The Na+-K+-ATPase alpha subunit is an entry receptor for white spot syndrome virus

Abstract

White spot syndrome virus (WSSV) is a debilitating viral pathogen that poses a significant threat to the global crustacean farming industry. It has a wide host tropism because it uses several receptors to facilitate its attachment and entry. Thus far, not all the receptors have been identified. Here, we employed a BioID-based screening method to identify the Na⁺-K⁺-ATPase alpha subunit (PvATP1A) as a potential receptor in Penaeus vannamei. Although during the early stages of WSSV infection, PvATP1A was induced and underwent oligomerization, clustering, and internalization, knockdown of PvATP1A inhibited viral entry and replication. PvATP1A interacted with the WSSV envelope protein VP28 through its multiple extracellular regions, whereas synthetic PvATP1A extracellular region peptides blocked WSSV entry and replication. We showed that PvATP1A did not affect WSSV attachment but facilitated internalization via caveolin-mediated endocytosis and macropinocytosis. These findings provide a robust receptor screening approach that identified PvATP1A as an entry receptor for WSSV, presenting a novel target for the development of anti-WSSV therapeutics.

Importance: Cell surface receptors are crucial for mediating virus entry into host cells. Identification and characterization of virus receptors are fundamental yet challenging aspects of virology research. In this study, a BioID-based screening method was employed to identify the Na⁺-K⁺-ATPase alpha subunit (PvATP1A) as a potential receptor for white spot syndrome virus (WSSV) in the shrimp Penaeus vannamei. We demonstrated that PvATP1A interacted with the WSSV envelope protein VP28 via its multiple extracellular regions, thereby promoting viral internalization through caveolin-mediated endocytosis and macropinocytosis. Importantly, compared with previously identified WSSV receptors such as β-integrin, glucose transporter 1 (Glut1), and polymeric immunoglobulin receptor (pIgR), PvATP1A demonstrated significantly enhanced viral entry, indicating that PvATP1A is a crucial entry receptor of WSSV. This study not only presents a robust approach for screening virus receptors but also identifies PvATP1A as a promising target for the development of anti-WSSV therapeutics.

Keywords: Na+-K+-ATPase; WSSV; cell receptor; crustaceans; virus entry.

Fig 1

Identification of transmembrane proteins potentially interacting with WSSV VP28 protein by BioID screening. (A) Flow chart of the BioID screening approach. Recombinant proteins GST-BirA* and GST-BirA*-VP28 were incubated with primary cultured hemocytes for 2 h. Following washing to remove unbound proteins, the cells underwent in vitro biotinylation, followed by denaturing lysis, biotin pull-down, and LC-MS/MS analysis. (B and C) Expression and purification of the recombinant proteins GST-BirA* and GST-BirA*-VP28. These proteins were expressed in Escherichia coli BL21 cells (B) and purified using glutathione Sepharose 4B (C). M: protein marker. (D) Analysis of biotinylation activity of GST-BirA* and GST-BirA*-VP28 proteins. Hemocytes treated with GST-BirA* or GST-BirA*-VP28 were lysed and analyzed by a western blot using an HRP-streptavidin antibody. (E) Venn diagram showing the numbers of identified proteins in GST-BirA* and GST-BirA*-VP28 treatments by LC-MS/MS analysis. (F) Summary of transmembrane proteins that potentially interacted with VP28 protein. Proteins uniquely identified in the GST-BirA*-VP28 group were assessed using TMHMM-2.0 software, and those predicted to possess transmembrane helices were considered candidate virus receptors.

Fig 2

Upregulation of PvATP1A in WSSV-challenged shrimp. (A–D) Quantification of WSSV load and PvATP1A mRNA expression in hemocytes and intestines. Hemocytes and intestines were collected at 0, 4, 12, 24, 48, and 72 h post-WSSV or PBS injection for subsequent DNA and RNA extraction. Quantification of WSSV copy numbers (A and B) and PvATP1A mRNA expression (C and D) was performed using qPCR. PvATP1A mRNA expression of the PBS group at each time point was set to 1.0. (E–H) Detection of PvATP1A protein expression in hemocytes and intestines. The hemocytes and intestines collected at the indicated time points following WSSV (E and F) or PBS injection (G and H) were lysed for western blot analysis using an anti-ATP1A antibody. The gray values obtained from the western blot results were analyzed utilizing ImageJ software and subsequently normalized to the PBS group at 0 h. Statistical significance between groups was determined by two-tailed Student’s t-test. *P < 0.05, ***P < 0.01, and ***P < 0.001; ns: not significance.

Fig 3

Suppression of WSSV infection in hemocytes following PvATP1A knockdown. (A and B) Knockdown efficiency analysis of PvATP1A in hemocytes. Shrimps were individually injected with dsEGFP and dsPvATP1A, then infected with WSSV at 48 h post-dsRNA injection. Hemocytes were harvested at 24 and 48 h post-viral infection to assess PvATP1A mRNA and protein levels using qPCR (A) and western blot (B) analyses. (C–E) Quantification of the mRNA and protein expression of WSSV genes. The mRNA levels of the WSSV genes IE1 (C) and VP28 (D) following PvATP1A knockdown were determined by qPCR, whereas the VP28 protein level was measured by western blot using a VP28 antibody. (F) Quantification of WSSV copy numbers following PvATP1A knockdown by qPCR. (G) Shrimp survival rate after PvATP1A knockdown followed by WSSV or PBS injection. Statistical significance was assessed using a two-tailed Student’s t-test for comparisons between two groups (A, C, D, and F) and the log-rank test in GraphPad Prism for survival rate analysis. *P < 0.05, ***P < 0.01, and ***P < 0.001.

Fig 4

Oligomerization, clustering, and internalization of PvATP1A following WSSV infection. (A and B) Oligomerization analysis of PvATP1A. Hemocytes were collected at 0, 1, 2, and 4 h post-PBS (A) or WSSV (B) injection, treated with BS3, and analyzed by western blot analysis with an anti-ATP1A antibody. M: protein marker. (C and D) Subcellular localization analysis of PvATP1A. Hemocytes collected at 0, 1, 2, and 4 h after WSSV or PBS injection were subjected to immunofluorescence staining with an anti-ATP1A antibody (B). The percentage of PvATP1A subcellular localization in hemocytes was quantified in panel C with 50 hemocytes counted and analyzed per treatment. (E and F) Colocalization analysis of PvATP1A and WSSV particles. Hemocytes collected at 0, 2,nucleus and membrane. and 4 h post-infection were stained with anti-ATP1A and anti-VP28 antibodies. Representative immunofluorescence images are shown in panel E, and the colocalization rate is quantified in panel F. Data were from three independent experiments, with at least 200 hemocytes counted per replicate. ND: not detected.

Fig 5

PvATP1A facilitates WSSV entry. (A–D) In vitro assessment of WSSV entry following PvATP1A knockdown. Hemocytes collected from shrimp before and after PvATP1A knockdown and cultured in insect-XPRESS medium were either mock-infected or exposure to WSSV, and virus entry was assessed by western blot (A). The efficiency of PvATP1A knockdown was confirmed via qPCR (B) and western blot (C), and virus entry was evaluated by Western blot using an anti-VP28 antibody (D). (E–H) In vivo analysis of WSSV entry following PvATP1A knockdown. FITC-labeled or unlabeled WSSV particles were injected into shrimp before and after PvATP1A knockdown. Hemocytes collected at 4 h post-infection were used to assess knockdown efficiency (E) and determine viral entry by qPCR (F) and flow cytometry (G). Entry rate was quantified in panel H based on data from three independent flow cytometry experiments. (I and J) Immunofluorescence evaluation of WSSV entry in vivo. Unlabeled WSSV particles were injected into shrimp before and after PvATP1A knockdown, and hemocytes were harvested at 4 h post-infection for immunofluorescence staining with an anti-VP28 antibody. Representative immunofluorescence images were shown in panel I, with the WSSV entry index quantified in panel J. Data were obtained from three independent experiments, with a minimum of three hundred hemocytes counted per replicate. (K and L) WSSV entry in non-permissive cells after PvATP1A overexpression. Zebrafish PAC2 fibroblast cells transfected with PvATP1A or EGFP expression plasmids (K) were infected with WSSV for 1 h. Following the removal of the uninfected virions, WSSV entry was assessed by qPCR (L). Statistical significance was determined using a two-tailed Student’s t-test. *P < 0.05, ***P < 0.01, and ***P < 0.001; ns: not significance.

Fig 6

PvATP1A binds to WSSV VP28 protein via its multiple extracellular regions. (A–C) Homology modeling of PvATP1A. The 3D structure of PvATP1A was predicted using homology modeling (A) and validated by Ramachandran plot analysis (B), where dark green and yellow dots indicate residues in favored regions, respectively. The primary structure of PvATP1A determined from its 3D model is illustrated in panel C. (D and E) Crystal structure of WSSV VP28. The VP28 structure was obtained from the RCSB Protein Data Bank (D) and evaluated by Ramachandran plot analysis (E). (F) Molecular docking of PvATP1A and VP28. PvATP1A (cyan) and VP28 (yellow) are shown with their residues represented as sticks, whereas hydrogen bonds are indicated by red dashes and salt bridges by blue dashes. (G–I) Interaction analysis of PvATP1A and VP28 via biotin pull-down. Recombinant VP28 protein was expressed and purified from E. coli BL21 cells (G) and confirmed by western blot with an anti-VP28 antibody (H). The purified VP28 was incubated with five biotin-labeled synthetic peptides corresponding to the extracellular regions of PvATP1A, with a synthetic EGFP peptide (pEGFP) as a negative control (I). M: protein marker. (J) Binding affinity between PvATP1A extracellular region peptides and VP28 as determined by SPR analysis. ND indicates not detected.

Fig 7

Synthetic PvATP1A extracellular region peptides inhibit WSSV infection. (A–E) Impact of peptide blocking on WSSV entry. WSSV particles were pre-incubated with synthetic peptides from the extracellular regions of PvATP1A or a control peptide (pEGFP) for 2 h, followed by infection of primary cultured hemocytes for 1 h. Virus entry was assessed by qPCR (A), flow cytometry (B), and immunofluorescence (D). Data from three independent experiments are summarized in panels C and E for flow cytometry and immunofluorescence, respectively. (F–J) Effect of peptide blocking on WSSV replication. Hemocyte viability post-treatment with synthetic PvATP1A extracellular peptides was measured using the CCK-8 assay (F). WSSV particles, pre-treated with synthetic PvATP1A peptides (pATP1A-ER1-5) or varying concentrations of pATP1A-ER4, were used to infect hemocytes in vitro or injected into shrimp in vivo. After 8 h of in vitro infection or 48 h in vivo, hemocytes were collected to quantify WSSV copy numbers and VP28 levels by qPCR (G–I) and western blot (J). (K) Time-of-peptide-addition analysis with pATP1A-ER4. Hemocytes were treated concurrently with pATP1A-ER4 and WSSV, pre-treated with pATP1A-ER4 prior to WSSV infection, or infected with WSSV before pATP1A-ER4 treatment. After infection for 8 h, hemocytes were collected for DNA extraction, and WSSV copy numbers were quantified by qPCR. Statistical significance was determined using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant.

Fig 8

PvATP1A facilitates WSSV internalization via caveolin-mediated endocytosis and macropinocytosis. (A–C) Role of PvATP1A in WSSV attachment and internalization. WSSV particles were used to infect shrimp hemocytes and zebrafish PAC2 fibroblast cells before and after PvATP1A knockdown (A) or overexpression (C), respectively. Viral attachment and internalization were assessed by qPCR. Additionally, WSSV particles pre-treated with synthetic peptides (pATP1A-ER or pEGFP) were used to analyze viral attachment and internalization in hemocytes (B). (D–I) Impact of PvATP1A on endocytic marker uptake. Hemocytes from shrimp pre- and post-PvATP1A knockdown were incubated with endocytic markers (TFN-AF555, CTB-AF555, and DTN-AF568) in the presence of WSSV or PBS. After 1 h of infection, uptake rates were evaluated by flow cytometry. Representative images for TFN-AF555, CTB-AF555, and DTN-AF568 uptake are shown in panels D, E, and F with quantification provided in panels G, H, and I from three independent replicates. (J–K) Effect of endocytic inhibitors on WSSV entry in PvATP1A-overexpressing cells. Zebrafish PAC2 cells treated with inhibitors (AMR, CPZ, and GEN) were evaluated for cell viability using the CCK8 assay (J). Subsequently, cells overexpressing PvATP1A were pre-treated with these inhibitors or DMSO (negative control) before WSSV infection. After 1 h of infection, the cells were rinsed and WSSV copy numbers were quantified by qPCR (K). Statistical significance was determined using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant.

Fig 9

Schematic representation of PvATP1A mediating WSSV entry in shrimp. This study illustrates that during the early stages of WSSV infection; PvATP1A was upregulated and underwent oligomerization, clustering, and internalization. PvATP1A interacts with the WSSV envelope protein VP28 through multiple extracellular regions, facilitating viral internalization via caveolin-mediated endocytosis and macropinocytosis.