J Vet Sci.  2018 Jul;19(4):528-535. 10.4142/jvs.2018.19.4.528.

Identification of determinants that mediate binding between Tembusu virus and the cellular receptor heat shock protein A9

• Zhao D
• Liu Q
• Huang X
• Wang H
• Han K
• Yang J
• Bi K
• Liu Y
• Zhang L
• Li Y

• KMID: 2417568
• DOI: http://doi.org/10.4142/jvs.2018.19.4.528

Abstract

Heat shock protein A9 (HSPA9), a member of the heat shock protein family, is a putative receptor for Tembusu virus (TMUV). By using Western blot and co-immunoprecipitation assays, E protein domains I and II were identified as the functional domains that facilitate HSPA9 binding. Twenty-five overlapping peptides covering domain I and domain II sequences were synthesized and analyzed by using an HSPA9 binding assay. Two peptides showed the capability of binding to HSPA9. Dot blot assay of truncated peptides indicated that amino acid residues 19 to 22 and 245 to 252 of E protein constitute the minimal motifs required for TMUV binding to HSPA9. Importantly, peptides harboring those two minimal motifs could effectively inhibit TMUV infection. Our results provide insight into TMUV-receptor interaction, thereby creating opportunities for elucidating the mechanism of TMUV entry.

Keyword

Tembusu virus; binding; envelope protein; heat shock protein A9

MeSH Terms

Blotting, Western
Heat-Shock Proteins*
Hot Temperature*
Humans
Immunoprecipitation
Peptides
Protein Structure, Tertiary
Heat-Shock Proteins
Peptides

Figure

• 

  Fig. 1 Identification of the HSPA9 binding domains in E protein. (A) Determination of HSPA9 binding domains in E protein by Western blot. The membrane containing transferred HSPA9 protein was incubated with purified recombinant protein of PBST (lane 1), DIII (lane 2), DII (lane 3), or DI (lane 4) and subsequently detected by adding anti-TMUV serum; lane M, molecular weight marker. (B) SDS-PAGE analysis of co-immunoprecipitation assay. The purified recombinant DI (lane 1; black arrow), DII (lane 2; black arrow), or DIII (lane 4) proteins were incubated with purified recombinant HSPA9 protein followed by incubation with anti-HSPA9 antibody. The co-immunoprecipitated complexes were then separated by SDS-PAGE. As controls, purified recombinant DI (lane 3), DII (lane 5), or DIII (lane 6) proteins were incubated without purified recombinant HSPA9 protein followed by incubation with anti-HSPA9 antibody. Lane M, molecular weight marker. PBST, phosphate buffered saline with Tween 20 (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 4.3 mM, KH2PO4 1.4 mM, pH to 7.4, with 0.05% Tween 20); TMUV, Tembusu virus; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DI, E protein domain I; DII, extended domain II; DIII, globular domain III.

• 

  Fig. 2 Recombinant HSPA9 interaction with overlapping peptides derived from E protein domain I (DI) and extended domain II (DII). Peptides were allowed to bind to HSPA9 (4 µg/mL) and tested for their HSPA9 binding capability by using indirect enzyme-linked immunosorbent assay. OD, optical density.

• 

  Fig. 3 Identification of the minimal HSPA9-binding motif by using truncated peptides. Truncated peptides were probed for reactivity with recombinant HSPA9 by Dot blot assay. Full-length E protein and DIII protein were used as positive and negative controls, respectively. (A) Identification of the HSPA9-binding motif by using truncated peptides. (B) Validation of minimal HSPA9-binding motif by Dot blot assay.

• 

  Fig. 4 Toxicity assay results. After 1 h pre-incubation with 100 µg/mL of peptides 2, 21, F, G, EWID, AHATKQSV, and bovine serum albumin (BSA) as a control, Cell Counting Kit-8 (CCK-8) assays to determine cell viability were performed. Medium without DF-1 cells was used as the blank control. Data presented are from three independent experiments. OD, optical density.

• 

  Fig. 5 Inhibition of Tembusu virus (TMUV) binding by peptides. DF-1 cells were pre-incubated with peptides 2, 21, F, G, EWID, AHATKQSV, and bovine serum albumin (BSA) as a control. TMUV was added and assayed for virus binding by using quantitative reverse transcriptase polymerase chain reaction. Data presented are from three independent experiments and statistical analysis was done with SPSS software (USA). Asterisks designate a statistically significant difference (p < 0.05) between a peptide pre-incubated group and the BSA pre-incubated group.

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