Structural and functional analysis of the hemagglutinin-esterase of infectious salmon anaemia virus

Abstract

Infectious salmon anaemia virus (ISAV) is a piscine orthomyxovirus causing a serious disease in farmed Atlantic salmon (Salmo salar L.). The virus surface glycoprotein hemagglutinin-esterase (HE) is responsible for both viral attachment and release. Similarity to bovine and porcine torovirus hemagglutinin-esterase (BToV HE, PToV HE), bovine coronavirus HE (BCoV HE) and influenza C hemagglutinin-esterase-fusion (InfC HEF) proteins were exploited in a computational homology-based structure analysis of ISAV HE. The analysis resolved structural aspects of the protein and identified important features of relevance to ISAV HE activity. By recombinant expression and purification of secretory HE (recHE) proteins, receptor-binding and quantitative analyses of enzymatic activities displayed by ISAV HE molecules are presented for the first time. Three different recHE molecules were constructed: one representing a high virulent isolate, one a low virulent, while in the third a Ser(32) to Ala(32) amino acid substitution was introduced in the enzymatic catalytic site as inferred from the model. The three amino acid differences between the high and low virulent variants, of which two localized to the putative receptor-binding domain and one in the esterase domain, had no impact on receptor-binding or -release activities. In contrast, the Ser(32) amino acid substitution totally abolished enzymatic activity while receptor binding increased, as observed by agglutination of Atlantic salmon red blood cells. This demonstrates the essential role of a serine in the enzyme's catalytic site. In conclusion, structural analysis of ISAV HE in combination with selected recHE proteins gave insights into structure-function relationships and opens up for further studies aiming at dissecting molecular determinants of ISAV virulence.

Fig. 1

Multiple alignment of hemagglutinin sequences, showing the FFAS (Rychlewski et al., 2000) alignment of ISAV HE (Q911Y4) against the structure-based alignment of porcine torovirus HE (PToV HE; PDB id code 3I1K), bovine torovirus HE (BToV HE; 3I26), bovine coronavirus HE (BCoV HE; 3CL5) and influenza C virus HEF (InfC HEF; 1FLC). The upper part shows the sequence alignment, the lower part shows the corresponding alignment of secondary structure elements determined from the experimental protein structures (β-strands: green arrows, α-helices: red coils). The qual line indicates alignment quality based on the degree of consensus between FFAS alignments against each of the structural templates, ranging from full consensus in black to no consensus in light grey. The cons line shows completely conserved positions in the alignment. The anno line shows relevant annotation of the alignment, including residues involved in active site (A), oxyanion hole (O), ligand binding (L), substrate coordination (S) and Cys–Cys pairing (1–8). The anno line also shows domain organisation (MP: membrane-proximal (red); E1, E2: esterase (green); R: receptor (blue)) taken from Langereis et al. (2009). The receptor domain is frequently annotated as a lectin domain in other publications, e.g. in Langereis et al. (2009). The numbering of the alignment is according to the full-length ISAV HE sequence. The alignment includes part of the signal peptide for ISAV HE (LLAPVYS from position 10 to 16), but no part of the signal peptide for any of the other sequences. This shows a clear sequence similarity between part of the ISAV HE signal peptide and the structural templates.

Fig. 2

(A) Visualization of ISAV HE based on the experimental structure of porcine torovirus HE (PToV HE; PDB id code 3I1L). The structure includes an analog of the natural ligand in the receptor-binding pocket. The right monomer of the dimer structure is shown in grey. The left monomer shows the sequence alignment overlap with ISAV HE, color coded according to domain (membrane-proximal: red; esterase: green; receptor: blue). Seven residues of the first membrane-proximal sequence overlap with the ISAV HE signal peptide (see Fig. 1 caption). Non-overlapping regions are shown in white. Also shown are the active site residues in the esterase domain (see B), ligand binding residues in the receptor domain (white spheres) and the three amino acid residues differing between ISAV4 HE and ISAV7 HE (yellow spheres; see C). In (B) the active site region is magnified. The side chains for the active site residues, Ser₃₂-His₂₆₄-Asp₂₆₁, are shown, as well as the oxyanion hole residues Gly₅₉ and Asn₈₉, and the substrate coordinating Arg (not conserved in ISAV HE). In (C) the ligand binding region of HE is magnified, showing the three amino acid differences between ISAV4 HE and ISAV7 HE (yellow spheres) as well as the residues involved in ligand binding in any of the known 3D structures (white spheres). The ligand of the 3I1L PToV HE structure is also shown.

Fig. 3

(A) Recombinant (rec) protein samples (4 μg each) were separated by SDS-PAGE and stained with Coomassie blue; lane 1: recHE4, lane 2: recHE7, lane 3: recHE4_S32A; M: MagicMark™ XP protein standard (5 μl). (B) Immunostaining of recHEs coupled to Dynabeads^®TALON™ using a mixture of two monoclonal anti-ISAV HE antibodies as a primary antibody and Alexa405^® goat anti-mouse IgG as a secondary antibody. The negative controls included the unrelated His₆-tagged protein drNimA, rec empty-1 and recHE4-coupled beads stained with secondary antibody (sec. ab. ctrl.) only.

Fig. 4

Agglutination of red blood cells (RBCs) with recHEs. RecHE4, recHE7 and recHE4_S32A coupled to Dynabeads^®TALON™ were pre-treated with DCIC in DMSO, or DMSO only, and mixed with RBCs. (A) The mixtures of Atlantic salmon RBCs (AsRBCs) and beads were observed under the microscope between 1–3 h and 5–7 h after adding AsRBCs. Negative controls included beads coupled with the His₆-tagged protein drNimA, rec empty-1 and beads only. (B) Aliquots from the AsRBC-bead mixtures in (A) were taken out between 1–3 h and 5–7 h after addition of AsRBCs, and free beads (not associated with AsRBCs) were counted using a Bürker chamber. These experiments were repeated three times, and data from one representative experiment is presented. (C) The mixtures of rabbit RBCs (rRBCs) and beads were observed under the microscope 1 h after adding of rRBCs. Beads coupled with the His₆-tagged protein drNimA served as a negative control. All photographs presented were taken at 40× magnification.

Fig. 4

Agglutination of red blood cells (RBCs) with recHEs. RecHE4, recHE7 and recHE4_S32A coupled to Dynabeads^®TALON™ were pre-treated with DCIC in DMSO, or DMSO only, and mixed with RBCs. (A) The mixtures of Atlantic salmon RBCs (AsRBCs) and beads were observed under the microscope between 1–3 h and 5–7 h after adding AsRBCs. Negative controls included beads coupled with the His₆-tagged protein drNimA, rec empty-1 and beads only. (B) Aliquots from the AsRBC-bead mixtures in (A) were taken out between 1–3 h and 5–7 h after addition of AsRBCs, and free beads (not associated with AsRBCs) were counted using a Bürker chamber. These experiments were repeated three times, and data from one representative experiment is presented. (C) The mixtures of rabbit RBCs (rRBCs) and beads were observed under the microscope 1 h after adding of rRBCs. Beads coupled with the His₆-tagged protein drNimA served as a negative control. All photographs presented were taken at 40× magnification.

Fig. 5

Acetylesterase activities of recHEs. (A) The acetylesterase activities of recHEs coupled to Dynabeads^®TALON™ were determined using increasing concentrations of the substrate 4MUAc ([S]). The data (each data point is the average of at least three parallels) are presented by Michaelis–Menten and Lineweaver–Burk plots. The experiment was repeated twice, but only one is shown. Beads coupled to the His₆-tagged protein drNimA served as negative control. RFU: relative fluorescence units; 1/v: reciprocal values of RFU min⁻¹. (B) The influence of DCIC on acetylesterase activities of the recHEs coupled to Dynabeads^®TALON™ using 4MUAc was studied. End-point data following 40 min incubation with substrate are shown. Beads coupled with the His₆-tagged protein drNimA served as negative control. RFU: relative fluorescence units. (C) The acetylesterase activities of recHEs in solution were determined using increasing concentrations of the substrate pNPA ([S]). Rec empty-1 and drNimA served as negative controls (not shown). The data (each data point is the average of at least three parallels) are presented by Michaelis–Menten and Lineweaver–Burk plots. The experiment was repeated twice, but only one is shown. 1/v: reciprocal values of OD min⁻¹ μg⁻¹ protein.