Sialoadhesin and CD163 join forces during entry of the porcine reproductive and respiratory syndrome virus

Hanne Van Gorp,Wander Van Breedam,Peter L Delputte,Hans J Nauwynck

doi:10.1099/vir.0.2008/005009-0

Abstract

The porcine reproductive and respiratory syndrome virus (PRRSV) shows a restricted tropism for subsets of porcine macrophages in vivo. To date, two PRRSV receptors have been identified on primary macrophages, heparan sulphate for binding and sialoadhesin for binding and internalization. However, additional factors are needed because the expression of both receptors in non-permissive cells results in virus internalization but not in virus uncoating and productive infection. Recently, CD163 was described as a PRRSV receptor on Marc-145 cells that renders non-permissive cells susceptible to PRRSV. Therefore, the potential role of CD163 in PRRSV entry in macrophages and its potential interplay with sialoadhesin were studied. Incubation of macrophages at 37 degrees C with either sialoadhesin- or CD163-specific antibodies reduced PRRSV infection by up to 75 %, while infection was completely blocked by a combination of both antibodies. When incubated at 4 degrees C, only sialoadhesin- and not CD163-specific antibodies reduced PRRSV infection. In addition, confocal analysis of PRRSV entry in non-permissive cells expressing only sialoadhesin showed PRRSV internalization but no uncoating. In contrast, when both sialoadhesin and CD163 were expressed, PRRSV was uncoated upon internalization, resulting in productive infection. Virus internalization was not observed when only CD163 was expressed; although, cells became productively infected. Thus, sialoadhesin is confirmed as a PRRSV internalization receptor and CD163 is shown to be involved in PRRSV entry, probably during uncoating. Co-expression of recombinant sialoadhesin and CD163 in non-permissive cells increased virus production 10-100 times compared with cells expressing only CD163, sustaining the requirement of both for efficient PRRSV infection.

Highlights

A ‘mystery swine disease’ appeared in the 1980s and has been present ever since in the pig industry, causing important economical losses worldwide (Neumann et al, 2005)
Primary alveolar macrophages were obtained from 4- to 6-week-old conventional Belgian Landrace pigs from a porcine reproductive and respiratory syndrome virus (PRRSV)-negative herd as described by Wensvoort et al (1991), and cultivated in RPMI 1640 supplemented with 10 % fetal bovine serum (FBS), 2 mM L-glutamine, 1 % non-essential amino acids, 1 mM sodium pyruvate and a mixture of antibiotics
Analysis of sialoadhesin and CD163 expression in primary macrophages, the in vivo target cells of PRRSV. Both sialoadhesin and CD163 expression are restricted to the monocyte–macrophage lineage (Duan et al, 1998b; Sanchez et al, 1999)

Summary

Introduction

A ‘mystery swine disease’ appeared in the 1980s and has been present ever since in the pig industry, causing important economical losses worldwide (Neumann et al, 2005). The African green monkey kidney cells MA-104 and cells derived thereof (Marc-145 and CL2621) are shown to sustain PRRSV infection, they are not from porcine origin, they do not belong to the monocyte– macrophage lineage and they do not express sialoadhesin (Duan et al, 1998b; Kim et al, 1993; Mengeling et al, 1995; Wissink et al, 2003). Despite this very restricted cell tropism of PRRSV, the virus is able to replicate in several non-

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