Journal: mBio

Article Title: A novel host-targeted inhibitor of clathrin-mediated endocytosis limits spring viremia of carp virus infection

doi: 10.1128/mbio.00453-26

Figure Lengend Snippet: CPT preserves mitochondrial integrity and inhibits Bcl-2-associated X protein (Bax)-dependent intrinsic apoptosis in SVCV-infected EPC cells. ( A ) Calcein/CoCl 2 quenching assay of EPC cells at 24 and 48 hpi showing that CPT restores mitochondrial calcein fluorescence reduced by SVCV infection. Quantification of gray values is shown on the right. ( B ) Immunoblot analysis of cytochrome c (Cyt-C) and Bax in cytosolic and mitochondrial fractions. SVCV promotes Cyt-C release and Bax accumulation in mitochondria, whereas CPT reverses these changes. Densitometric analysis is shown on the right. ( C ) Immunoblot analysis of pro- and cleaved caspase-3 and caspase-9. SVCV activates the mitochondrial caspase cascade, which is suppressed by CPT. Quantification of cleaved caspases is shown on the right. ( D ) Annexin V/PI flow-cytometric analysis of apoptosis. CPT markedly reduces SVCV-induced apoptosis and increases the proportion of viable cells at 24 and 48 hpi. ( E ) Transmission electron microscopy of EPC cells. SVCV causes mitochondrial fragmentation, cristae disruption, and nuclear abnormalities, whereas CPT-treated infected cells largely retain normal ultrastructure. Scale bars, 1 μm. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with appropriate post hoc tests; * P < 0.05, ** P < 0.01.

Article Snippet: Fluorescence signals were acquired on a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec), and at least 10,000 events were collected per sample for quantitative analysis.

Techniques: Infection, Fluorescence, Western Blot, Transmission Assay, Electron Microscopy, Disruption

Journal: mBio

Article Title: A novel host-targeted inhibitor of clathrin-mediated endocytosis limits spring viremia of carp virus infection

doi: 10.1128/mbio.00453-26

Figure Lengend Snippet: Virion pre-incubation with CPT impairs subsequent SVCV internalization. ( A ) Schematic diagram of the experimental design. SVCV particles were incubated with CPT for the indicated times, purified by ultracentrifugation, and then used to infect EPC cells for binding, internalization, or replication assays. ( B–D ) RT-qPCR analysis of SVCV- N gene expression after infection with DMSO-treated (SVCV DMSO ) or CPT-treated (CPT + SVCV) virions. Effects of CPT pre-incubation on viral replication ( B ), attachment at 4°C ( C ), and internalization after shift to 25°C ( D ) are shown. ( E ) Confocal images of EPC cells infected with DiO-labeled SVCV (green) after pre-incubation of virions with DMSO or CPT for 1, 2, or 4 h. Nuclei were stained with DAPI (blue), and differential interference contrast (DIC) images are shown. Quantification of intracellular DiO fluorescence intensity is shown on the right. ( F ) Colocalization of DiO-labeled SVCV (green) with DiD-labeled plasma membrane (red) in cells infected with DMSO- or CPT-treated virions. Line-scan profiles of membrane and viral fluorescence along the indicated lines are shown to the right. ( G ) Representative motion tracks of DiD-labeled SVCV particles in live EPC cells after virion pre-incubation with DMSO or CPT. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with appropriate post hoc tests; * P < 0.05, ** P < 0.01.

Article Snippet: Fluorescence signals were acquired on a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec), and at least 10,000 events were collected per sample for quantitative analysis.

Techniques: Incubation, Purification, Binding Assay, Quantitative RT-PCR, Gene Expression, Infection, Labeling, Staining, Fluorescence, Clinical Proteomics, Membrane

Journal: mBio

Article Title: A novel host-targeted inhibitor of clathrin-mediated endocytosis limits spring viremia of carp virus infection

doi: 10.1128/mbio.00453-26

Figure Lengend Snippet: CPT pretreatment of EPC cells blocks SVCV internalization and replication in a reversible manner. ( A ) Effect of CPT pretreatment on SVCV binding. Nucleoprotein mRNA level was measured by RT-qPCR and expressed as fold change relative to SVCV DMSO . ( B ) Effect of CPT pretreatment on SVCV internalization. After CPT pretreatment for the indicated times, cells were infected with SVCV, shifted to 25°C for 2 h, treated with trypsin, and SVCV- N gene expression was quantified. ( C ) Effect of CPT pretreatment on SVCV replication. EPC cells were pre-incubated with CPT for the indicated times, infected with SVCV, and SVCV- N gene expression was determined at 48 hpi (left). Representative bright-field images at 8, 16, and 24 hpi (right) show that CPT pretreatment ≥4 h greatly reduces CPE. ( D ) Reversibility of CPT-mediated inhibition. After 24 h CPT pretreatment, cells were washed and cultured in drug-free medium for the indicated periods before SVCV infection. SVCV- N gene expression (left) and CPE (right) were evaluated at 48 hpi. ( E ) Colocalization of DiO-labeled SVCV (green) with DiD-labeled plasma membrane (red) in DMSO- or CPT-pretreated cells. Line-scan plots to the right show overlapping fluorescence peaks in CPT-pretreated cells, indicating virions remaining at the membrane. ( F ) Representative motion traces of DiD-labeled SVCV particles on live EPC cells pretreated with DMSO or CPT. In control cells, virions move inward from the cell surface, whereas in CPT-pretreated cells, they remain largely immobilized at the plasma membrane. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with appropriate post hoc tests; * P < 0.05, ** P < 0.01.

Article Snippet: Fluorescence signals were acquired on a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec), and at least 10,000 events were collected per sample for quantitative analysis.

Techniques: Binding Assay, Quantitative RT-PCR, Infection, Gene Expression, Incubation, Inhibition, Cell Culture, Labeling, Clinical Proteomics, Membrane, Fluorescence, Control

Journal: mBio

Article Title: A novel host-targeted inhibitor of clathrin-mediated endocytosis limits spring viremia of carp virus infection

doi: 10.1128/mbio.00453-26

Figure Lengend Snippet: CPT inhibits clathrin-mediated endocytosis in EPC cells. ( A ) Confocal images of Tfn-555 uptake in EPC cells treated with DMSO, CPT, or CPZ in the absence or presence of SVCV. Nuclei were stained with DAPI. ( B ) Quantification of intracellular Tfn-555 fluorescence intensity in the indicated groups. Both CPT and CPZ significantly reduce Tfn-555 uptake, with or without SVCV infection. ( C ) Immunofluorescence staining of CHC showing its distribution in Control DMSO , SVCV DMSO , CPT, CPT + SVCV, CPZ, and CPZ + SVCV cells. CPT and CPZ promote peripheral clustering of CHC at the plasma membrane. ( D and E ) Immunoblot analysis of CHC in plasma membrane (D; Na/K-ATPase as marker) and cytosolic (E; α-tubulin as marker) fractions. CPT and CPZ increase membrane-associated CHC while decreasing cytosolic CHC. Data are presented as mean ± SD. ** P < 0.01.

Article Snippet: Fluorescence signals were acquired on a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec), and at least 10,000 events were collected per sample for quantitative analysis.

Techniques: Staining, Fluorescence, Infection, Immunofluorescence, Control, Clinical Proteomics, Membrane, Western Blot, Marker