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Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis.

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.

SUBMITTER: Levican J

PROVIDER: S-EPMC5465193 | biostudies-other | 2017 Jun

REPOSITORIES: biostudies-other

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Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis.

Levican Jorge J Miranda-Cárdenas Camila C Soto-Rifo Ricardo R Aguayo Francisco F Gaggero Aldo A León Oscar O

Scientific reports 20170608 1

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-l ...[more]

PMID: 28596575

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Project description:Edwardsiella piscicida is an important pathogen that infects a wide range of hosts from fish to human. Recent studies demonstrated that E. piscicida can invade and survive within multiple nonphagocytic cells, but the internalization mechanism remains poorly understood. Here, we used HeLa cells as a nonphagocytic cell model to investigate the endocytic strategy used by the pathogenic E. piscicida isolate EIB202. Using a combination of optical and electron microscopy, we observed obvious membrane ruffles and F-actin rearrangements in HeLa cells after EIB202 infection. We also revealed that EIB202 internalization significantly depended on the activity of Na+/H+ exchangers and multiple intracellular signaling events related to macropinocytosis, suggesting that E. piscicida utilizes the host macropinocytosis pathway to enter HeLa cells. Further, using inhibitory drugs and shRNAs to block specific endocytic pathways, we found that a caveolin-dependent but not clathrin-dependent pathway is involved in E. piscicida entry and that its entry requires dynamin and membrane cholesterol. Together, these data suggest that E. piscicida enters nonphagocytic cells via macropinocytosis and caveolin-dependent endocytosis involving cholesterol and dynamin, improving the understanding of how E. piscicida interacts with nonphagocytic cells.IMPORTANCE Bacterial internalization is the first step in breaking through the host cell defense. Therefore, studying the mechanism of bacterial internalization improves the understanding of the pathogenic mechanism of bacteria. In this study, the internalization process on nonphagocytic cells by Edwardsiella piscicida was evaluated. Our results showed that E. piscicida can be internalized into nonphagocytic cells via macropinocytosis and caveolin-mediated endocytosis, and that cholesterol and dynamin are involved in this process. These results reveal a new method for inhibiting E. piscicida infection, providing a foundation for further studies of bacterial pathogenicity.

Project description:We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of the Birnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3'-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3' end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.

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Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis.

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Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis.

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