Betanodavirus-like particles enter host cells via clathrin-mediated endocytosis in a cholesterol-, pH- and cytoskeleton-dependent manner

Abstract

Betanodavirus, also referred to nervous necrosis virus (NNV), is the causative agent of the fatal disease, viral nervous necrosis and has brought significant economic losses in marine and freshwater cultured fish, especially larvae and juveniles. Here, we used an established invasion model with virus-like particle (VLP)-cells, mimicking orange-spotted grouper nervous necrosis virus (OGNNV), to investigate the crucial events of virus entry. VLP were observed in the perinuclear regions of Asian sea bass (SB) cells within 1.5 h after attachment. VLP uptake was strongly inhibited when cells were pretreated with biochemical inhibitors (chlorpromazine and dynasore) blocking clathrin-mediated endocytosis (CME) or transfected with siRNA against clathrin heavy and light chains. Inhibitors against key regulators of caveolae/raft-dependent endocytosis and macropinocytosis had no effect on VLP uptake. In contrast, disruption of cellular cholesterol by methyl-β-cyclodextrin or reduction of cholesterol fluidity by Cholera toxin B subunit significantly decreased VLP entry. Furthermore, VLP entry is dependent on low pH and cytoskeleton, demonstrated by inhibitor (chloroquine, ammonia chloride, cytochalasin D, wiskostatin, and nocodazole) perturbation. Therefore, OGNNV VLP enter SB cells via CME depending on dynamin-2, cholesterol and its fluidity, low pH, and cytoskeleton. In addition, ten more cell lines were screened for VLP entry and VLP can only enter NNV-sensitive cells, GB and SSN-1, via CME, indicating that CME is the common endocytosis pathway for VLP. These results may provide the data for NNV entry without the influence of the viral genome, an ideal model for exploring the behaviour of betanodavirus in cells, and valuable references to vaccine development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-017-0412-y) contains supplementary material, which is available to authorized users.

Betanodavirus and VER Disease: A 30-year Research Review

“…To date, it has not been possible to identify the neuronal receptors involved in NNV entry and the available data on cell receptors comes from studies using cell lines. Viral entry is believed to occur through clathrin-mediated endocytosis [169] and interaction with different cell receptors has been proposed. Consequently, sialic acid seems to be involved in NNV binding to SNN-1 cells [170] and therefore also to E-11, which are a clone of SSN-1 [171].…”

“…Consequently, sialic acid seems to be involved in NNV binding to SNN-1 cells [170] and therefore also to E-11, which are a clone of SSN-1 [171]. In grouper fin cells (GF-1), the grouper heat shock cognate protein 70 (GHSC70) has been proposed as an NNV receptor or co-receptor protein [172] and in SB cells, receptors have been reported to probably be proteins located at lipid rafts or even specific lipids [169]. Recently, Nectin-4/PVRL4, belonging to the family of immunoglobulin-like cell adhesion molecules, has been identified as a potential cellular receptor for NNV in both seven-band grouper and transfected SSN-1 cells [173,174].…”

Specific Aptamer-Based Probe for Analyzing Biomarker MCP Entry Into Singapore Grouper Iridovirus-Infected Host Cells via Clathrin-Mediated Endocytosis

“…The MCP entry efficiency was assessed with the protocol described by Huang et al (2017), with some modifications. GS cells were cultured to 100% confluence in 12-well plates or 35mm glass-bottom dishes at 28 • C for 24 h and then infected FIGURE 5 | MCP endocytosis depends on dynamin.…”

“…The inhibitor MβCD depletes cholesterol from the cell plasma membrane, and cholesterol plays a critical role in caveolae/raft-dependent endocytosis (Huang et al, 2017). Therefore, we used MβCD to analyze the role of cholesterol in the entry of the Q5-MCP complex into SGIV-infected cells.…”

“…Briefly, aptamer Q5 first binds to the target protein (MCP) on the cell surface, and the Q5-MCP interaction activates the cellular signaling pathways, which internalize Q5-MCP through one of several endocytotic mechanisms. These include clathrin-mediated endocytosis, caveolae/raft-dependent endocytosis, macropinocytosis, and non-clathrin-caveolae/raftdependent endocytosis (Wang et al, 2014;Huang et al, 2017). In this study, an aptamer (Q5)-based specific probe was used to investigate the trafficking mechanism and endocytotic pathway of MCP in host cells during SGIV infection.…”