Voltage-Dependent Anion Channel Protein 2 (VDAC2) and Receptor of Activated Protein C Kinase 1 (RACK1) Act as Functional Receptors for Lymphocystis Disease Virus Infection

Abstract

In previous research, a 27.8-kDa protein in flounder Paralichthys olivaceus gill (FG) cells was identified as a putative cellular receptor (27.8R), which mediated lymphocystis disease virus (LCDV) infection via interaction with a 32-kDa viral attachment protein (VAP) of LCDV, and monoclonal antibodies (MAbs) against 27.8R and 32-kDa VAP were developed. In this study, the 27.8R was identified as voltagedependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1) of flounder. Recombinant VDAC2 (rVDAC2) and RACK1 (rRACK1) were obtained by prokaryotic expression, and rabbit anti-VDAC2/RACK1 polyclonal antibodies were prepared. The rVDAC2, rRACK1, and 27.8-kDa proteins in FG cells were recognized by anti-27.8R MAbs and anti-VDAC2/RACK1 polyclonal antibodies simultaneously. Preincubation of FG cells with anti-VDAC2/RACK1 polyclonal antibodies significantly decreased the percentages of LCDV-infected cells and LCDV copy numbers, blocked virus infection, and delayed the development of cytopathic effect. The mRNA expressions of VDAC2 and RACK1 in FG cells were upregulated to maximum levels 12 h and 48 h after LCDV infection, respectively. VDAC2/RACK1 knockdown through short interfering RNA (siRNA) significantly reduced VDAC2/RACK1 expression and LCDV copy numbers in FG cells compared with negative controls, while VDAC2/ RACK1 expression on LCDV-nonpermissive epithelial papillosum cells (EPCs) conferred susceptibility to LCDV infection, indicating the VDAC2 and RACK1 were sufficient to allow LCDV entry and infection. All these results collectively showed that VDAC2 and RACK1 function as receptors for LCDV entry and infection. IMPORTANCE Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease in fish, which has caused huge economic losses to the aquaculture industry worldwide, but the molecular mechanism underlying the LCDV-host interaction remains unclear. Here, the 27.8-kDa putative cellular receptor for LCDV was identified as voltagedependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1), and our results revealed that VDAC2 and RACK1 expression was sufficient to allow LCDV entry and that they are functional receptors that initiate LCDV infection for the first time, which leads to a better understanding of the molecular mechanism underlying LCDV infection and virus-host interactions.KEYWORDS 27.8-kDa cellular receptor, 32-kDa viral attachment protein, lymphocystis disease virus, receptor of activated protein C kinase 1, virus-host interaction, voltage-dependent anion channel protein 2 F ish lymphocystis disease (LCD) is a chronic viral disease with typical symptoms of clusters of enlarged hypertrophied dermal cells on the skin, fins, and internal organs of affected fish (1). Lymphocystis disease virus (LCDV) is the causative agent of LCD and Downloaded from der, was determined by qPCR, and VDAC2/RACK1 and LCDV were doubly stained in EPCs by immunofluorescence. RESULTSIdentification of the 27.8R protein. FG cell membrane protei...

Lymphocystis Disease Virus (Iridoviridae) Enters Flounder (Paralichthys olivaceus) Gill Cells via a Caveolae-Mediated Endocytosis Mechanism Facilitated by Viral Receptors

“…In LCDV infection, we previously observed that the LCDV 32 kDa VAP interacts with VDAC2 and RACK1 receptors to initiate virus infection. VDAC2/RACK1 knockdown through siRNA significantly reduced LCDV copy numbers, whereas VDAC2/RACK1 expression on LCDV-nonpermissive EPC conferred susceptibility to LCDV infection [ 15 ]. The LCDV 32 kDa 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 ].…”

“…For viruses that enter via caveolae-mediated endocytosis, receptor proteins are usually abundant in the caveolae of susceptible cells [ 67 ]. VDAC2 and RACK1 are widely distributed in caveolae [ 16 , 17 , 18 , 19 , 20 , 21 ], and VDAC2 and RACK1 knockdown or blocking inhibits LCDV infection of FG cells [ 15 ]. To elucidate the roles of VDAC2 and RACK1 in LCDV entry, we confirmed that LCDV could co-localize with VDAC2 and RACK1, and CTB also co-localized with VDAC2 and RACK1, while LCDV partially co-localized with CTB, indicating that VDAC2 and RACK1 mediate LCDV infection, and CTB might co-localize with VDAC2 and RACK1 during LCDV entry, even though the receptor protein for CTB in caveolae is ganglioside [ 68 ].…”

“…Rabbit anti-LCDV 32 kDa VAP polyclonal antibody, mouse anti-32 kDa VAP MAb, rabbit anti-RACK1 polyclonal antibody, and rabbit anti-VDAC2 polyclonal antibody were produced in the authors’ laboratory [ 13 , 14 , 15 ]. Alexa Fluor 647 conjugated goat anti-mouse or -rabbit Ig, Alexa Fluor 488 conjugated goat anti-mouse or -rabbit Ig, and Alexa Fluor 647 conjugated CTB (recombinant) or transferrin from human serum were purchased from Thermo Fisher (Waltham, MA, USA).…”

“…Moreover, 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 ]. Recently, 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 ]. However, the LCDV infection mechanism remains to be elucidated.…”

“…Cell membrane VDAC has also been reported to act as a scaffold protein [ 18 ]. Although VDAC2 and RACK1 are functional receptors for LCDV, infection by LCDV can be blocked by pre-incubation with anti-VDAC2 and anti-RACK1 antibodies or knockdown of VDAC2 and RACK1 expression through short interfering RNAs (siRNAs), and VDAC2/RACK1 expression on LCDV-nonpermissive epithelial papillosum cell (EPC) conferred susceptibility to LCDV infection [ 15 ]. Nonetheless, the underlying mechanism of VDAC2- and RACK1-mediated LCDV entry remains unclear, and further studies focusing on the entry pathway of LCDV facilitated by VDAC2 and RACK1 receptors are required.…”