Porcine arterivirus infection of alveolar macrophages is mediated by sialic acid on the virus

Abstract

Recently, we showed that porcine sialoadhesin (pSn) mediates internalization of the arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) in alveolar macrophages (Vanderheijden et al., J. Virol. 77:8207-8215, 2003). In rodents and humans, sialoadhesin, or Siglec-1, has been described as a macrophage-restricted molecule and to specifically bind sialic acid moieties. In the current study, we investigated whether pSn is a sialic acid binding protein and, whether so, whether this property is important for its function as a PRRSV receptor. Using untreated and neuraminidase-treated sheep erythrocytes, we showed that pSn binds sialic acid. Furthermore, pSn-specific monoclonal antibody 41D3, which blocks PRRSV infection, inhibited this interaction. PRRSV attachment to and infection of porcine alveolar macrophages (PAM) were both shown to be dependent on the presence of sialic acid on the virus: neuraminidase treatment of virus but not of PAM blocked infection and reduced attachment. Enzymatic removal of all N-linked glycans on the virus with N-glycosidase F reduced PRRSV infection, while exclusive removal of nonsialylated N-linked glycans of the high-mannose type with endoglycosidase H had no significant effect. Free sialyllactose and sialic acid containing (neo)glycoproteins reduced infection, while lactose and (neo)glycoproteins devoid of sialic acids had no significant effect. Studies with linkage-specific neuraminidases and lectins indicated that alpha2-3- and alpha2-6-linked sialic acids on the virion are important for PRRSV infection of PAM. From these results, we conclude that pSn is a sialic acid binding lectin and that interactions between sialic acid on the PRRS virion and pSn are essential for PRRSV infection of PAM.

FIG. 1.

Sheep erythrocyte (SE) binding to PAM. (A) Effect of different monoclonal antibodies on sheep erythrocyte attachment to PAM. PAM were incubated with sheep erythrocytes in the presence of pSn-specific monoclonal antibody 41D3, irrelevant isotype-matched monoclonal antibody 13D12, or monoclonal antibody 74-22-15, which reacts with SWC3 on PAM. (B) Effect of neuraminidase treatment of sheep erythrocytes (black bars) and PAM (gray bars) on sheep erythrocyte attachment to PAM. (C) A representative light microscopic image of the attachment of mock-treated (picture 1) and neuraminidase-treated (picture 2) sheep erythrocytes to PAM. Data represent means ± standard deviation (error bars) of triplicate assays. % cells binding > 4 SE = percentage of cells binding more than four sheep erythrocytes.

FIG. 2.

Effect of neuraminidase treatment of PRRSV LV (▪), 94V360 (), and VR-2332 (•) on infection of PAM. PRRSV was treated either with V. cholerae (A) or with A. urefaciens (B) neuraminidase, which remove sialic acids with α2-3,6,8 and α2-3,6,8,9 linkages, respectively. PAM were infected with the virus for 1 h at 37°C, washed to remove unbound virus and enzyme, and fixed 10 h postinoculation (C) PRRSV LV was incubated for 1 h at 37°C with beaded neuraminidase (▪) or with heat-inactivated beaded neuraminidase (○). The beaded enzyme was removed by low-speed centrifugation, PAM were inoculated with the virus, washed to remove unbound virus after 1 h, and fixed 10 h postinoculation Data represent means ± standard deviation (error bars) of triplicate assays.

FIG. 3.

Effect of N-linked glycan removal on PRRSV infection of PAM. PRRSV (▪) or PAM (□) were treated with N-glycosidase F (A) or endoglycosidase H (B) before infection of PAM. After treatment, PAM were inoculated for 1 h at 37°C with PRRSV, washed to remove unbound virus, and cells were fixed 10 h postinoculation Data represent means ± standard deviation (error bars) of triplicate assays.

FIG. 4.

Effect of sialic acid and sialylated (neo)glycoproteins on PRRSV infection. PAM were infected with PRRSV in the presence of (A) sialyllactose (black bars) or lactose (gray bars), (B) BSA neoglycoproteins containing sialyllactose (black bars) or lactose (gray bars), (C) the sialic acid-containing protein fetuin (black bars) or asialofetuin, which is devoid of sialic acid (gray bars). Cells were washed 1 h postinoculation to remove the inoculum and fixed 10 h postinoculation Data represent means ± standard deviation (error bars) of triplicate assays.

FIG. 5.

Effect of lectins on PRRSV infection of PAM. PRRSV was incubated for 1 h at 37°C with wheat germ agglutinin (□) and T. mobilensis lectin (□), which react with a broad range of sialic acids; MAA lectin (○), which recognizes α2-3-linked sialic acids; SNA lectin (□), which recognizes α2-6-linked sialic acids; and GNA lectin (▪), which does not react with sialic acids but does react with mannose residues. Virus-lectin mixtures were then inoculated on PAM. PAM were washed after 1 h of incubation at 37°C and fixed 10 h postinoculation Data represent means ± standard deviation (error bars) of triplicate assays.

FIG. 6.

Flow cytometric analysis of PRRSV attachment to PAM. Biotinylated PRRSV was incubated for 90 min at 37°C with different concentrations of V. cholerae neuraminidase. PAM were then incubated for 1 h at 4°C with enzyme-treated virus. After washing, bound virus particles were stained with fluorescein isothiocyanate-labeled streptavidin, and fluorescence intensity was analyzed by flow cytometry. Data represent means ± standard deviation (error bars) of triplicate assays.