Keck Foundation to support COVID-19 research to A

Keck Foundation to support COVID-19 research to A. studies further confirmed that GRP78 can directly bind to the RBD of SARS-2-S and ACE2. To investigate the role of GRP78 in this complex, we knocked down GRP78 in VeroE6-ACE2 cells. Loss of GRP78 markedly reduced cell surface ACE2 expression and led to activation of markers of the unfolded protein response. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159) selected for its safe clinical profile in preclinical models depleted cell surface GRP78 and reduced cell surface ACE2 expression, as well as SARS-2-S-driven viral entry and SARS-CoV-2 infection gene, is the major HSP70 family member in the endoplasmic reticulum (ER) serving critical protein folding functions (7, 8). In addition, GRP78 is a master regulator 11-hydroxy-sugiol of the unfolded protein response, which allows cells to adapt to adverse stress conditions targeting the ER (9, 10, 11). GRP78 is broadly expressed in many tissues including bronchial epithelial cells and the respiratory mucosa at levels significantly higher than that of ACE2 (12). In recent case-control studies, serum GRP78 levels were found to be elevated in SARS-CoV-2 cases (13). Under pathophysiological conditions such as cancer and viral infection, GRP78 can translocate from the ER to Rabbit Polyclonal to APOL4 the cell surface where it acts as a coreceptor for various signaling molecules, as well as for viral entry (10, 14, 15, 16, 17, 18, 19, 20, 21). For coronaviruses, GRP78 is known to interact with the bat coronavirus HKU9 and MERS-CoV Spike proteins, facilitating cell surface attachment and viral entry (22). Furthermore, virus infection leads to ER stress and increased total and cell surface GRP78 (csGRP78) expression further enhancing viral infection in a positive feedback cycle (15, 22). Here, utilizing biochemical and imaging approaches, we established GRP78 interactions with SARS-2-S and ACE2. We further demonstrated that a humanized monoclonal antibody (hMAb159) with high affinity and specificity against GRP78 and a safe clinical profile in preclinical models (23) depletes csGRP78 and reduces cell surface ACE2 (csACE2), SARS-CoV-2 entry, and infection. Results GRP78 forms complex with SARS-CoV-2 Spike protein and host receptor ACE2 To test GRP78 binding to SARS-2-S in cells, we expressed HA-tagged SARS-2-S (HA-Spike) and FLAG-tagged GRP78 (F-GRP78) in African green monkey kidney epithelial VeroE6 cells overexpressing human ACE2 (VeroE6-ACE2) as a model system. Co-immunoprecipitation (IP) 11-hydroxy-sugiol for the HA-epitope showed that F-GRP78 can be pulled down with HA-Spike suggesting potential interaction between the two proteins (Fig.?1pull-down assays (Fig.?1molecular docking study (4). Additionally, GRP78 can directly bind ACE2 and that binding to ACE2 requires both the SBD and the ATP-binding domain. Open in a separate window Figure?1 GRP78 interacts with SARS-CoV-2 Spike protein and ACE2.binding assays. GST (G) or GST-tagged GRP78 (G78) proteins affixed to Glutathione Sepharose resin were incubated with His-tagged recombinant SARS-CoV-2 Spike receptor-binding domain (RBD) or His-tagged recombinant human ACE2 protein. The input GST and G78 proteins, the bound and flow through (FT) fractions showing unbound proteins were subjected to western blot using antibodies against GST, His, or ACE2 as indicated. except lysates 11-hydroxy-sugiol from cells expressing WT or the indicated mutant forms of GRP78 were subjected to IP with anti-FLAG antibody, with GADPH serving as loading control for the WCL. and and represent permeabilized (Perm) and nonpermeabilized (non-Perm) cells respectively. The are enlarged on the indicate costaining. (Scale bars, 20?m). indicates colocalization (Scale bars, 10?m). except for IF staining for ACE2 (except VeroE6-ACE2 cells were subjected to PLA using anti-ACE2 and anti-GRP78 antibodies (Scale bars, 10?m). and and and XTT assay. Data are mean? SD (n?= 3). and pull-down assay, immunoprecipitation, cell surface biotinylation, immunoblot analysis, flow cytometric analysis, immunofluorescent staining, proximity ligation assay, generation of VSV pseudotype and transduction experiments, quantification of cell viability, and plaque inhibition assay. Statistical analysis All results are expressed as means. Error bars are reported as standard deviation. Differences between two group means were analyzed using a two-tailed unpaired Students t-test. A p-value less than 0.05 is statistically significant. Data availability The authors confirm that the data supporting the findings of this study are available within the article. Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. Supporting information This article contains supporting information (20, 21, 23, 27, 31, 32). Acknowledgments We.