key: cord-0983675-5szalmsz
authors: CHARLEY, B.; LAVENANT, L.; DELMAS, B.
title: Glycosylation is Required for Coronavirus TGEV to Induce an Efficient Production of IFNα by Blood Mononuclear Cells
date: 2006-06-29
journal: Scand J Immunol
DOI: 10.1111/j.1365-3083.1991.tb01792.x
sha: a31d6ea0972f8ce473d289cafcf5fe986346beb9
doc_id: 983675
cord_uid: 5szalmsz

Porcine peripheral blood mononuclear cells (PBMC) are induced to produce interferon alpha (IFNα) following in vitro exposure to coronavirus TGEV (transmissible gastroenteritis virus)‐infected glutaraldehyde‐fixed cell monolayers or lo TGEV virions. In the present report, we examined the possibility that glycosylation of viral proteins could play a major role in interactions with PBMC leading to the production of IFNα. Con A pretreatment of TGEV‐infected cell monolayers before fixation with glutaraldehyde and exposure to PBMC caused a dose‐dependent inhibition of IFNα induction, implying that masking of carbohydrates at the surface of infected cells lowered IFNα induction. Similarly, inhibition of N‐linked glycosylation by tunicamycin during viral infection of cell monolayers altered their ability to induce IFNα. In addition, complete cleavage of complex type' oligosaccharides by peptide‐N‐glycohydrolase Flowered the capacity of TGEV virions to induce IFNα. Thus, these findings strongly suggest that glycosylation of the viral proteins, and more precisely the presence of complex‐type oligosaccharides, is an important requirement for a completely efficient interaction with PBMC leading to the production of IFN‐α.

Various stimuli such as viruses, bacterial products or tumour cells can induce leucocytes to prodtice interferon alpha (IKNx) [7] . The demonstration thai inactivated vira! particles or gluiaraldchyde-fi.xcd virus-infected cells could induce IFNx [2. 4. 10. 12. 13, 17] led to the suggestion that the IFN-inducingcapacity wasnot related to virus replication but could rather reflect direct membrane interactions between leucocytes and viral proteins present at the surface of viral particles or virus-infected cells [7. 17] .

Studies conducted to characterize the nature of IFNx-produccr cells (IPC) in response to such stimuli, indicated that IPC were infrequent but highly efficient mononuciear cells [3, II. 23] . IPC could be characterized in several mammalian species as non-adherent, non-T. non-B cells. expressing MHC class II and CD4 molecules [5.9, 18.21.22.24] .

In contrast, few reports arc available about the nature of the viral component involved in membrane interactions with lymphocytes leading to the activation of IFN-a-coding genes. Thus, in the course of studies on IFNa induction by herpes simplex virus (HSV). Lebon [19] showed thai monoclonal antibodies (McAbs) directed at the major viral glycoprotein D could inhibit HSVinduced IF'ND: production. These data suggested that a defined glycoprotein could play a crucial role in IFNsi-induction. ln the case of transmissible gastroenteritis virus (TGEV). a coronavirus which induces acute diarrhoea and intense IFNa production in newborn piglets [14] 

Preparation of PBMC. Porcine periphenil blood mononuciear cells (PBMC) were prepared from heparini/cd hlood collected from 2 4-month old animals, by centrifugation on Ficoll (MSL. density 1.077, Eurobio. Paris. France). The PBMC were rcsuspended in RPMI-1640 medium supplemented with 10"" heat-inactivated fetal calf serum. 2 niM L-g!uiamine and antibiotics.

Viru.s. AsasourceofTGi-V. we used thecell-adapicd Purdue 115 strain. Methods for propagation and titration were as described by Laude et at. []5\. IFN induction. PBMC were indueed to produce IFNs hy overnight incubation on TGEV-infecled. glutaraldehyde-fixed cell monolayers as described previously [4] : brielly. pig kidney cells (PD5) were plated in 96-well microplates. infected by the coronavirus TGEV for 18h. then fixed with 0.25% glutaraldehyde (1 h at 4 C) and stored with 3% giycine. Monolayers were washed before addition of PBMC {100 fi\ per well at 5 x 10''. ml). Supernatants were colleeted after 18 h of incubation at 37 C and assayed for IFN activity.

Con .4 treatment tif TGEV-infected cett monotayers. Pig kidney cell (PD5) monolayers. infected for 18 h. were washed once with PBS, ihen incubated for I h al 37 C with various amounts of Con A (Miles-Yeda, Israel) before being fixed with glularaldehyde as described above.

Tuntiamycin treatment of 'tGEV-infected cett monotayers. At I h post-infectton, tunicamycin {Serva. Heidelberg. FRG) was added to the pig kidney cell tnonoiayers al a dosage of 2 /ig/ml for 18 h. until fixation by glutaraldehyde.

Gtyco.sida.se treatments. Endo-/J-N-acetylglLicosaminidase H (endo H). endo-/f-N-acetylgltieosaminidase F {endo F)and peptide-s-glycosidase F(PNGase F) were purchased from Boehringer-Mannheim-F""ranee (Meylan. France}. Aliquots of 20 /J1 of purified TGEV suspensions (500 /ig/ml) prepared as described by Laude et at. 115] , with the addition o\' 3 /d of "S-labellcd TGEV [6] . were incubaled with 20 ;jl of glycosidase and 20 ;il of 20 mM sodium phosphate (pH 7.2). 50 msi EDTA solution, for 18 h at 37 C. The final concentrations of enzymes were 60 U/ml for glyeosidase F, 15 U/ ml for endo F and 0.3 U/ml for endo H. The resulting preparations were used to induce PBMC as follows: serial 10-fold dilutions of 10 ii\ aliquots were added to PBMCCi X IO'vml) in a final volumeofO.2 ml for IS hat 37 C. In order to ascertain the en-^ymatic cleavage. 6 ;ii aliquots of each preparation were subjected to electrophoresis on ]O"' li polyacrylamide gels which were then processed for Huorography.

IEN hioa.ssav. Log3 dilutions of PBMC supernatants were assayed for IFN on bovine MDBK cells using vesicular stomatitis virus as a challenge 114|. A standard porcine IFNa was included in each assay. This standard was ealibraled on MDBK cells with the human international reference IFN B69/19 (NIH. Bethesda. MD. USA). In our results. I U is equivalent to 1 IU of human IFN.

PBMC exposed lo TGEV-infected glutaraldehyde-fixed cell monolayers are induced lo secrete IFNa: in the results shown in Table I . 5x 10'' PBMC per ml produced 1000 U/ml IFN following 18 h incubation at 37 C. In order to evaluate the role played by viral glycoproteins present at ihe surface of infected cells for the induction of IFNof, Con A was added to TGEV-infected cell tnonoiayers before fixalion wiih gtutaraldchyde. Table I shows that Con A treatment causes a dose-dependent inhibition o\' IFNx-induction. Control experiments showed that uninfected cell monolayers. with or without Con A treatment. ?. IFN produced by PBMC at 5 x IO*;ml exposed to cell monolayers for 18 h. did not induce PBMC to produce IFNx (not shown).

Pig kidney cell monolayers were ireaied by tunieamyein for 18 h following initiation of viral infeciion. then fixed with gttitaraldehyde. TunJeamyin-treated eell monotayers indueed PBMC to produee 10 IOO times less IKN than tnoektrcated eeil monolayers (Table 11 ; four independent experiments). It was shown previously that tunicamycin (at 2/ig/ml) inhibited N-tinked glyeosylation with slight eflects on ihe overall eell protein synthesis (Dehiias and Laude. submitted for publieation). Our present dala indicate that tunicamycin markedly affects the ability of TGEV-infected cells to induce IFNa production. 3. IEN (U ml) produLxd hy PBMC exposed to cell monolayers for IK h.

In order lo further analyse the role played by oligosaccharide moieties of TCiRV proteins for IFNa induclion. we examined the elTects of pretreating virus particles by gtycosidascs before incubation with PBMC. Three endoglyeosidases able to cleave N-linked oligosaccharides were used: endo H and endo F eleave predominantly high mannose oligosaccharide chains, whereas PNGase K cleaves both high mannose and eomptcx-lype saccharides [27] . Virus particles pretreated with endo H or endo F induced the same amounis of IFNa as control virions (Fig. I) . In contrast, PNGase F markedly decreased the virions* ability to induce PBMC production of IFN'DC Fig. I shows that the same amount of PNGase F-treated virus could induce 10-100 times less IFNi than other vira! preparations. SDS-polyacrylamide ge! electrophoresis control analysis, performed on the mixture of cold and .15S labelled TGEV. showed that endo H and endo Kcatised a Mr shift ofthe major 29 kDa M protein species to 26 kDa. while 30 36 kDa speeies remained unaffected (Fig. 2) . In contrast. PNGase F caused a complete carbohydrate cleavage leading to the presence ol' only one 26 kDa molecular species. The nucleoprotcin N was not affected by digestion which indicates that proteolytic cleavages of viral polypeptides were unlikely to occur during glycosidase treatments. When ciliquots of virus preparations were incubated with PBMC. a residual PNGase F activity might have directly acted on PBMC. thereby altering their ability to secrete IFNa. This did not appear to be the case since the induction of IFNa remained unchanged in a control experiment in which PNGase F was added to the virus just before incubation with PBMC (Fig. 3) . 

The resutts of the present investigation indicate that glycosytation of coronavirus TGEV proteins is of major importance for induction of IFNa production by blood mononuclcar cells. The fact that potent IFNa induction is achieved by lixed cells, as already described [2, 4, 13 . 17], strongly suggested that TFNx induction is independent of virus penetration into PBMC but could rather reflect direct inleraetion between membrane-associated viral antigens and PBMC membranes [7, 19] . In a lirst set of experiments, TGEV-infected cells were treated with Con A before fixation with glutaraldehyde: such treatments caused a dosc-dependcnt inhibition in the ability of cell tnonoiayers to induce PBMC to produce IFNa (Table I ). Beeause Con A has a high capacity to bind to complex-type carbohydrates [26] . these data imply that rnasking of carbohydrates present at the surface of infected cells will impede adequate interaction with PBMC leading to IFNa-production. However, the possibility that the Con A inhibitory effect might be due to steric hindrance of the relevant structure could not be excluded. In addition. Con A could bind to cell membrane glyeoproteins as well as viral glyeoproteins.

A more precise experimental approach consisted, therefore, in treating TGEV-infeeted cells with tunieamyein, an antibiotic which blocks Nlinked glycosylation [8] . Such a treatment was previously shown to have slight effects on ihe overall eell protein synthesis and M and S polypeptides were produced with Mr corresponding to those predicted for the apoproteins (Delmas and Latide. submitted for publication}. In our experiments, tunicamycin redueed theability of infected cell monolayers to Induce I FNa (Table  II) , which implies that inhibition of glycosylation in cell monolayers has negative effects on the expression of an "IFNa-inducing-signal* at the eell surface. However, since tunicamyein ean affect glycosylation ofcell membrane proteins in addition to virus-eoded proteins, it is not possible to determine from these data whether reduction of IFN-induetion is related to the absence of carbohydrate moieties on viral or on cellular proteins. Moreover, tunicamycin could also block the transport ofthe viral apoproleins to the cell surface (see for example. Ref. 20) .

For these reasons a last set of experiments was conducted in which virus particles were treated with glycosidases before being incubated with PBMC in order to induce IFNx. Three enzymes were used: two glycosidases (endo H and endo F) eleave predominantly high mannose oligosaeeharide chains whereas peplide-N-glycohydrolase V (PNGase F) cleaves both high mannose and 'complex-type' oligosaccharides [27] . Our results show thai PNGase F was the only enzyme which could reduce TGEV virions ability to induce IFNa (Fig. I) . A control experiment showed that this rcduciion was nol due lo a direct efiecl of PNGase F on PBMC (Fig. 3) . When treated virionaliquois were tritratedon PBMC (Fig. I) it appeared that a reduced IFNa-induetion was obtained with diluted PNGase F-treatcd viral preparations whereas high concentrations of PNGase F-treated virus (about 1.5 /ig virions for 5 X 10^ PBMC) induced as much IFNo: as control, endo H-or endo F-treated virus. We have already reported strong evidence for the role of glycoprotein M as the effector viral molecule for IFNxinduction ( [4] ; Laude ct at., submitted for publication). It is therefore interesting to notice that PNGase F was also the only glycosidase tested whieh could cleave completely oligosaecharidcs from the viral protein M. leading to the produetion of a 26 kDa molecule (Fig. 2) . corresponding to the size predicted for the apoprotein M [ 16] and found after tunicamycin treatment (Delmas and Laude. submitted for publication). Moreover, since PNGase F is able to cleave 'complex-type' oligosaccharides in contrast to endo H or endo F [27] . our results indieate that only a complete cleavage of'complex-type' oligosaeeharides from the viral protein M alters the virus" ability to induce PBMC to produee IFNa.

What is the role(s) ofearbohydrates, and more precisely eomplex-type oligosaeeharides. present at the surface of virus particles or virus-infected cells, in the induction of IFNa? It is unlikely that carbohydrates alone are the 'IFNa-inducing signal': in fact, we show here that deglyeosylated virus particles do induce IFNa when exposed at high concentrations to PBMC (Fig. I) . One possible explanation is that glycosylation of viral protein M is an itnportant structural characteristic creating Ihe adequate conformation required for it to interact with a putative receptor on PBMC membrane, leading to activation of IFNa genes. The need for adequate conformation for IFNj-induction was also suggested by our previous observalion that a mutation on the glycoprotein M, which should cause important confor-tnational changes, greatly impaired IFNotinduetion (Laude et at., submitted for publication). The existence of a membrane receptor involved in IFNa-induction was suggested by the results from Lebon [18] that IFNa-induetion was sensitive to lysosomotropic drugs.

Alternatively, carbohydrate moieties of viral polypeptides may influence IFN:(-induction by increasing the stability of complexes formed between virus panicles or virus-infeeted cells and PBMC. With that respect, adhesion receptors termed "seleetins" were recently demonstrated on lymphocytes [25] : these molecules contain an Nterminal lectin domain potentially able to interact with carbohydrates. Cell-surface molecules equivalent to such 'seleetins' could bind virus-associated carbohydrates, which in turn would facilitate stable interactions between PBMC and the actual "IFN-inducing domain' of the viral polypeptide, leading lo IFNa synlhesis. Obviously it would be interesting to know whether carbohydrates are important for induction of IFNa by viruses other than TGFV. and which of the mechanisms we suggested (i.e.. adequate conformation or adhesion requirement) is involved in the IFNa-induction process. A consequence of our findings is that synthetic peptides corresponding to the M protein or Esherichia coliprodueed unglycosytated reeombinant M proteins by themselves will be unlikely to efficiently induce IFNa, unless used at high concentrations. In addition, carbohydrates such as "complextype" oligosaccharides may be useful tools to probe the infrequent leucocyte subpopulation involved in the production of IFNa.

Structural determinants of Concanavalin A specilicity for oligosaccharides

Induction of alpha inlerferon by membrane iutcractioti between viral surface and peripheral blood tnononuciear cells

Infrequent btit efficient interferon-a-producing human mononuclcar leukocytes induced by Herpes Simplex Virus in titro studies hy immuno-pluqtte and limiting dilution assays

Induction of alpha interfcron by transmissible gastroenteritis coronavirus: Role of tran

Characterization of blood mononuciear cells producing IF'Ny following induction by corona virus-infected cells

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Mechanism of induction ot\ilpha interleron. Pp. 21 .10 in

Inhibitors of the hiosyrthesis and processing of N-linked oligosaecharide chains

Human mononuciear cells which produee interferon-alpha during NK (HSV-FS) assays are HLA-DR positive cells distinct irom cytolylic Natural Killer eflectors

Interferon synthesis indueed by Viral Hemorrhagic .Septiecmia Virus (VHSV) in rainbow trout leukocytes in vitro

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Human peripheral null lymphocytes. IL Producers of type 1 interferon upon stimulation with tumor cells, herpes simplex virus and Corynelmcterium parvutn

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A distinct population of nonphagocytic and low level CD4* null lymphocytes produce IFN-a after stitnulation by Herpes'Simplex Virus-infected cells

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Enzymatic approaches for studying the structure, synthesis, and processing of glycoproteins

We thank Drs R. Hedriek (Davis. Calif, USA) and H. Laude (Jouy en Josas. France) for critical reading of the manuscript.