Interactions of Severe Acute Respiratory Syndrome Coronavirus 2 Protein E With Cell Junctions and Polarity PSD-95/Dlg/ZO-1-Containing Proteins.

The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ-binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains, which is identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified 10 human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, and LNX2) and MPP5/PALS1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2, and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo, sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4, and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in the E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.

The ACE2 Receptor for Coronavirus Entry Is Localized at Apical Cell-Cell Junctions of Epithelial Cells.

Transmembrane proteins of adherens and tight junctions are known targets for viruses and bacterial toxins. The coronavirus receptor ACE2 has been localized at the apical surface of epithelial cells, but it is not clear whether ACE2 is localized at apical Cell-Cell junctions and whether it associates with junctional proteins. Here we explored the expression and localization of ACE2 and its association with transmembrane and tight junction proteins in epithelial tissues and cultured cells by data mining, immunoblotting, immunofluorescence microscopy, and co-immunoprecipitation experiments. ACE2 mRNA is abundant in epithelial tissues, where its expression correlates with the expression of the tight junction proteins cingulin and occludin. In cultured epithelial cells ACE2 mRNA is upregulated upon differentiation and ACE2 protein is widely expressed and co-immunoprecipitates with the transmembrane proteins ADAM17 and CD9. We show by immunofluorescence microscopy that ACE2 colocalizes with ADAM17 and CD9 and the tight junction protein cingulin at apical junctions of intestinal (Caco-2), mammary (Eph4) and kidney (mCCD) epithelial cells. These observations identify ACE2, ADAM17 and CD9 as new epithelial junctional transmembrane proteins and suggest that the cytokine-enhanced endocytic internalization of junction-associated protein complexes comprising ACE2 may promote coronavirus entry.

Bioinformatics analyses reveal cell-barrier junction modulations in lung epithelial cells on SARS-CoV-2 infection.

Cell junctions maintain the blood-tissue barriers to preserve vascular and tissue integrity. Viral infections reportedly modulate cell-cell junctions to facilitate their invasion. However, information on the effect of COVID-19 infection on the gene expression of cell junction and cytoskeletal proteins is limited. Using the Gene Expression Omnibus and Reactome databases, we analyzed the data on human lung A549, NHBE, and Calu-3 cells for the expression changes in cell junction and cytoskeletal proteins by SARS-CoV-2 (CoV-2) infection. The analysis revealed changes in 3,660 genes in A549, 100 genes in NHBE, and 592 genes in Calu-3 cells with CoV-2 infection. Interestingly, EGOT (9.8-, 3- and 8.3-fold; p < .05) and CSF3 (4.3-, 33- and 56.3-fold; p < .05) were the only two genes significantly elevated in all three cell lines (A549, NHBE and Calu-3, respectively). On the other hand, 39 genes related to cell junctions and cytoskeleton were modulated in lung cells, with DLL1 demonstrating alterations in all cells. Alterations were also seen in several miRNAs associated with the cell junction and cytoskeleton genes modulated in the analysis. Further, matrix metalloproteinases involved in disease pathologies, including MMP-3, -9, and -12 demonstrated elevated expression on CoV-2 infection (p < .05). The study findings emphasize the integral role of cell junction and cytoskeletal genes in COVID-19, suggesting their therapeutic potential. Our analysis also identified a distinct EGOT gene that has not been previously implicated in COVID-19. Further studies on these newly identified genes and miRNAs could lead to advances in the pathogenesis and therapeutics of COVID-19.