Characterization and interactome study of white spot syndrome virus envelope protein VP11

Abstract

White spot syndrome virus (WSSV) is a large enveloped virus. The WSSV viral particle consists of three structural layers that surround its core DNA: an outer envelope, a tegument and a nucleocapsid. Here we characterize the WSSV structural protein VP11 (WSSV394, GenBank accession number AF440570), and use an interactome approach to analyze the possible associations between this protein and an array of other WSSV and host proteins. Temporal transcription analysis showed that vp11 is an early gene. Western blot hybridization of the intact viral particles and fractionation of the viral components, and immunoelectron microscopy showed that VP11 is an envelope protein. Membrane topology software predicted VP11 to be a type of transmembrane protein with a highly hydrophobic transmembrane domain at its N-terminal. Based on an immunofluorescence assay performed on VP11-transfected Sf9 cells and a trypsin digestion analysis of the virion, we conclude that, contrary to topology software prediction, the C-terminal of this protein is in fact inside the virion. Yeast two-hybrid screening combined with co-immunoprecipitation assays found that VP11 directly interacted with at least 12 other WSSV structural proteins as well as itself. An oligomerization assay further showed that VP11 could form dimers. VP11 is also the first reported WSSV structural protein to interact with the major nucleocapsid protein VP664.

Figure 1. Time course analysis of WSSV vp11 transcripts by RT-PCR.

(A)–(E) Total RNAs were extracted from the gill of WSSV-infected shrimp and subjected to RT-PCR analysis with the indicated primers. Shrimp elongation factor 1-α was included as a template control. Lane headings show times post-infection (hours). M: 100 bp DNA ladder. The arrows indicate the size of the amplicon for each gene.

Figure 2. Determination of VP11’s location in the WSSV virion.

Western blot analysis of the full-length recombinant WSSV VP11 (VP11-V5) expressed in Sf9 cells using either (A) anti-VP11 antibody or (B) anti-V5 antibody. Lane 1: cell lysate of pDHsp/V5-His transfected Sf9 cells. Lane 2: cell lysate of pDHsp/VP11-V5-His transfected Sf9 cells. (C) Intact WSSV virions were subjected to detergent and NaCl treatment as indicated. After fractionation, the pellet (P) and supernatant (S) fractions were separated on SDS-PAGE and detected by Western blotting to produce profiles that are characteristic of envelope, tegument and nucleocapsid proteins (6). Three representative WSSV structural proteins are shown for comparison. Lane V is the untreated purified virus.

Figure 3. Immunoelectron microscopy of WSSV VP11.

Localization of VP11 in the WSSV virion by immunogold assay using a rabbit anti-VP11 antibody probe followed by a gold-labeled secondary antibody. IEMs of (A) the envelope integrity disrupted WSSV virion (B) purified intact virions and (C) the viral nucleocapsids. (D) IEM of the envelope integrity disrupted virions with pre-immune rabbit serum was used as the probe instead of the anti-VP11 antibody. The gold particle signals (arrows) were detected only on the envelope integrity disrupted virion. Scale bars equal 100 nm.

Figure 4. Membrane topology of WSSV VP11.

(A) V5-tagged recombinant VP11 (VP11-V5) was transiently expressed in Sf9 cells that were fixed with paraformaldehyde and either permeabilized with Triton X-100 (panels a–c) or not permeabilized (panels d–f). Due to the failure to detect the C-terminal V5 tag in the non-permeabilized cells, we conclude that VP11’s C-terminal must be located inside the cell membrane (cf the PmSTAT assay used by Liu et al. [53]). VP11-V5 was visualized with rabbit anti-V5 antibody and Cy3-conjugated donkey anti-rabbit IgG antibody (panels a and d). Nuclei were visualized by counterstaining with DAPI (panels b and e). Panels c and f show the merged Cy3 and DAPI signals. Scale bar equals 10 µm. (B) Western blot analysis showing trypsin digestion of VP11 and two other proteins for comparison: VP28 (an envelope protein with most of its C-terminal exposed outside the envelope) and VP26 (a tegument protein) in intact and detergent-treated virions. (C) Schematic of the proposed transmembrane topology of VP11. (This schematic was modified from the SOSUI program prediction by transposing the inside and outside of the virion.).

Figure 5. Yeast two-hybrid screening results for all of the confirmed WSSV VP11 interaction proteins.

(A) to (M) VP11’s interactions with VP24, VP26, VP28, VP37, VP38A, VP38B, VP51B, VP60B, VP75, VP95, VP160B, VP664-7 and itself, respectively. (a) Yeast growth on medium lacking both Leu and Trp (DDO) indicates the presence of each respective pair of plasmids. (b) and (c) Yeast growth on low stringency (DDO/X/A) and high stringency (QDO/X/A) medium, respectively. The blue signals in (b) and (c) are due to the presence of X-α-Gal. The positive signals represent protein-protein interactions. The numbers around the plates indicate the bait and prey plasmids of the transformed yeast: 1, pGBKT7-53/pGADT7-T; 2, pGBKT7-Lam/pGADT7-T; 3, pGBKT7-VP11/pGADT7-VPxx; 4, pGBKT7-VP11/pGADT7; 5, pGBKT7/pGADT7-VPxx; 6, pGBKT7/pGADT7, with VPxx respectively standing for each of the WSSV structural proteins listed above.

Figure 6. Co-immunoprecipitation confirmation of WSSV VP11 interaction proteins.

(A) to (M) co-immunoprecipitation of FLAG-tagged VP11 (VP11-FLAG) with V5-tagged interaction proteins VP24, VP26, VP28, VP37, VP38A, VP38B, VP51B, VP60B, VP75, VP95, VP160B, VP664-7, and VP11. Sf9 cells were co-transfected with FLAG-tagged VP11 plasmids, V5-tagged WSSV structural protein plasmids and empty plasmids (vector) as indicated. At 6 h after heat shock, the cell lysates were harvested. (a) After separation by SDS-PAGE, input expression was confirmed by Western blotting using either anti-V5 antibody or anti-FLAG antibody as a probe. Arrows indicate the expressed VP11-FLAG and respective V5-tagged WSSV structural protein. (b) The cell lysates were immunoprecipitated with anti-FLAG M2 affinity resins and then the immunoprecipitated complexes were subjected to Western blot analysis with an anti-V5 antibody probe.

Figure 7. An interactome diagram showing the relationships among the indicated WSSV structural proteins.

Although these interactions are not yet fully understood, VP11, VP24, VP26, and VP28 seem to act as the core of the virion protein complex. VP11, VP26, and VP28 interact directly with the nucleocapsid proteins VP664, VP60B, VP75, VP51C, and VP37, respectively, and may serve to anchor the entire envelope protein complex to the nucleocapsid. The interactions shown here were established either in the present study or else were based on previous results as indicated. The location of the unshaded protein (i.e. VP160B) on the virion has not yet been determined. EP: envelope proteins. TP: tegument proteins. NP: nucleocapsid proteins.

Figure 8. Homotypic interaction of VP11.

Immunoblot analysis of glutaraldehyde-treated VP11 proteins immobilized on the Ni-NTA beads. Lane 1: VP11 bound on Ni-NTA beads. Lanes 2–4: First, second, and third elutions from the column of VP11 protein after glutaraldehyde treatment. Monomers and dimers of VP11 proteins are indicated.