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-dystroglycan is not critical for lymphocytic choriomeningitis virus receptor function in vivo
1 Institute for Microbiology, ETH Zurich, 8093 Zürich, Switzerland
2 Institute of Genetics, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK
Correspondence
Annette Oxenius
oxenius{at}micro.biol.ethz.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-Dystroglycan (-DG) is a ubiquitously expressed molecule that has been identified as a cellular receptor for lymphocytic choriomeningitis virus (LCMV) and other arenaviruses. Recently, it was demonstrated that LCMV receptor function is critically dependent on post-translational modifications, namely glycosylation. In particular, it was shown that O-mannosylation, a rare type of mammalian O-linked glycosylation, is important in determining the binding of LCMV to its cellular receptor. All studies carried out so far showed a dependence on glycosylation in LCMV receptor function in vitro. This work extended these studies to two in vivo models of -DG hypoglycosylation. The results confirm earlier findings on the in vitro dependence of carbohydrate modifications in LCMV receptor function. However, experiments in animal models showed that this dependence was only very weak in vivo. It is likely that alternative receptors or alternative entry pathways may account for this attenuated in vivo phenotype.
Present address: Ospedale San Giovanni, Laboratorio di Chimica Clinica, 6500 Bellinzona, Switzerland. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Riviere et al., 1985; Salvato & Shimomaye, 1989; Singh et al., 1987). The two single-stranded RNA segments encode four proteins. Whereas the short segment encodes the nucleoprotein (NP) and the glycoprotein (GP) precursor (Riviere et al., 1985; Salvato & Shimomaye, 1989), the long segment encodes the viral RNA-dependent RNA polymerase and a small zinc-binding protein (Z) (Salvato et al., 1989, 1992). The GP precursor is cleaved post-translationally into the peripheral GP1 and the transmembrane GP2 proteins by the protease SKI-1/S1P (Beyer et al., 2003; Buchmeier & Oldstone, 1979; Kunz et al., 2003). Upon GP-1-mediated receptor binding, arenaviruses are internalized in endosomal compartments via uncoated vesicles.
-Dystroglycan (-DG) has been described as a cellular receptor for LCMV and other arenaviruses, as well as for Mycobacterium leprae (Cao et al., 1998; Rambukkana et al., 1998; Spiropoulou et al., 2002). Initially expressed as a pro-peptide, DG is processed into the peripheral -DG subunit and the membrane-spanning β-DG subunit, which remain non-covalently associated (Ibraghimov-Beskrovnaya et al., 1992). -DG is responsible for the binding of extracellular matrix molecules such as laminin, whereas the β-DG subunit represents the transmembrane-spanning protein that connects to the actin-based cytoskeleton, for example via dystrophin (Ervasti & Campbell, 1991, 1993; Holt et al., 2000; Ibraghimov-Beskrovnaya et al., 1992).
-DG consists of N- and C-terminal globular domains that are connected via a highly glycosylated, mucin-like region rich in prolines, serines and threonines (Brancaccio et al., 1995; Wilson et al., 1991). The -DG molecule is subject to extensive post-translational modifications, especially glycosylation. Notably, more than 50 % of the apparent molecular mass of -DG originates from O-linked sugars, including a rare type of O-mannosyl-linked carbohydrate; these are mainly concentrated within the mucin-like region (Chiba et al., 1997). The O-mannosylation pathway is initiated in the endoplasmic reticulum, where a mannose is transferred to serine/threonine residues, a process that is catalysed by protein O-mannosyltransferase-1 and -2 (POMT-1 and POMT-2). The protein O-linked mannose β-1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) then catalyses the transfer of N-acetylglucosamine to O-linked mannose in a pathway that continues in the Golgi apparatus and involves a series of other glycosyltransferases that catalyse the transfer of galactose and sialic acid. This gives rise to a tetrasaccharide forming the core structure of O-mannosyl-linked sugars. Although not directly implicated in this O-mannosylation pathway, the putative glycosyltransferase Large seems to play a critical role in the synthesis of a functional DG molecule (Kanagawa et al., 2004). Large recognizes the N-terminal domain of the -DG molecule, initiating the functional glycosylation of DG within the first half of the mucin-like region (Kanagawa et al., 2004). The LCMV-binding site on -DG has been mapped between aa 313 and 408 of the mucin-like region, which is subject to Large-dependent glycosylation. It was further shown that Large-dependent -DG modifications are crucial in LCMV receptor function (Kunz et al., 2005). In addition to Large, the enzyme POMGnT1 has been shown to be essential for LCMV receptor function (Imperiali et al., 2005). Collectively, these studies clearly show that glycosylation of -DG is essential for LCMV receptor function in vitro. However, to date there is no evidence for the implication of -DG glycosylation in LCMV receptor function in vivo.
Here, we analysed the impact of carbohydrates on LCMV receptor function in vivo. We showed that glycosylation of -DG is essential for LCMV receptor function in vitro but only to a small degree in vivo. Using animal models including Largemyd/myd mice expressing a non-functional Large glycosyltransferase and POMGnT1-deficient mice (Liu et al., 2006), we demonstrated that cells from these dystrophic animals were less susceptible to LCMV infection in vitro. However, the in vivo dependence of LCMV infection on a functional Large protein or on POMGnT1 was less stringent, possibly due to compensatory mechanisms for O-mannosylation or the use of alternative receptors, or perhaps the presence of natural antibodies that may target LCMV to phagocytic cells, thereby promoting their infection independently of -DG.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Generation of radiation chimeras.
For the generation of chimeric mice, C57BL/6 Ly5.1 host mice were lethally irradiated with 950 rad and reconstituted with bone marrow cells from C57BL/6, POMGnT1–/– or POMGnT1+/– mice. Bone marrow cells from POMGnT1–/– mice and heterozygous controls were a kind gift from Dr Huaiyu Hu, Upstate Medical University, USA. Eight weeks after reconstitution, the chimeric mice were used for experiments. Blood flow cytometric analysis showed 2–9 % cells remaining of host origin.
Peritoneal macrophages and bone marrow-derived dendritic cells (BM-DCs).
Peritoneal macrophages were harvested 3 days after intraperitoneal injection of 1 ml 4 % thioglycolate by washing the peritoneal cavity with ice-cold PBS. BM-DCs were generated as described previously (Inaba et al., 1992).
Viruses and peptides.
The LCMV isolates Clone 13 and Docile were propagated on BHK-21 or MDCK cells at a low m.o.i. Mice were infected intravenously with 200 p.f.u. or 1x106 p.f.u. (low and high dose, respectively) LCMV Clone 13 or Docile for 4 or 8 days, as indicated. Quantification of infectious virus titre was performed as described previously (Battegay et al., 1991).
The peptides gp33 (LCMV GP aa 33–41, KAVYNFATM) and np396 (LCMV NP aa 396–404, FQPQNGQFI) were purchased from NeoMPS.
Antibodies.
The following antibodies and antisera were used in this study: anti--dystroglycan (clone VIA4-1; Upstate), fluorescein isothiocyanate (FITC)-conjugated anti-LCMV NP (VL-4) (Battegay et al., 1991) and phycoerythrin (PE)-conjugate donkey anti-mouse IgG (Milian Analytica). In addition, anti-CD107a, anti-IFN-
, anti-TNF-, anti-CD4, anti-CD8, anti-CD45.1, anti-B220, anti-CD49b (clone DX5), anti-CD11b, anti-CD11c and anti-I-A/I-E were purchased from Becton Dickinson. Monoclonal antibody 2.4G, specific for CD16/CD32, was produced as a hybridoma supernatant.
Leukocyte purification and stimulation.
Lymphocytes were harvested from spleen, stimulated in vitro with 1 µg gp33 or np396 peptide ml–1 and stained for degranulation and intracellular cytokine production as described previously (Wolint et al., 2004). For ex vivo analysis of LCMV-infected DCs, spleens were removed, cut into small pieces and digested in RPMI 1640 containing 10 % heat-inactivated fetal calf serum (FCS), 2.4 mg collagenase (Invitrogen) ml–1 and 0.2 mg DNase I (Roche Diagnostics) ml–1 for 30 min at 37 °C. The samples were then passed through an 18-gauge needle and further digested as above. The single-cell suspension obtained was passed again through an 18-gauge needle, filtered through a sterile 100 µm cell strainer (BD Biosciences), washed once with RPMI/10 % FCS and stained intracellularly for LCMV NP.
Astrocyte cultures.
Primary brain cells were obtained from newborn mice (day 2) as described previously (Waldburger et al., 2001). Briefly, brain tissue was passed twice through a 100 µm cell strainer and the resulting single-cell suspension was incubated in Dulbecco's modified Eagle's medium (DMEM) with 20 % FCS for 7 days. The medium was replaced with fresh DMEM/10 % FCS and the cells were further cultivated for 1–2 weeks before being used for -DG staining and infection experiments. Astrocytes and macrophages were identified as CD11bnegative and CD11bhigh cells, respectively.
Flow cytometric analysis of cell-surface and intracellular protein expression.
Cells were surface stained with directly labelled antibodies for 20 min on ice. For -DG cell-surface staining, cells were first stained with VIA4-1 antibody at 4 °C, washed and stained with PE-conjugated donkey anti-mouse secondary antibody at 4 °C. If cells were adherent (e.g. astrocytes and peritoneal macrophages), they were harvested using PBS containing 5 mM EDTA and incubation at 37 °C (astrocytes) or on ice (peritoneal macrophages). Cell-surface staining of cells isolated from peripheral blood was carried out as described above, followed by erythrocyte lysis with 500 µl FACSLyse solution (Becton Dickinson).
Intracellular staining of gamma interferon (IFN-) and tumour necrosis factor (TNF)- was performed as described previously (Wolint et al., 2004). For the intracellular detection of LCMV NP in CD11c+ and CD11b+ cells, FcRIII/II was blocked using antibody 2.4G before staining with the VL-4 antibody. Data were acquired on an LSRII flow cytometer (Becton Dickinson) and analysed using FlowJo software (Tree Star).
In vitro infection experiments.
Peritoneal macrophages (1x106) and brain cells (1.5x105) were infected (m.o.i.=0.01) with the indicated viral strains for 1 h at 37 °C. Cells were washed and incubated for 2 days at 37 °C in RPMI 1640/10 % FCS or DMEM/10 % FCS, respectively.
BM-DCs (2x106) were infected (m.o.i.=0.01) in a polypropylene tube in a volume of 150 µl with the indicated viral strain, washed once and incubated for 2 days at 37 °C in RPMI 1640/10 % FCS supplemented with 1 µg granulocyte–macrophage colony-stimulating factor ml–1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-DG is essential for LCMV receptor function in non-murine cells. In particular, we demonstrated that a genetic defect in POMGnT1, a glycosyltransferase responsible for attachment of N-acetylglucosamine to the core mannose, severely reduces the ability of LCMV to infect primary human fibroblasts (Imperiali et al., 2005). To analyse further the role of -DG glycosylation in LCMV infection of natural murine host cells, we assessed the role of the glycosyltransferase Large. Mutations in Large lead to hypoglycosylation of -DG in humans (Longman et al., 2003) and in mice (Grewal et al., 2001; Holzfeind et al., 2002; Levedakou et al., 2005; Michele et al., 2002). We generated BM-DCs from Largemyd/myd mice and Largewt/myd littermate control animals and assessed their ability to be infected by LCMV. DCs are preferentially infected by LCMV isolates that bind with high affinity to -DG (Sevilla et al., 2000). In parallel with BM-DCs, we also isolated peritoneal macrophages from the corresponding animals. Cell-surface expression of -DG on these cells was analysed by flow cytometry using an antibody that specifically recognizes a carbohydrate moiety of -DG (Ervasti & Campbell, 1991; Ibraghimov-Beskrovnaya et al., 1992). Reduced -DG cell-surface staining was observed in both cell types derived from Largemyd/myd mice in comparison with control animals (Fig. 1a) and was associated with reduced susceptibility to LCMV infection, in particular in the case of peritoneal macrophages (Fig. 1b). These results indicated that, in BM-DCs and peritoneal macrophages in particular, -DG glycosylation is impaired if the glycosyltransferase Large is non-functional, resulting in decreased permissiveness for LCMV infection.
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; Grewal et al., 2005). Large2 also represents a putative glycosyltransferase that may be able to glycosylate DG even more efficiently than Large itself (Fujimura et al., 2005; Grewal et al., 2005). Large and Large2 show little overlap in their expression pattern. Large transcripts are found primarily in neural and muscle tissues, whilst Large2 expression is high in epithelial tissues. However, in the CNS, Large2 expression is strongly reduced (Grewal et al., 2005). We therefore measured the impairment of DG glycosylation in cells from the CNS derived from Largemyd/myd mice. We analysed DG glycosylation in astrocytes (CD11bnegative) and macrophages (CD11bhigh) generated from neonate brain cell cultures. -DG cell-surface staining revealed that the sugar epitope recognized by the VIA4-1 antibody was virtually absent in astrocytes and was strongly reduced in macrophages derived from Largemyd/myd mice in comparison with cells from heterozygous controls (Fig. 2a), confirming the results we obtained with peritoneal macrophages (Fig. 1a
). Moreover, cells isolated from the CNS of Largemyd/myd mice were more resistant to LCMV infection, as we observed reduced infection susceptibility with both of the LCMV strains tested (Fig. 2b). These results further support the notion that impaired -DG glycosylation due to a defect in Large correlates with reduced infection susceptibility.
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). To evaluate further the impact of a defective glycosylation pathway in LCMV viral infection in vivo, we used mice deficient in POMGnT1, a glycosyltransferase involved in the synthesis of O-mannosyl-linked glycans. As POMGnT1–/– mice suffer from muscular problems, we generated chimeric mice by reconstitution of lethally irradiated hosts with bone marrow from POMGnT1–/– animals, which limits the glycosylation defect to the haematopoietic system. As controls, chimeric mice were generated using bone marrow cells from POMGnT1+/– or wild-type C57BL/6 mice. Eight weeks after the reconstitution, chimeric mice were infected with LCMV Clone 13. Analysis of viral titres in spleen, liver, lung and kidney 4 days after infection revealed a phenotype comparable to that of the Largemyd/myd mice, showing only minor reductions in viral titres (Fig. 4). Interestingly, the viral titre in kidneys appeared to be the most affected as no virus was detected in that organ in POMGnT1–/– chimeric mice; however, it should be noted that the kidney virus titres were also low in control animals. The relatively mild phenotype in POMGnT1–/– chimeras could be explained by the remaining 2–9 % of host-derived, wild-type haematopoietic cells in these chimeras, and the fact that all non-haematopoietic cells in these animals were wild type. Some of these cells are susceptible to LCMV infection and might therefore contribute to the overall organ viral titres. Taken together, our results from the Largemyd/myd and the POMGnT1-deficient chimeric animals suggested that (-DG) glycosylation defects have a mild effect on the LCMV burden in vivo.
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-DG glycosylation would result in altered T-cell responses following LCMV infection. We analysed the lymphocyte composition in Largewt/myd and Largemyd/myd mice prior to infection by assessing the frequencies of the most important lymphocyte populations such as CD4+ T cells, CD8+ T cells, B cells (B220+) and natural killer cells (DX5+). We observed a normal percentage of all analysed lymphocyte subsets in Largemyd/myd mice when compared with controls (Fig. 5). Next, we characterized the LCMV-specific immune response in these mice. To this end, Largemyd/myd and control mice were infected with LCMV Clone 13 at two different doses, which induce either an acute or a persistent infection (200 p.f.u. and 1x106 p.f.u., respectively). At day 8 post-infection, we measured degranulation and cytokine production of LCMV-specific CD8+ T cells. The majority of gp33- and np396-specific CD8+ T cells from both control and Largemyd/myd mice were able to degranulate and to produce IFN- and TNF- following a low-dose infection (Fig. 6a, b). Splenocytes from Largemyd/myd and control mice infected with a high dose of LCMV Clone 13 exhibited reduced frequencies of cells able to produce IFN- and TNF- (Fig. 6a, b), as described previously (Barber et al., 2006). These results indicated that the CD8+ T-cell response to LCMV is normal in mice with a non-functional Large protein.
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). However, as (i) APCs are crucial for the priming of CD8+ T-cell responses, and (ii) the CD8+ T-cell response to LCMV in Largemyd/myd mice was normal (Fig. 6a, b), this suggests that APCs are either readily infected in vivo or the LCMV antigens are efficiently cross-presented. To address the former possibility, we immunized Largemyd/myd and POMGnT1–/– chimeric mice intravenously with 1x106 p.f.u. LCMV Clone 13. At 4 days post-infection, APCs that were productively infected with LCMV in vivo were quantified by intracellular staining for LCMV NP. DCs and macrophages in infected Largemyd/myd mice and POMGnT1–/– chimeric animals showed a similar infection frequency to those from the respective control animals (Fig. 7a and b, respectively). Importantly, intracellular staining for LCMV NP allowed us to gate on specific cell subsets, in particular in POMGnT1 chimeras in which 2–9 % of the leukocytes were of wild-type host origin, so we could specifically gate on the POMGnT1-deficient donor-derived leukocytes. Using this stringent gating procedure, we were also unable to find any difference in the frequency of productively infected DCs or macrophages of POMGnT1-deficient and wild-type origin. These results demonstrated that, during a systemic LCMV infection in vivo, APCs are infected by the virus, even if cells originate from mice with genetic defects in the O-mannosylation pathway.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The -DG molecule has been described to be a receptor for LCMV, Lassa fever virus and clade C New World arenaviruses (Cao et al., 1998; Spiropoulou et al., 2002). The DG molecule is subject to extensive post-translational modifications such as glycosylation, which are tissue- and cell-specific. We and others have recently shown that -DG glycosylation is essential for LCMV receptor function in vitro (Imperiali et al., 2005; Kunz et al., 2005). A number of enzymes are involved in the process of post-translational protein glycosylation. Among the best-characterized glycosyltransferases are Large and POMGnT1; these play a critical role in catalysing the sugar modifications of -DG that are involved in recognition by LCMV (Imperiali et al., 2005; Kunz et al., 2005). To gain a better insight into in vitro and in vivo LCMV–DG interactions, we took advantage of two animal models in which the DG molecules are hypoglycosylated. We used Largemyd/myd mice, which carry a spontaneous mutation in the Large gene resulting in a non-functional protein. As a result, DG is hypoglycosylated (Grewal et al., 2001; Michele et al., 2002). The second model involved mice deficient in the glycosyltransferase POMGnT1, which also results in -DG hypoglycosylation (Liu et al., 2006).
The specific role of Large in -DG glycosylation is not yet fully understood. Sequence homology studies have proposed that Large is a putative glycosyltransferase with two distinct domains that are homologous to bacterial -glycosyltransferase and mammalian β-1,3-N-acetylglucosaminyltransferase (Grewal et al., 2001; Peyrard et al., 1999). Nevertheless, these classes of glycosyltransferase have so far not been shown to modify -DG. It is therefore possible that Large affects the DG glycosylation pathway by modulating the activity of other enzymes. In a recent report, it was proposed that Large might have a dual, concentration-dependent function. At physiological concentrations, Large might regulate DG glycosylation, whilst at high experimental concentrations it might mediate alternative pathways for -DG glycosylation (Barresi et al., 2004).
DCs and macrophages are the most important APCs and are prominent target cells for LCMV infection. We therefore generated BM-DCs and thioglycolate-induced peritoneal macrophages from Largemyd/myd mice. In comparison with controls, both cell types derived from mutant mice showed significantly reduced -DG staining with a carbohydrate-specific antibody (VIA4-1), confirming impaired -DG glycosylation. This reduction in DG glycosylation was paralleled by a decreased in vitro infection susceptibility of cells from homozygous Largemyd/myd mice when infected with LCMV strains that show high-affinity binding to DG, such as Docile (Imperiali et al., 2005) and Clone 13 (Smelt et al., 2001).
Recently, the Large homologue Large2 has been described. Similar to Large, Large2 is localized in the Golgi apparatus and is a putative glycosyltransferase (Fujimura et al., 2005; Grewal et al., 2005). In mice, at the mRNA level, the expression patterns of Large and Large2 differ among tissues. Whereas Large transcripts are preferentially found in neuronal and muscular tissues, Large2 transcripts are detected in epithelial tissues. However, Large2 transcripts are barely detected in the CNS (Grewal et al., 2005). Whether Large2 co-operates with Large or can compensate for Large defects is still unclear. In situ hybridization analyses indicate that Large and Large2 mRNAs are not co-expressed in the same cell types, indicating that the two proteins are unlikely to co-operate (unpublished data). When co-expressed in HEK 293 T cells together with -DG, Large2 is more efficient at glycosylating -DG than Large (Fujimura et al., 2005). As these two proteins may have partially redundant functions, it is not possible to exclude a compensatory role of Large2 when Large is defective. As Large2 expression is absent in the CNS, we analysed cells derived from the brains of Largemyd/myd mice so that we had a system where -DG glycosylation is likely to be strongly impaired, as possible compensatory mechanisms by Large2 are likely to be absent. Using an -DG carbohydrate-specific antibody, astrocytes from Largemyd/myd brains showed strongly decreased DG staining in comparison with control cells derived from Largewt/myd mice. Accordingly, astrocytes derived from Largemyd/myd mice exhibited severely reduced LCMV infection susceptibility in comparison with controls. This further supports the notion that -DG glycosylation is important in LCMV receptor function, corroborating our results from experiments with human fibroblasts derived from muscle–eye–brain (MEB) disease patients (Imperiali et al., 2005). However, in the previous study, the genetic backgrounds of MEB disease patients and healthy donors were not matched and thus could not be accounted for. By using inbred mice homozygous or heterozygous for the myd Large allele, we circumvented this problem in the present study.
In contrast with the in vitro experiments in which we observed reduced infection of cells from Largemyd/myd mice, in vivo infection with LCMV Clone 13 did not reveal marked differences in viral titres in spleen, kidney, liver and lung when Largemyd/myd or Largewt/myd mice were compared. Of note, we observed a very small difference (overall twofold reduction) in viral titres in the liver and lung from Largemyd/myd mice, which might be explained by the Large2 expression pattern. In spleen and kidney, Large2 mRNA is present at high levels compared with in liver and lung where it is barely detectable (Grewal et al., 2005). Large2 expression in cells of spleen, kidney, and potentially other organs might efficiently compensate for a defect in Large. However, in organs where Large2 expression is low, such as the liver and lung, fewer such compensatory effects may occur, hence leading to the reduced LCMV titres in these organs. Thus, it is conceivable that, in vivo, Large2 expression is more important overall than Large for LCMV receptor function. In this context, it is interesting to note that a recent genome-wide analysis of positive selection in human populations showed strong positive selection for a LARGE gene allele within the West African population of Nigeria, a region in which Lassa fever virus infection is endemic (Sabeti et al., 2007).
Irradiated mice reconstituted with bone marrow cells derived from POMGnT1–/– mice showed a more pronounced phenotype than Largemyd/myd mice when immunized with LCMV. In this case, a reduction in viral titre was observed in all tested organs, with the exception of the spleen. In contrast to Large mutant mice, where the O-mannosyl core sugar chain is present on the -DG molecule, cells derived from POMGnT1–/– mice are likely to lack any complete core structure. The O-mannosylation pathway is interrupted after the transfer of the first mannose. As -DG is severely hypoglycosylated in POMGnT1–/– cells, this may explain the slightly stronger phenotype in the POMGnT1–/– chimeras compared with Largemyd/myd mice. We have shown previously that fibroblasts from MEB disease patients are largely resistant to LCMV infection (Imperiali et al., 2005). As MEB disease fibroblasts and POMGnT1–/– mice share the same glycosyltransferase defect, we expected a similar phenotype in POMGnT1–/– chimeras. However, despite a slight reduction in in vivo viral titres in POMGnT1–/– and POMGnT1+/– chimeras, the in vivo effects were clearly less prominent than the in vitro effects caused by the absence of POMGnT1 (Imperiali et al., 2005). As -DG is probably not the only receptor for LCMV, it seems likely that the virus can infect cells via alternative receptor(s) (Kunz et al., 2004) and therefore can replicate more or less efficiently in organs of dystrophic mice. It is also conceivable that the dependence of LCMV infectability on DG glycosylation differs among different cell types. We limited our in vitro infection experiments to BM-DCs and peritoneal macrophages with a mutant or control background. It is possible that other cell types that are permissive for LCMV replication, such as stromal cells and hepatocytes, may be less dependent on -DG glycosylation for LCMV infection. In the case of the POMGnT1–/– chimeras, about 95 % of the haematopoietic cells were POMGnT1-deficient, but all non-haematopoietic cells were POMGnT1-sufficient. It is therefore conceivable that a significant part of LCMV replication occurred in the wild-type non-haematopoietic cells, thus resulting in only moderate overall reductions in viral titres in different organs. However, when we restricted our analysis of productive in vivo LCMV infection to DCs of either POMGnT1-deficient or POMGnT1-sufficient origin, we still did not observe significant differences in the percentage of infected cells. This further suggests that infection of DCs in vivo can be mediated by alternative receptor(s) and pathways. For example, naturally occurring antibodies can bind to LCMV and promote its transport to secondary lymphoid organs such as the spleen (Ochsenbein et al., 1999). Natural antibodies are not neutralizing but are likely to increase the viral particle size and thus enhance phagocytosis, which leads eventually to infection of the phagocyte (Lamers et al., 1981; Weigle, 1973). Therefore, LCMV might infect DCs in an -DG-independent manner (Borrow & Oldstone, 1994; Di Simone & Buchmeier, 1995; Di Simone et al., 1994). Taken together, these findings support the notion that -DG glycosylation plays a crucial role in LCMV receptor function in vitro. However, the in vitro findings do not translate into a comparable in vivo phenotype. Alternative LCMV receptor(s), natural antibodies and possibly compensatory mechanisms for -DG glycosylation may account for the mild in vivo phenotype following LCMV infection.
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ACKNOWLEDGEMENTS |
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Received 7 June 2008;
accepted 19 July 2008.
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), by flow cytometric analysis of cells derived from Largewt/myd (wt/myd) or Largemyd/myd (myd/myd) mice. (a) Surface staining (%) for 
