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Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
Correspondence
Peter L. Delputte
peter.delputte{at}ugent.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Keffaber, 1989; Wensvoort et al., 1991). The causative agent, PRRSV, belongs to the family Arteriviridae, order Nidovirales (Cavanagh, 1997). In vivo, the virus infects a subpopulation of macrophages characterized by expression of sialoadhesin (Duan et al., 1997b; Vanderheijden et al., 2003). In vitro, PRRSV can be propagated in porcine alveolar macrophages and cells derived from African green monkey kidney cells, such as MA-104, Marc-145 and CL2126 (Benfield et al., 1992; Duan et al., 1997a; Kim et al., 1993; Wensvoort et al., 1991, 1992). So far, two PRRSV receptors have been identified on primary macrophages: heparan sulphate and sialoadhesin (Delputte et al., 2002, 2005; Duan, 1998; Vanderheijden et al., 2003). Recently, vimentin has been suggested as a putative PRRSV receptor on Marc-145 cells (Kim et al., 2006), but the role of this molecule as a PRRSV receptor on the in vivo target cell, the macrophage, has not been established.
The PRRSV virion is composed of three major proteins, glycoprotein (GP) 5, matrix protein (M) and nucleocapsid protein (N), and three minor proteins, GP2a, E and GP4 (Dea et al., 2000; Meulenberg & Petersen-Den Besten, 1996; Meulenberg et al., 1995; Wu et al., 2005). The structural nature of GP3 remains controversial, as GP3 of the PRRSV isolate Lelystad virus (LV) is incorporated into the virus particle, whereas it has been found in the medium as a secreted protein for some North American strains (Gonin et al., 1998; Mardassi et al., 1998; van Nieuwstadt et al., 1996). As demonstrated previously for Equine arteritis virus (EAV) (de Vries et al., 1995), the prototype virus of the Arteriviridae, PRRSV GP5 and M occur as disulphide-linked heterodimers that are formed in the endoplasmic reticulum (ER) (Mardassi et al., 1996; Meulenberg et al., 1995; Verheije et al., 2002). It has been demonstrated for EAV and PRRSV that N, M and GP5 are essential and sufficient for particle formation, but that the minor proteins are required for virus infectivity (Wieringa et al., 2003, 2004; Wissink et al., 2005). As the EAV and PRRSV minor envelope proteins are interdependent for their incorporation into virions, it has been suggested that they form heteromultimeric complexes prior to or during virion assembly (Wieringa et al., 2003, 2004; Wissink et al., 2005).
PRRSV is able to persist in pigs for several weeks to several months after initial infection (Duan et al., 1997b; Labarque et al., 2000; Rowland et al., 2003; Wills et al., 1997). The mechanisms by which PRRSV persists are not well known. PRRSV-infected pigs develop a humoral immune response consisting of non-neutralizing antibodies starting from 7 days post-infection, with neutralizing antibodies appearing at 25–35 days post-infection (Delputte et al., 2004; Labarque et al., 2000; Loemba et al., 1996; Yoon et al., 1995). Several reports indicate that virus-neutralizing antibodies are correlated with virus elimination from infected pigs and protection against PRRSV replication and virus-induced disease (Labarque et al., 2000; Osorio et al., 2002). Virus-specific antibodies can help to resolve infection, not only by direct virus neutralization, but also by antibody-mediated cytotoxicity. For many viruses, newly synthesized viral glycoproteins are incorporated in the plasma membrane, rendering infected cells visible to the host humoral immune system. Virus-specific antibodies can then bind to the cell surface, which can result in antibody-dependent, complement-mediated cell lysis (ADCML) or cell lysis by activation of natural killer cells, neutrophils, monocytes or macrophages (antibody-dependent, cell-mediated cytotoxicity) (reviewed by Burton, 2002). The absence of viral proteins in the plasma membrane can make virus-infected cells invisible to the humoral immune system (Favoreel et al., 2003) and can be caused by intracellular retention or internalization from the cell surface. Retention of viral proteins within the cell has been described for coronaviruses (Lim & Liu, 2001; Locker et al., 1994; Machamer et al., 1993), adenoviruses (Nilsson et al., 1989; Pääbo et al., 1987), flaviviruses (Cocquerel et al., 1998; Drummer et al., 2003), togaviruses (Hobman et al., 1997) and bunyaviruses (Andersson et al., 1997). Clearance of viral glycoproteins from the plasma membrane by internalization has been reported for retroviruses and herpesviruses (Alconada et al., 1996; Bu et al., 2004; Favoreel et al., 1999, 2002; Ficinska et al., 2005; Fultz et al., 2001; Heineman & Hall, 2001; Marsh & Pelchen-Matthews, 2000; Olson & Grose, 1997; Van de Walle et al., 2003). In the case of Pseudorabies virus (PRV), this results in protection of virus-infected cells against ADCML (Van de Walle et al., 2003). In this study, we investigated whether PRRSV proteins were incorporated into the plasma membrane of primary macrophages infected in vitro and in vivo and whether infected cells could be lysed by the action of antibody and complement.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Marc-145 cells were maintained as described by Kim et al. (1993). LV, the prototype European PRRSV isolate (Wensvoort et al., 1991), was cultivated in porcine alveolar macrophages (13th passage) or in Marc-145 cells (4th passage). For some experiments, PRRSV was semipurified by ultracentrifugation at 100 000 g for 3 h through a 30 % sucrose cushion using an SW41Ti rotor (Beckman Coulter). The Kaplan strain of PRV was cultivated in swine testicle cells (second passage) (Kaplan & Vatter, 1959). To investigate the presence of viral proteins in the plasma membrane of in vivo PRRSV-infected macrophages, two 3-week-old PRRSV-negative pigs were inoculated with 106 TCID50 LV in 3 ml PBS (1.5 ml in each nostril). The pigs were euthanized at 5 and 6 days post-inoculation (p.i.) and alveolar macrophages were obtained by bronchoalveolar lavage.
Antibodies
Polyclonal antibodies.
Polyclonal antibodies (pAbs) A [immunoperoxidase monolayer assay (IPMA) antibody titre, 10240; serum neutralization assay (SN) antibody titre, 4] were derived from a pig that was subsequently inoculated with LV and the Belgian 98V145 PRRSV isolate with an interval of 6 weeks. pAbs B (IPMA antibody titre, 10240; SN antibody titre, 24) were obtained from a pig that was infected with LV (Delputte et al., 2004). PRRSV-negative pAbs were derived from a PRRSV-negative pig. The PRV-specific pAbs used in this study have been described by Favoreel et al. (1997). All pAbs were purified by using protein A–Sepharose (Amersham Biosciences) and, for some experiments, they were biotinylated by using a protein biotinylation module (Amersham Biosciences). As established by Western immunoblotting, pAbs A and B recognized LV GP3, GP4, GP5, M, N and possibly GP2a and E. It was not possible to confirm whether the pAbs recognized GP2a and E, as monoclonal antibodies (mAbs) against GP2a and E were not available.
PRRSV-negative serum (used in the ADCML assay) was derived from a PRRSV-negative pig. PRRSV-specific serum (used in the ADCML assay) was derived from an LV-infected pig at 7, 10, 14, 21 and 141 days p.i. The sera collected at 7, 10, 14, 21 and 141 days p.i. had IPMA titres of 10240; only the serum collected at 141 days p.i. showed neutralizing activity (SN antibody titre, 24). Sera were heat-inactivated before use.
mAbs.
The mAbs used were: 126.02 (Meulenberg & Petersen-Den Besten, 1996), 122.29 (Meulenberg et al., 1997), 4BE12 (Rodriguez et al., 2001), 126.3 (Meulenberg et al., 1995) and P3/27 (Wieczorek-Krohmer et al., 1996), which recognized LV GP3, GP4, GP5, M and N, respectively.
Effect of m.o.i. and macrophage cultivation on susceptibility to PRRSV infection.
Before the presence of viral proteins in the plasma membrane of PRRSV-infected cells was analysed, we first optimized the conditions to obtain a high number of infected cells during the first infection cycle. Macrophages were cultivated for 24 or 66 h before inoculation at an m.o.i. of 1 or 10. The m.o.i. was calculated as the ratio between the TCID50 value and the number of cells. TCID50 values were obtained by performing titrations on macrophages that had been cultivated for 24 h. The batch of macrophages used for the titration was not the same as the batch used in the experiments. At 12 h p.i., macrophages were fixed with 3 % paraformaldehyde (Vel Chemicals), permeabilized with 0.1 % saponin (Sigma) and stained with biotinylated PRRSV-specific pAb A and fluorescein isothiocyanate (FITC)-labelled streptavidin (Molecular Probes). PRRSV-infected cells were counted by using fluorescence microscopy.
Confocal-microscopic analysis of plasma membrane incorporation of viral proteins in PRRSV-infected macrophages and Marc-145 cells.
Before investigating the presence of viral proteins on the cell surface, the presence of intracellular GP3, GP4, GP5, M and N was examined in PRRSV-infected cells. Macrophages or Marc-145 cells were inoculated with PRRSV (m.o.i.=10), fixed with 3 % paraformaldehyde at 1, 3, 6, 9 and 12 h p.i., permeabilized with 0.1 % saponin and stained with mAb 126.02, 122.29, 4BE12, 126.3 or P3/27 and FITC-labelled goat anti-mouse IgG (Molecular Probes). To determine whether viral proteins were incorporated into the plasma membrane of PRRSV-infected cells, macrophages or Marc-145 cells were inoculated with PRRSV (m.o.i.=10) and at 6, 9 and 12 h p.i., the cells were washed and kept on ice during the staining. Surface staining was performed either with mAb against GP3, GP4, GP5 or M and FITC-labelled goat anti-mouse IgG or with biotinylated PRRSV-specific pAb (A or B) and FITC-labelled streptavidin. Dead cells were excluded by staining with 0.05 mg ethidium monoazide bromide ml–1 (EMA; Molecular Probes) before fixation with 3 % paraformaldehyde. PRRSV-infected cells were identified after permeabilization with 0.1 % saponin by staining either with biotinylated PRRSV-specific pAb and Alexa Fluor 350-labelled streptavidin (Molecular Probes) or with N protein-specific mAb P3/27 and Alexa Fluor 350-labelled goat anti-mouse IgG (Molecular Probes). Finally, cells were mounted in glycerin/1,4-diazabicyclo[2.2.2]octane (DABCO) and analysed with a Bio-Rad MRC 1024 confocal laser-scanning system connected to a Nikon Eclipse TE300 inverted microscope.
To investigate whether viral proteins were present in the plasma membrane of in vivo-infected macrophages by confocal microscopy, macrophages were obtained by bronchoalveolar lavage from a PRRSV-infected pig at 5 days p.i. and fixed and stained as described for macrophages infected in vitro.
Flow-cytometric analysis of plasma membrane incorporation of viral proteins in PRRSV-infected macrophages.
Macrophages were inoculated with PRRSV (m.o.i.=10) after 66 h cultivation. At 1, 3, 6, 9 and 12 h p.i., the cells were cooled on ice and incubated for 1 h at 4 °C with biotinylated PRRSV-specific pAb A, followed by FITC-labelled streptavidin. Background FITC fluorescence was determined by surface staining of PRRSV-infected macrophages with PRRSV-negative biotinylated pAb and FITC-labelled streptavidin. Before flow-cytometric analysis, macrophages were incubated for 5 min with 0.02 mg propidium iodide ml–1 (Molecular Probes) to label and exclude dead cells from the analysis. Analysis was performed with a Becton Dickinson FACSCalibur equipped with an argon ion laser. Cells were counted at a speed of 100–200 cells s–1 and a minimum of 10 000 cells were analysed for each sample. FITC fluorescence was detected by using the FL1 channel and propidium iodide fluorescence was detected by using the FL2 channel.
ADCML assay.
Macrophages were inoculated with PRRSV (m.o.i.=10) after 66 h cultivation in suspension. At 9 h p.i., the cells were incubated for 1 h at 4 °C with various concentrations of pAb (purified PRRSV-negative pAb, purified PRRSV-specific pAb A, purified PRRSV-specific pAb B or 25 % inactivated serum derived from a PRRSV-infected pig at 0, 7, 10, 14, 21 and 141 days p.i.). Macrophages were washed twice and incubated for 1 h at 37 °C with various concentrations of porcine complement (non-inactivated serum of a PRRSV-negative pig). After incubation with complement, macrophages were stained with EMA to label dead cells, fixed with 3 % paraformaldehyde, permeabilized with 0.1 % saponin and stained with N-specific mAb P3/27 and FITC-labelled goat anti-mouse IgG to identify PRRSV-infected macrophages. Dead, infected cells were counted by using fluorescence microscopy. To confirm the activity of the porcine complement, an ADCML assay was performed on PRV-infected macrophages, as described previously (Van de Walle et al., 2003). Therefore, alveolar macrophages were inoculated with PRV Kaplan (m.o.i.=10). At 12 h p.i., the cells were incubated with 50 µg genistein ml–1 (Sigma) for 45 min at 37 °C. Genistein inhibits tyrosine kinase activity and, as a consequence, also inhibits antibody-induced internalization of viral proteins from the cell surface (Van de Walle et al., 2003). Cells were incubated with 1 mg purified PRV-specific pAb ml–1, washed twice and incubated with 5 % porcine complement. Thereafter, cells were stained with EMA to label dead cells, fixed with 3 % paraformaldehyde, permeabilized with 0.1 % saponin and stained with FITC-labelled PRV-specific pAb.
To investigate the effect of antibody and complement on in vivo-infected macrophages, the ADCML assay was performed on fresh macrophages obtained by bronchoalveolar lavage of PRRSV-infected pigs at 5 and 6 days p.i.
Statistical analyses of data were based on the non-parametric Kruskal–Wallis test (rejection level 0.05), using SPSS version 12.0.
Internalization of viral proteins from the surface of PRRSV-infected macrophages.
After 66 h cultivation, macrophages were inoculated with PRRSV at a m.o.i. of 10. The viral inoculum was removed by washing at 1 h p.i. At 9 h p.i., macrophages were incubated for 1 h at 37 °C in the presence of 100 µg biotinylated PRRSV-specific pAb A or B ml–1, fixed with 3 % paraformaldehyde and permeabilized with 0.1 % saponin. Antibodies were visualized by using FITC-labelled streptavidin. PRRSV-infected cells were identified with mAb P3/27 and Texas red-labelled goat anti-mouse IgG (Molecular Probes). To assess the specificity of this internalization assay, mock-inoculated macrophages were used or PRRSV-inoculated macrophages were incubated with biotinylated pAb derived from a PRRSV-negative pig. To confirm that internalized antibodies could be visualized by using the technique described, PRV Kaplan-inoculated macrophages (9 h p.i.) were incubated with 100 µg biotinylated PRV-specific pAb ml–1 and visualized by using FITC-labelled streptavidin.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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showed that the susceptibility of macrophages to PRRSV infection increases by cultivating the cells for 24 h. Therefore, macrophages were cultivated for 24 h, inoculated with PRRSV at increasing m.o.i.s and fixed at 12 h p.i. The percentages of infected macrophages were 11.9, 13.3, 14.1, 26.4, 44.6, 46.2 and 45.9 % at m.o.i.s of 1, 3, 6, 12.5, 25, 50 and 100, respectively. The maximum level of infection was reached at m.o.i.s of 25, 50 and 100, suggesting that the susceptibility of macrophages was restricted at 24 h p.i. We also investigated whether cultivation of macrophages for 66 h instead of 24 h before infection increased the number of infected cells (Fig. 1). Using an m.o.i. of 1, a 24 h cultivation time resulted in 0.9±0.3, 2.6±1.0, 11.8±1.4 and 40.9±9.6 % infected cells at 6, 9, 12 and 24 h p.i., whereas 66 h of cultivation resulted in 3.6±1.2, 8.6±0.6, 30.8±4.1 and 95.1±1.1 % infected cells, indicating that ageing makes macrophages more susceptible to PRRSV. Using an m.o.i. of 10, after 66 h macrophage cultivation, >90 % infection was obtained at 12 h p.i. These conditions were used throughout the study, except where indicated otherwise.
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shows representative images of surface-stained PRRSV-infected macrophages at 9 h p.i. In dead, permeable cells, identified by positive EMA staining, surface staining with PRRSV-specific pAb resulted in cytoplasmic fluorescence.
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).
Viral proteins cannot be detected on the surface of PRRSV-infected macrophages by using flow cytometry
PRRSV-infected macrophages were surface-stained with PRRSV-specific pAb at 1, 3, 6, 9 and 12 h p.i. and analysed by flow cytometry. Fig. 3(a) shows the median fluorescence intensities (MFI) of surface-stained, infected macrophages at the different time points. Compared with the MFI values of surface staining with PRRSV-negative pAb, a small, non-significant increase in fluorescence intensity was observed for surface staining with PRRSV-specific pAb. This shift could be observed before 6 h p.i., when newly synthesized viral proteins were not yet detected. This was probably due to virus or viral proteins that were bound but not internalized by the macrophages, as surface staining of mock-infected macrophages with PRRSV-specific pAb did not result in such a shift. Fig. 3(b) shows histograms of the results at 9 h p.i. Histograms produced from the results at 1, 3, 6 and 12 h p.i. were comparable. Permeabilization of infected macrophages before staining with PRRSV-specific pAb resulted in a strong fluorescence intensity shift for the whole population, indicating that most cells were infected.
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). The background mortality of PRV-infected cells was 11.7±4.7 %, whereas incubation of PRV-infected cells with 1 mg PRV-specific pAb ml–1 and 5 % complement resulted in 83.1±4.5 % cell death. This corresponded to the results of Van de Walle et al. (2003).
To investigate whether macrophages infected in vivo were sensitive towards elimination by antibody and complement, an ADCML assay was performed on macrophages derived by bronchoalveolar lavage of PRRSV-infected pigs at 5 and 6 days p.i. (Table 3). Incubation of the cells with 1 mg purified PRRSV-specific pAb A or B ml–1 together with 5 or 10 % complement did not result in a significant increase in cell lysis.
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; Ficinska et al., 2005; Fultz et al., 2001; Marsh & Pelchen-Matthews, 2000; Olson & Grose, 1997). As neither confocal microscopy nor flow cytometry revealed viral proteins on the surface of PRRSV-infected macrophages, we investigated whether there was spontaneous or antibody-induced internalization of PRRSV proteins.
PRRSV-infected macrophages were incubated with PRRSV-specific pAb (A or B) that were visualized after fixation and permeabilization (Fig. 4). If internalization of viral proteins occurred, PRRSV-specific antibody would be co-internalized and intracellular fluorescent vesicles would be visualized by using FITC-labelled streptavidin. Compared with PRRSV-infected macrophages incubated with pAb derived from a PRRSV-negative pig or with mock-infected macrophages incubated with PRRSV-specific pAb, no difference in fluorescence pattern or intensity was seen. As a positive control for this internalization assay, PRV Kaplan-infected macrophages were incubated with PRV-specific pAb, as spontaneous as well as antibody-mediated endocytosis of viral proteins from the plasma membrane of infected monocytes has been described for this virus (Favoreel et al., 1999, 2002; Ficinska et al., 2005; Van de Walle et al., 2003). In this study, the PRV-infected macrophages also internalized their viral proteins from the plasma membrane when PRV-specific antibodies were added.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Labarque et al., 2000; Murtaugh et al., 2002; Rowland et al., 2003; Wills et al., 1997). The reason for this ineffective virus elimination is not fully understood. A remarkable delay in the onset of some arms of the cellular and humoral immune response may explain the ineffective clearance of PRRSV. PRRSV-specific cell-mediated immunity can only be detected from 4 weeks post-infection, with a peak at 7 weeks post-infection (Bautista & Molitor, 1997; López Fuertes et al., 1999; Molitor et al., 1997). PRRSV-specific non-neutralizing antibodies are induced quickly starting from 7 days post-infection, but virus-neutralizing antibodies are only detected starting from 25 days post-infection, and even then only at low titres (Labarque et al., 2000; Loemba et al., 1996; Lopez & Osorio, 2004; Yoon et al., 1995). In general, antibodies can act against free virus or against virus-infected cells, mediating several antiviral activities. Binding of antibody to free virus can result in direct virus neutralization, complement-mediated virolysis and/or phagocytosis, leading to a reduction in viral load (reviewed by Burton, 2002). For PRRSV, the role of virus-neutralizing antibody in protection against PRRSV replication has been studied extensively and a clear correlation has been shown between the appearance of PRRSV-specific neutralizing antibody and a strong reduction in PRRSV or elimination of PRRSV from the body (Delputte et al., 2004; Labarque et al., 2000; Lopez & Osorio, 2004; Osorio et al., 2002). In contrast to what is known about virus-neutralizing antibodies, the effect of antibodies on PRRSV-infected cells has not been studied previously. In this study, the role of PRRSV-specific antibody in lysing PRRSV-infected cells was investigated.
Enveloped mammalian viruses use the biosynthetic transport pathway of the host cell to synthesize and process their envelope proteins. For many viruses, newly synthesized viral proteins are transported to and incorporated into the plasma membrane, rendering infected cells visible to the antibody-dependent immune response (reviewed by Burton, 2002; Favoreel et al., 2003). Until now, whether viral proteins are incorporated into the plasma membrane of PRRSV-infected macrophages has not been investigated. In this study, surface stainings using PRRSV-specific antibodies revealed no viral proteins on the cell surface of macrophages inoculated in vitro and in vivo with PRRSV. This observation was not cell type-dependent, as viral proteins were not detected on the surface of PRRSV-infected Marc-145 cells. The absence of viral proteins from the cell surface is known to make infected cells invisible to virus-specific antibodies, protecting them against antibody-dependent lysis via complement, phagocytosis and destruction by cell-mediated cytotoxicity (Favoreel et al., 1999; Van de Walle et al., 2003; van der Meulen et al., 2003). To investigate whether PRRSV-infected cells were protected against ADCML, an ADCML assay was performed on macrophages infected with PRRSV in vitro. PRRSV-infected macrophages were incubated with purified porcine PRRSV-specific antibody and porcine complement, but only a small, non-significant increase in cell lysis was observed. As the pAbs were purified from sera of PRRSV-inoculated pigs at 141 or 165 days p.i., a total reliance on polyclonal porcine IgG as the specific detection reagent could be suggested. Therefore, ADCML assays were also performed with 25 % inactivated serum (derived from an LV-infected pig at 0, 7, 10, 14, 21 and 141 days p.i.) as the source of pAb, but ADCML was not observed, indicating that macrophages infected with PRRSV in vitro are protected against ADCML. An ADCML assay was performed on macrophages infected with PRRSV in vivo and no significant increase in the number of dead cells was observed, indicating that macrophages infected with PRRSV in vivo are also protected against ADCML. The protection of PRRSV-infected macrophages against ADCML is consistent with the fact that viral proteins were not observed in the plasma membrane of macrophages infected with PRRSV in vitro and in vivo, and points towards a limited role for PRRSV-specific antibodies in eliminating PRRSV-infected cells.
When viral proteins are absent from the surface of infected cells, viral proteins can either (i) be cleared from the plasma membrane, for example by internalization (spontaneous or antibody-mediated), or (ii) be retained inside infected cells. The envelope glycoproteins of Human immunodeficiency virus and Simian immunodeficiency virus are spontaneously internalized and cleared from the plasma membrane, thereby protecting infected cells from the antibody-mediated immune response (Bu et al., 2004; Fultz et al., 2001; Marsh & Pelchen-Matthews, 2000). Spontaneous and antibody-induced endocytosis of viral glycoproteins from the plasma membrane has also been described for alphaherpesviruses, such as PRV, Human herpesvirus 3 (varicella-zoster virus) and Human herpesvirus 1 (herpes simplex virus) (Alconada et al., 1996; Favoreel et al., 1999, 2002; Ficinska et al., 2005; Heineman & Hall, 2001; Olson & Grose, 1997; Van de Walle et al., 2003), and in the case of PRV, this was shown to protect infected monocytes against ADCML (Favoreel et al., 1999, 2002; Ficinska et al., 2005; Van de Walle et al., 2003). However, when PRRSV-infected macrophages were incubated with PRRSV-specific pAb, no internalization of viral proteins from the cell surface was observed, suggesting that the viral proteins are retained within the cells. This is consistent with electron microscopy studies showing that PRRSV particles, like the other arteriviruses, assemble within the host cell by budding of nucleocapsids into the lumen of the ER and/or Golgi compartments (Dea et al., 1995; Pol et al., 1997; Snijder & Meulenberg, 1998; Weiland et al., 1995). For viruses that bud through intracellular membranes, it has been shown that viral proteins are retained in and accumulate at the budding site, which might be the ER and/or Golgi complex in the case of arteriviruses. The suggestion of retention of PRRSV proteins in the ER for intracellular budding is also supported by several findings (Mardassi et al., 1996, 1998; Wissink et al., 2005). Wissink et al. (2005) demonstrated that PRRSV minor proteins are retained in the ER when expressed individually or with one of the other minor proteins and that transport competence through the Golgi complex was only acquired when the minor proteins were expressed together. Intracellular retention of proteins can be established by retention motifs, as described for Hepatitis C virus (Cocquerel et al., 1998; Drummer et al., 2003; Okamoto et al., 2004), Rubella virus (Hobman et al., 1997), Coronaviridae (Lim & Liu, 2001; Locker et al., 1994; Machamer et al., 1993; Vennema et al., 1992), Adenoviridae (Nilsson et al., 1989; Pääbo et al., 1987) and Bunyaviridae (Andersson et al., 1997). Sequences of LV GP2, GP3, GP4, GP5, M and N were screened for the presence of motifs known to be involved in ER or Golgi localization. One putative ER-retention motif was found in GP2: LVXXXL (aa 23–28). In contrast to the North American strain IAF-Klop, where no ER-targeting sequences were detected in GP3 (Mardassi et al., 1998), two putative ER-retention motifs were found in LV GP3: LVXXXL (aa 19–24) and HDEL (aa 87–90), the latter being a motif for ER lumen retention. In LV GP3, one putative Golgi-retention motif was also detected: CXXH (aa 144–147). So far, it is not known whether retention motifs are responsible for the localization of PRRSV proteins in the ER or Golgi, or whether retention is due to other mechanisms, such as interactions with chaperone proteins, as suggested by Mardassi et al. (1998).
In conclusion, this study indicated that viral proteins are not incorporated into the plasma membrane of PRRSV-infected macrophages, which masks the infected cells from PRRSV-specific antibodies and could explain their protection against antibody-dependent cell lysis, both in vitro and in vivo. PRRSV-specific antibodies clearly have the potential to clear free virus from the circulation, but their role in eliminating PRRSV-infected cells is limited, as shown in this study. Cell-mediated immunity may be a necessary component for the elimination of PRRSV-infected cells and its importance will be investigated in the near future.
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ACKNOWLEDGEMENTS |
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), a cultivation time of 66 h and an m.o.i. of 1 (
) and a cultivation time of 66 h and an m.o.i. of 10 (
). The data are means±SD of three experiments.
