A 32 kDa viral attachment protein of lymphocystis disease virus (LCDV) specifically interacts with a 27.8 kDa cellular receptor from flounder (Paralichthys olivaceus)

Abstract

The 27.8 kDa protein in flounder gill (FG) cells was previously proved to be a receptor specific for lymphocystis disease virus (LCDV) entry and infection. In this paper, a 32 kDa viral attachment protein (VAP) of LCDV specifically binding to the 27.8 kDa receptor (27.8R) was found by far-Western blotting coupled with monoclonal antibodies (MAbs) against 27.8R. The 32 kDa protein was confirmed to be encoded by the open reading frame (ORF) 038 gene in LCDV-C, and predicted to contain a putative transmembrane region, multiple N-myristoylation and glycosylation sites and phosphorylation motifs. The expression plasmid of pET-32a-ORF038 was constructed and the recombinant VAP (rVAP) was obtained. Rabbit polyclonal antibodies against the rVAP were prepared and could recognize the rVAP and 32 kDa protein in LCDV. Immunogold electron microscopy showed that the 32 kDa protein was located on the surface of LCDV particles. Immunofluorescence assay demonstrated that the rVAP could bind to the 27.8R on the cell membrane of the FG monolayer and the anti-27.8R MAbs could block the rVAP binding. Pre-incubation of the rVAP with FG cells before LCDV infection, or pre-incubation of LCDV with the antibodies against the rVAP, could significantly decrease the LCDV copy numbers (P<0.05) and delay the emergence of cytopathic effects in FG cells in a dose-dependent manner. These results indicated for the first time that the 32 kDa protein functioned as an attachment protein for the initial attachment and entry of LCDV, and the interaction of the 32 kDa VAP with the 27.8R-initiated LCDV infection.

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

“…Research on virus cellular receptors is critical for elucidating the molecular mechanisms of virus infection, replication, and pathogenesis (34,35). Previously, a 27.8-kDa protein in FG cells was found to be the putative receptor specific for LCDV entry and infection (14), which could interact with a 32-kDa envelope protein of LCDV functioning as a VAP (19). In the present study, we expanded the investigation to identify 27.8R, and 27.8R was identified as VDAC2 and RACK1 through two-dimensional (2D) Western blotting, 2D far-Western blotting, a 2D virus overlay protein binding assay (2D VOPBA), and mass spectrometry (MS).…”

“…FG cell membrane proteins were prepared using gradient centrifugation as described in our previous study (14). Escherichia coli strain BL21(DE3) expressing the 32-kDa VAP of LCDV was grown in LB broth, and recombinant VAP (rVAP) protein was purified by using a Ni-NTA column (19). White spot syndrome virus (WSSV) particles and recombinant pET28a-VP26 of WSSV were produced previously by our laboratory (52,53).…”

“…After three washes with PBS, the percentages of LCDV-infected FG cells were analyzed by FACS analysis using a flow cytometer (Beckman Coulter, USA). The DNA of FG cells in each treatment group was extracted using a TIANamp marine animal DNA kit (Qiagen, Germany), and LCDV copy numbers were detected by qPCR using specific primers LCDV038F and LCDV038R ( Table 2) and calculated according to a standard curve as described in a previous study (19). Furthermore, FG cells grown on circular coverslips in 24-well plates were incubated with 1.6 g/ml anti-VDAC2 polyclonal antibodies or 16 g/ml anti-RACK1 polyclonal antibodies and then infected with LCDV as described above.…”

“…To confirm whether VDAC2/RACK1 knockdown can reduce LCDV infection, 24 h and 48 h after transfection with VDAC2-siRNA, RACK1-siRNA, and NC-siRNA, FG cells were infected with LCDV particles at 4 TCID 50 /ml at 22°C for 1 h. Forty-eight hours after LCDV infection, the cells were collected, DNA was extracted using a TIANamp marine animal DNA kit (Qiagen, Germany), and LCDV copy numbers were detected by qPCR and calculated by using a standard curve according to the threshold cycle (C T ) values (19). The experiments were carried out in triplicate.…”

“…Besides, LCDV infection could induce the upregulation of 27.8R expression, which in turn increased the susceptibility and availability of FG cells for LCDV entry (17), and 27.8R was widely detected on tissues and leukocytes of flounder and turbot (Scophthalmus maximus) (7,18). In the following research, a 32-kDa envelope protein of LCDV, encoded by the open reading frame (ORF) 038 gene of LCDV-C, was found to function as a viral attachment protein (VAP) and initiate LCDV infection via interaction with 27.8R (19), and anti-32-kDa VAP MAbs were prepared (20). However, further studies are required to determine the biological function of 27.8R and the specific mechanism of 27.8R-mediated LCDV infection.…”