Abstract
Infectious pancreatic necrosis virus (IPNV) is a non-enveloped virus belonging to the Birnaviridae family. IPNV produces an acute disease in salmon fingerlings, with high mortality rates and persistent infection in survivors. Although there are reports of IPNV binding to various cells, the viral receptor and entry pathways remain unknown. The aim of this study was to determine the endocytic pathway that allows for IPNV entry. We observed that IPNV stimulated fluid uptake and virus particles co-localysed with the uptake marker dextran in intracellular compartments, suggesting a role for macropinocytosis in viral entry. Consistent with this idea, viral infection was significantly reduced when the Na+/H+ exchanger NHE1 was inhibited with 5-(N-Ethyl-N-isopropyl) amiloride (EIPA). Neither chlorpromazine nor filipin complex I affected IPNV infection. To examine the role of macropinocytosis regulators, additional inhibitors were tested. Inhibitors of the EGFR pathway and the effectors Pak1, Rac1 and PKC reduced viral infection. Together, our results indicate that IPNV is mainly internalized into CHSE-214 cells by macropinocytosis.
Highlights
Infectious pancreatic necrosis virus (IPNV), a dsRNA virus belonging to the Birnaviridae family, is the etiological agent of infectious pancreatic necrosis, which affects several salmonid species
Our results show that IPNV stimulates fluid uptake by CHSE-214 cells and that inhibition of the Na+/H+ pump with 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) reduces IPNV entry and infection Macropinocytosis inhibitors that do not affect clathrin-mediated endocytosis (CME) or lipid raft/caveolin-mediated endocytosis impede viral infection, suggesting that macropinocytosis is a major pathway for IPNV entry into CHSE-214 cells
This assay was used in the following experiments to examine the impact of inhibitors of macropinocytosis, CME and lipid raft-/caveolin-mediated endocytosis in IPNV entry
Summary
Infectious pancreatic necrosis virus (IPNV), a dsRNA virus belonging to the Birnaviridae family, is the etiological agent of infectious pancreatic necrosis, which affects several salmonid species This disease has great economic impact in all salmon-farming countries. To identify the cellular pathway that allows for IPNV entry into CHSE-214 cells, we took advantage of several well-characterized inhibitors of known components of these endocytosis mechanisms and analyzed their impact on viral internalisation. In this communication, we examined the roles of macropinocytosis, CME and lipid raft-mediated endocytosis in IPNV entry into CHSE-214 cells. Our results show that IPNV stimulates fluid uptake by CHSE-214 cells and that inhibition of the Na+/H+ pump with 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) reduces IPNV entry and infection Macropinocytosis inhibitors that do not affect CME or lipid raft/caveolin-mediated endocytosis impede viral infection, suggesting that macropinocytosis is a major pathway for IPNV entry into CHSE-214 cells
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