Nandhini M. Sundaram

Several trafficking pathways are operational at the plasma membrane, and both clathrin-dependent, and clathrin-independent endocytosis (CIE), can serve as virus entry portals. Our research has shown that the neurotropic flavivirus : Japanese encephalitis virus (JEV), infects neuronal cells via CIE. Here we have identified and characterized two essential host-factors for JEV trafficking in neuronal cells: Endophilin & Epidermal Growth Factor Receptor (EGFR). Through quantitative estimation of viral RNA copy number, we demonstrate that JEV entry in neuronal cells, was blocked by knock-down of Endophilin A isoforms, while activation of Endophilin-mediated endocytosis using a specific inhibitor of GSK-3β enhanced virus entry. Deletion mutants of Endophilin showed an essential role of SH ₃ , BAR, H ₀ domains for virus entry. High resolution fluorescence imaging of virions showed overlap with Endophilin A2 puncta. Virus entry led to rearrangements of the actin cytoskeleton, and was highly sensitive to any pharmacological actin perturbation. Virus endocytosis activated EGFR, and the specific kinase inhibitors Erlotinib, and Gefitinib, reduced virus entry and replication in cultured cells and mouse primary cortical neurons. Silencing of EGFR, competitive inhibition with receptor ligand EGF, and EGFR specific antibodies significantly impaired JEV binding and entry, indicating the crucial role of its ligand binding domain for virus attachment/receptor interaction. EGFR colocalized with virions at early time-points of infection, and the ED3 domain of the JEV-envelope protein showed specific interaction with EGFR through Bio-layer interferometry (BLI). Our study provides evidence for JEV entry in neuronal cells through an endocytic pathway involving Endophilin and EGFR.

Several trafficking pathways are operational at the plasma membrane, and both clathrin-dependent, and clathrin-independent endocytosis (CIE), can serve as virus entry portals. Our research has shown that the neurotropic flavivirus : Japanese encephalitis virus (JEV), infects neuronal cells via CIE. Here we have identified and characterized two essential host-factors for JEV trafficking in neuronal cells: Endophilin & Epidermal Growth Factor Receptor (EGFR). Through quantitative estimation of viral RNA copy number, we demonstrate that JEV entry in neuronal cells, was blocked by knock-down of Endophilin A isoforms, while activation of Endophilin-mediated endocytosis using a specific inhibitor of GSK-3β enhanced virus entry. Deletion mutants of Endophilin showed an essential role of SH ₃ , BAR, H ₀ domains for virus entry. High resolution fluorescence imaging of virions showed overlap with Endophilin A2 puncta. Virus entry led to rearrangements of the actin cytoskeleton, and was highly sensitive to any pharmacological actin perturbation. Virus endocytosis activated EGFR, and the specific kinase inhibitors Erlotinib, and Gefitinib, reduced virus entry and replication in cultured cells and mouse primary cortical neurons. Silencing of EGFR, competitive inhibition with receptor ligand EGF, and EGFR specific antibodies significantly impaired JEV binding and entry, indicating the crucial role of its ligand binding domain for virus attachment/receptor interaction. EGFR colocalized with virions at early time-points of infection, and the ED3 domain of the JEV-envelope protein showed specific interaction with EGFR through BLI. Our study provides evidence for JEV entry in neuronal cells through an endocytic pathway involving Endophilin and EGFR.

Pancreatic β‐cells secrete insulin stored in large dense core vesicles (LDCV) by fusion of vesicle and plasma membrane during a process called insulin exocytosis. Insulin secretion is biphasic with a fast first phase and a sustained second phase. Previous studies have pointed out that exocytosis of insulin can occur via (1) single LDCVs fusing with the plasma membrane to release their content or (2) multiple vesicles are involved during a process called compound exocytosis. Compound exocytosis represents a specialized form of secretion in which vesicles undergo homotypic fusion either before (multi‐vesicular exocytosis) or continuous fusion in a sequential manner from (sequential exocytosis) within the same site at the plasma membrane. We see that the number of multi‐vesicles is few and not localized in the vicinity of the plasma membrane. Studying the kinetics of this process and correlating it with biphasic insulin secretion is not possible since there are no specific probes to detect them. It is challenging to identify compound exocytosis with probes that exist for simple exocytosis. To advance our understanding, we need a fluorescent probe that could detect secretory vesicles undergoing compound exocytosis and allow us to distinguish it from other modes of exocytosis. Here, we used two cargo proteins (NPY and tPA) labeled with different fluorescent proteins (mCherry GFP and eGFP) and employed total internal reflection fluorescence microscopy (TIRF‐M) to capture distinct single‐granule and multi‐granular fusion events. We identified tPA‐GFP as a better probe for studying compound exocytosis, as it can detect both simple and sequential exocytosis reliably. Using these probes, we have studied the kinetics of compound exocytosis in human β‐cells. These observations, with additional experiments, may open a whole new field to study the impact of compound exocytosis on biphasic secretion of insulin. Identifying targets to increase the compound exocytosis process can help potentiate insulin secretion in diabetics.