Summary auto-generated
This study investigated how Japanese encephalitis virus (JEV) enters Vero cells by testing three endocytosis inhibitors. Chlorpromazine, which blocks clathrin-dependent endocytosis, reduced JEV antigen-positive cells to approximately one-fifth of control levels. Nystatin and cytochalasin D, which inhibit caveola-dependent endocytosis and macropinocytosis respectively, produced minimal inhibitory effects. Subcellular fractionation demonstrated that in chlorpromazine-treated cells, JEV remained at the cell membrane rather than translocating to endosome compartments as occurred in untreated cells. Immunofluorescence microscopy confirmed that chlorpromazine prevented intracellular clathrin accumulation and JEV uptake. Importantly, chlorpromazine did not affect initial JEV binding to cells or cell viability, indicating its effects were specific to the endocytic entry process. These findings establish that JEV utilizes the clathrin-dependent endocytic pathway for cellular entry in Vero cells, with virus particles subsequently trafficking through acidic endosomal compartments critical for successful infection.
Key findings
- Japanese encephalitis virus enters Vero cells primarily through clathrin-dependent endocytosis, as chlorpromazine treatment reduced infection to less than 20% of control levels while sparing cell viability
- Chlorpromazine prevents viral translocation from the plasma membrane to endosomal compartments, blocking a critical early step in productive infection
- Caveola-dependent endocytosis and macropinocytosis are not required for JEV entry, as nystatin and cytochalasin D had minimal inhibitory effects
- Viral antigen accumulates at the cell membrane in chlorpromazine-treated cells rather than in endosomal compartments, confirming blockade of the clathrin-dependent pathway
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Abstract
Entry of Japanese encephalitis virus (JEV) into cells was analysed by using the vertebrate cell line Vero. Vero cells were treated with chlorpromazine, nystatin or cytochalasin D, which inhibit clathrin- and caveola-dependent endocytosis, and macropinocytosis of the cells, respectively. Productive JEV infection was inhibited by pretreatment with chlorpromazine; the number of JEV antigen-positive cells was less than one-fifth of that in untreated cultures, but was not significantly decreased by pretreatment with nystatin or cytochalasin. Viral antigens were detected in the membrane fractions, but not in the endosome fractions from chlorpromazine-treated JEV-inoculated cells. When the cells were treated with chlorpromazine, clathrin heavy chain antigen and JEV antigen were not detected in cytoplasm by indirect immunofluorescence staining. These results indicate that JEV is taken up by cells through the clathrin-dependent endocytic pathway, and this process leads to infection.