein NS1 of influenza A in a PDZ-dependent manner in dendritic cells. Our findings suggest that these proteins might be involved in the mechanisms of innate immunity and/or antigen processing and presentation that can be hijacked by viral pathogens.

Interference of HTLV-1 Tax Protein with Cell Polarity Regulators: Defining the Subcellular Localization of the Tax-DLG1 Interaction.

Human T cell leukemia virus (HTLV)-1 Tax (Tax) protein is very important in viral replication and cell transformation. Tax localizes in the nucleus and cytoplasm in association with organelles. Some activities of Tax depend on interactions with PDZ (PSD-95/Discs Large/Z0-1) domain-containing proteins such as Discs large protein 1 (DLG1) which is involved in cell polarity and proliferation. The DLG1 interaction results in a cytoplasmic co-localization pattern resembling vesicular aggregates, the nature of which is still unknown. To further explore the role of PDZ proteins in HTLV-1 cell transformation, we deeply investigated the Tax-DLG1 association. By fluorescence resonance energy transfer (FRET), we detected, for the first time, the direct binding of Tax to DLG1 within the cell. We showed that the interaction specifically affects the cellular distribution of not only DLG1, but also Tax. After studying different cell structures, we demonstrated that the aggregates distribute into the Golgi apparatus in spatial association with the microtubule-organizing center (MTOC). This study contributes to understand the biological significance of Tax-PDZ interactions.

DLG1 polarity protein expression associates with the disease progress of low-grade cervical intraepithelial lesions.

Human Discs large tumour suppressor (DLG1) participates in regulating cell polarity and proliferation, suggesting an important connection between epithelial organization and cellular growth control. However, it was demonstrated that DLG1 could acquire oncogenic attributes in some specific contexts. In this work, we evaluated the expression of DLG1 and its contribution to the progress of cervical lesions in order to investigate a potential role of this polarity protein in human oncogenic processes. We analyzed cervical biopsies from women with low-grade squamous intraepithelial lesion (LSIL) diagnosis (n=30), for DLG1 expression by immunohistochemistry. These results were correlated with the clinical monitoring of the patients during a 24-month follow-up period. Our data indicate that while all LSIL patients with a DLG1 staining pattern similar to normal tissues are significantly more likely to regress (n=23, Pattern I), all LSIL biopsy specimens showing a diffuse and intense DLG1 staining likely progress to high-grade lesions (n=4, Pattern II). Finally, all persistent LSIL analyzed showed an undetermined DLG1 staining, with a diffuse distribution without a strong intensity (n=3, Pattern III). We found a significant association between the expression pattern of DLG1 and the evolution of the lesion (p<0.00001). This work contributes to the knowledge of DLG1 biological functions, suggesting that its expression may have an important role in the progression of early dysplastic cervical lesions, giving prognostic information.