Abstract
In previous research, voltage-dependent anion channel protein 2 (VDAC2) and the receptor of activated protein C kinase 1 (RACK1) in flounder (Paralichthys olivaceus) were confirmed as functional receptors for lymphocystis disease virus (LCDV) entry; however, the underlying mechanism of VDAC2- and RACK1-mediated LCDV entry remains unclear. In this study, we elucidated the endocytosis pathway of LCDV entry into flounder gill (FG) cells by treatment with specific inhibitory agents, siRNAs, and co-localization analysis. LCDV entry was significantly inhibited by the disruption of caveolae-mediated endocytosis, dynamin, and microtubules, and the knockdown of caveoline-1 and dynamin expression, but was not inhibited by the disruption of clathrin-mediated endocytosis, micropinocytosis, or low-pH conditions. The disruption of caveolae-mediated and clathrin-mediated endocytosis was verified by the internalization of cholera toxin subunit B (CTB) and transferrin, respectively. Confocal immunofluorescence assay demonstrated that LCDV was co-localized with VDAC2 and RACK1, CTB was co-localized with VDAC2 and RACK1 and partially with LCDV, but transferrin was not co-localized with LCDV, VDAC2, or RACK1, indicating that LCDV utilized the same pathway as CTB, i.e., caveolae-mediated endocytosis. This was different from the pathway of transferrin, which used clathrin-mediated endocytosis. Furthermore, caveolin-1 was co-localized with LCDV, VDAC2, and RACK1, suggesting that caveolin-1 was involved in LCDV entry. These results revealed for the first time that LCDV entered into FG cells via caveolae-mediated endocytosis facilitated by VDAC2 and RACK1 receptors, relying on dynamin and microtubules in a pH-independent manner, which provided new insight into the molecular mechanisms of LCDV entry and potential for the development of antiviral agents, expanding our understanding of iridovirus infection.
Highlights
Lymphocystis disease virus (LCDV), an enveloped DNA virus belonging to the genus Lymphocystivirus within the family Iridoviridae [1,2], is the causative agent of lymphocystis disease, which infects more than 140 wild and cultured species of marine, brackish, and freshwater fish worldwide [3,4]
Frog virus 3 (FV3), which is a type species of the genus Ranavirus and recognized as a model for iridoviruses, enters into mammalian cells (BHK-21) via the clathrin-mediated endocytosis [48], while tiger frog virus (TFV) enters into HepG2 cells via the caveola-mediated endocytosis pathway in a pH-dependent manner [49], and Singapore grouper iridovirus (SGIV) enters into the grouper spleen (GS) cells by clathrin-mediated endocytosis and micropinocytosis [45]
The LCDV 32 kDa viral attachment protein (VAP) is encoded by the ORF038 gene of LCDV isolated in China (LCDV-C) [14,50], and the LCDV-C ORF038 gene had homologues with genes encoding SGIV VP19 and rana grylio virus envelope protein 2L [50,51]
Summary
Lymphocystis disease virus (LCDV), an enveloped DNA virus belonging to the genus Lymphocystivirus within the family Iridoviridae [1,2], is the causative agent of lymphocystis disease, which infects more than 140 wild and cultured species of marine, brackish, and freshwater fish worldwide [3,4]. We have identified 27.8 kDa protein as a cellular receptor for LCDV from flounder (Paralichthys olivaceus) gill (FG) cells [11], a cell line derived from the gill tissue of a flounder [12]. A 32 kDa envelope protein of LCDV was found to function as a viral attachment protein (VAP), and the interaction of the 32 kDa VAP with the 27.8 kDa putative receptor protein initiates LCDV infection in FG cells [13]; monoclonal antibodies (MAbs) against the 32 kDa VAP can effectively neutralize LCDV infection [14]. Voltage-dependent anion channel protein 2 (VDAC2) and the receptor of activated protein C kinase 1 (RACK1) have been identified among the putative 27.8 kDa receptor protein as the functional receptor for LCDV entry [15].
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