The M/GP(5) glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner - PubMed (original) (raw)

The M/GP(5) glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner

Wander Van Breedam et al. PLoS Pathog. 2010.

Abstract

The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP(3), GP(4) and GP(5) envelope glycoproteins, only the M/GP(5) glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP(5). These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Structural characterization of pSn-Fc proteins.

(A) Samples of purified pSn4D-Fc and pSn4DRE-Fc proteins were subjected to non-reducing and reducing SDS-PAGE, after which Coomassie Blue staining was performed. (B) (Non-)reducing SDS-PAGE and Western Blot analysis of purified pSn4D-Fc and pSn4DRE-Fc proteins using Fc-specific antibodies and pSn-specific mAb 41D3. (C) pSn4D-Fc and pSn4DRE-Fc proteins were treated with specific glycosidases or control treated and subjected to reducing SDS-PAGE and Western Blot analysis using Fc-specific antibodies.

Figure 2

Figure 2. Evaluation of the functionality of pSn4D-Fc as a sialic acid-binding (A & B) and PRRSV-binding (C & D) protein.

Qualitative (A) and quantitative (B) analysis of sialic acid-binding activity using a solid phase red blood cell binding assay. ELISA plates were coated with dilution series of pSn4D-Fc, pSn4DRE-Fc or mSiglecE-Fc protein, after which human RBCs were added and RBC binding was evaluated. pSn4D-Fc + RBCs (black square); pSn4D-Fc + sialidase-treated RBCs (white square); mSiglecE-Fc + RBCs (black diamond); mSiglecE-Fc + sialidase-treated RBCs (white diamond); pSn4DRE-Fc + RBCs (black circle). Values represent means±SEM of 3 experiments. (C) Evaluation of PRRSV-binding activity via immunoprecipitation experiments. Protein A beads were either coated with the pSn4D-Fc or pSn4DRE-Fc protein or not coated and incubated with MARC-145-grown PRRSV at 37°C to allow binding. The bound and unbound fractions were collected and subjected to non-reducing SDS-PAGE and Western Blot analysis using PRRSV N- and GP5-specific mAbs. (D) Evaluation of PRRSV-binding activity via infection-inhibition experiments. A 3-fold dilution series of pSn-Fc protein was mixed with a constant amount of MARC-145-grown PRRSV, incubated for 1 h at 37°C to allow binding and transferred to 105 alveolar macrophages. After 1 h of incubation at 37°C, pSn-Fc - virus mixtures were removed, fresh medium was added to the cells and cells were incubated for 9 h at 37°C, after which they were fixed. Infected cells were then visualized via nucleocapsid-specific immunoperoxidase staining and counted. pSn4D-Fc (white square); pSn4DRE-Fc (black square). Values represent means±SEM of 3 experiments.

Figure 3

Figure 3. Identification of pSn-binding (glyco)protein(complexe)s of MARC-145-grown PRRSV and characterization of the pSn-ligand interaction.

(A) Protein A beads were coated with the pSn4D-Fc or pSn4DRE-Fc proteins and incubated with a lysate of MARC-145-grown PRRSV at 37°C to allow binding. The bound and unbound fractions were collected and subjected to non-reducing SDS-PAGE and Western Blot analysis using virus-specific mAbs. (B) MARC-145-grown PRRSV was either treated with sialidase or control-treated and subjected to reducing SDS-PAGE and Western Blot analysis using virus-specific mAbs. (C) MARC-145-grown PRRSV was either treated with sialidase or control-treated, lysed and incubated with pSn4D-Fc-coated protein A beads at 37°C to allow binding. The bound and unbound fractions were collected and subjected to non-reducing SDS-PAGE and Western Blot analysis using a PRRSV GP5-specific mAb.

Figure 4

Figure 4. Identification of pSn-binding (glyco)protein(complexe)s of macrophage-grown PRRSV.

Protein A beads were coated with the pSn4D-Fc or pSn4DRE-Fc protein and incubated with a lysate of macrophage-grown PRRSV at 37°C to allow binding. The bound and unbound fractions were collected and subjected to non-reducing SDS-PAGE and Western Blot analysis using virus-specific mAbs.

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