A putative cell surface receptor for white spot syndrome virus is a member of a transporter superfamily

Abstract

White spot syndrome virus (WSSV), a large enveloped DNA virus, can cause the most serious viral disease in shrimp and has a wide host range among crustaceans. In this study, we identified a surface protein, named glucose transporter 1 (Glut1), which could also interact with WSSV envelope protein, VP53A. Sequence analysis revealed that Glut1 is a member of a large superfamily of transporters and that it is most closely related to evolutionary branches of this superfamily, branches that function to transport this sugar. Tissue tropism analysis showed that Glut1 was constitutive and highly expressed in almost all organs. Glut1's localization in shrimp cells was further verified and so was its interaction with Penaeus monodon chitin-binding protein (PmCBP), which was itself identified to interact with an envelope protein complex formed by 11 WSSV envelope proteins. In vitro and in vivo neutralization experiments using synthetic peptide contained WSSV binding domain (WBD) showed that the WBD peptide could inhibit WSSV infection in primary cultured hemocytes and delay the mortality in shrimps challenged with WSSV. These findings have important implications for our understanding of WSSV entry.

Figure 1. VP53A interacted with Y455 clone in yeast.

(A) Yeast representing protein-protein interactions grew on high-stringency (–Leu/–Trp/–His/–Ade) medium. Plasmids used in this figure are described in the Materials and Methods. The blue signal in image was due to the presence of X-α-Gal. (B) Far western blot. Upper image: the yeast lysates were transferred on PVDF membrane and then detected with anti-HA antibody. Lower image: the yeast lysates were transferred on PVDF membrane, incubated with recombinant VP53A and the detected with anti-VP53A antibody. The yeast transformed with pGADT7-Rec only was set as control.

Figure 2. Primary structure of Y455 (Glut1) and major structural features.

The gene product of full-length of Glut1 contains 569 amino acids with 12 hydrophobic potential TM sequences. TM sequences are identified by the Kyte-Doolittle algorithm (Kyte and Doolittle, 1982) and are indicated by underlining the amino acid sequence. Signature sequences for MFS transporter are boxed. Potential N-linked glycosylation sites are shown in shaded boxes. The putative WSSV binding domain (WBD) between TM11 and TM12 is shown in bold font.

Figure 3. Hypothetcal model for the structure of the gene product of full-length of Y455 (Glut1).

The protein was predicted to contain 12 transmembrane (TM) sequences (TM1-12), with both the N- and C-termini intracellularly disposed. N-linked glycosylation can occur in the extracellular loop between TM1 and TM2 as shown. Conserved amino acids are indicated by the appropriate single-letter code; filled circles indicate conservative substitutions. The extracellular portion between TM1 and TM2 was selected to produce the recombinant protein to generate antibody and analyze the protein-protein interaction.

Figure 4. Tissue tropism analysis of Glut1.

Lane 1–9: PCR amplified Glut1 and α-actin fragments in pleopod, gill, stomach, midgut, heart, lymphoid organ, nervous tissue, hepatopancreas, and hemolymph. C: PCR reaction control which is set up in the same way as the experimental PCRs, but without template DNA added.

Figure 5. Recombinant expression in E. coli purification and western blot of shrimp Glut1.

(A) SDS-PAGE of expressed and purified of partial protein. Lanes M, pre-stained molecular weight marker; 1, non-induced; 2, induced; 3, purified Glut1-6×His partial fusion protein. (B) SDS-PAGE and western blotting of Glut1 in heart tissue lysate. Lanes M, pre-stained molecular weight marker; lane 1, coomassie blue stain; lane 2: heart tissue was incubated with rabbit pre-immune antiserum; lane 3: heart tissue was incubated with anti-Glut1 antibody. Glut1 was indicated with arrowhead.

Figure 6. Cellular localizations of Glut1 in L. vannamei hemocytes by confocal microscopy.

(A) Hemocytes were treated with rabbit anti-Glut1 antibody. (B) Hemocytes were treated with rabbit pre-immune antiserum. Scale bar: 7.5 µm.

Figure 7. Protein-protein interaction between Glut1 and PmCBP.

(A) Schematic diagram of Glut1 and PmCBP. Transmenbrane regions are shown in black boxes. The two solid lines indicated the portions of Glut1 and PmCBP for far western blot. The dot line indicated the portion interacting with VP53A in the yeast-2-hybrid (Y-2-H) assay. (B) SDS-PAGE analysis of the specificity of anti-Glut1 and anti-PmCBP antibodies. The short His tag containing protein purified from E. coli transformed with pET28 only was set as control. (C) Far western blot. Upper image: the recombinant PmCBP was transferred on PVDF membrane and then incubated with recombinant Glut1. Lower image: the recombinant Glut1 was transferred on PVDF membrane and then incubated with recombinant PmCBP.

Figure 8. In vitro and in vivo neutralization.

(A) In vitro neutralization. Hemocytes infected with WSSV or WSSV plus WBD were collected to identify virus infection. hpi: hours post-infection. (B) In vivo neutralization.