Abstract
Biomarkers have important roles in various physiological functions and disease pathogenesis. As a nucleocytoplasmic DNA virus, Singapore grouper iridovirus (SGIV) causes high economic losses in the mariculture industry. Aptamer-Q5-complexed major capsid protein (MCP) in the membrane of SGIV-infected cells can be used as a specific molecular probe to investigate the crucial events of MCP endocytosis into SGIV-infected host cells during viral infection. Chlorpromazine blocks clathrin-mediated endocytosis, and MCP endocytosis into SGIV-infected cells decreased significantly when the cells were pretreated with chlorpromazine. The disruption of cellular cholesterol by methyl-β-cyclodextrin also significantly reduced MCP endocytosis. In contrast, inhibitors of key regulators of caveolae/raft-dependent endocytosis and macropinocytosis, including genistein, Na+/H+ exchanger, p21-activated kinase 1 (PAK1), myosin II, Rac1 GTPase, and protein kinase C (PKC), had no effect on MCP endocytosis. The endocytosis of the biomarker MCP is dependent on low pH and cytoskeletal actin filaments, as shown with various inhibitors (chloroquine, ammonia chloride, cytochalasin D). Therefore, MCP enters SGIV-infected host cells via clathrin-mediated endocytosis, which is dependent on dynamin, cholesterol, low pH, and cytoskeletal actin filaments. This is the first report of a specific aptamer-based probe used to analyze MCP endocytosis into SGIV-infected host cells during viral infection. This method provides a convenient strategy for exploring viral pathogenesis and facilitates the development of diagnostic tools for and therapeutic approaches to viral infection.
Highlights
Iridoviruses are large icosahedral cytoplasmic DNA viruses with circularly permuted, terminally redundant, double-stranded DNA genomes (Qin et al, 2003)
Confocal fluorescence imaging showed that the Cy5-labeled aptamer Q5 (Cy5-Q5) bound to the surfaces of SGIVinfected cells at 4◦C, but not to normal Grouper spleen (GS) cells
When Cy5-Q5 was incubated with Singapore grouper iridovirus (SGIV)-infected cells at 4◦C, the fluorescent signal was only visible around the cell plasma membrane, but not inside the cells (Figure 1A)
Summary
Iridoviruses are large icosahedral cytoplasmic DNA viruses with circularly permuted, terminally redundant, double-stranded DNA genomes (Qin et al, 2003). Singapore grouper iridovirus (SGIV) was first isolated from the grouper Epinephelus tauvina It belongs to the genus Ranavirus and currently causes high economic losses in the mariculture industry (Qin et al, 2003; Xiao et al, 2019; Liu et al, 2020). Modifications appear in the host cell membranes (Verdaguer et al, 2014; Abós et al, 2015; Seeger and Mason, 2015; Yu et al, 2019a), which can potentially be used as important biomarkers of infection. Membrane proteins account for about 30% of the total cellular proteins and have important roles in various physiological functions (Shangguan et al, 2008) Knowledge of these biomarkers will extend our understanding of viral pathogenesis.
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