Muhammad K Salamat

Michel Dron
email: michel.dron@jouy.inra.fr.

Je ´ro ˆme Chapuis

Christelle Langevin

Hubert Laude
email: hubert.laude@jouy.inra.fr

Prion Propagation in Cells Expressing PrP Glycosylation Mutants ᰔ

Infection by prions involves conversion of a host-encoded cell surface protein (PrP C ) to a disease-related isoform (PrP Sc ). PrP C carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP C glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP Sc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP Sc , while PrP C with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP C , were able to form infectious PrP Sc . Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

Transmissible spongiform encephalopathies (TSE), or prion diseases, are fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease and related diseases in humans, scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in cervids. The prion, the transmissible agent, is thought to be made essentially of PrP Sc , a misfolded form of the host protein PrP C . PrP Sc displays a novel conformation enriched in ␤-sheets, conferring increased protease resistance and a tendency to form amyloid-like multimers. Prion propagation is believed to occur by recruitment and conversion of new PrP molecules within PrP Sc seeds. The three-dimensional structure of PrP Sc multimers and the conformational dynamic changes underlying PrP C conversion remain unresolved [START_REF] Caughey | Getting a grip on prions: oligomers, amyloids, and pathological membrane interactions[END_REF]48).

PrP C is a glycoprotein that is localized predominantly to detergent-resistant membrane microdomains via a glycosylphosphatidylinositol (GPI) anchor. Two sites for Asn-linked glycosylation are present in the structured, C-proximal half of the protein, both of which are variably occupied, thus producing di-, mono-, and unglycosylated molecules [START_REF] Ermonval | Evolving views in prion glycosylation: functional and pathological implications[END_REF]. PrP C is exposed at the cell surface, from which it is subjected to endocytosis and recycled between the endocytic compartment and the plasma membrane [START_REF] Harris | Trafficking, turnover and membrane topology of PrP[END_REF]. While several roles have been proposed, the biological function of PrP C remains elusive [START_REF] Aguzzi | The prion's elusive reason for being[END_REF]. In infected cells, PrP Sc accumulates at the cell surface and intracellularly, mainly in the endolysosomal compartment, but the subcellular site(s) where conversion takes place remains a subject of debate [START_REF] Caughey | Getting a grip on prions: oligomers, amyloids, and pathological membrane interactions[END_REF][START_REF] Marijanovic | Identification of an intracellular site of prion conversion[END_REF].

Natural and rodent-adapted prion strains can be differentiated based on the neuropathological changes they induce in a defined host and the biochemical features of the PrP Sc accumulating in the infected tissues [START_REF] Beringue | Prion agent diversity and species barrier[END_REF]. Thus, while all PrP glycotypes can acquire protease resistance, the ratio of bi-, mono-, and unglycosylated resistant PrP Sc species (PrPres) stably differs among prion strains, a feature commonly used for their molecular typing [START_REF] Hill | Molecular classification of sporadic Creutzfeldt-Jakob disease[END_REF]. Whether and how the PrP glycan chains influence the strain specificity of prion formation remain to be clarified.

Cell culture experiments have unambiguously established that not only the strain but also the cells in which the prion replication takes place can determine the PrPres glycotype [START_REF] Birkett | Scrapie strains maintain biological phenotypes on propagation in a cell line in culture[END_REF][START_REF] Paquet | Efficient dissemination of prions through preferential transmission to nearby cells[END_REF][START_REF] Vorberg | Molecular basis of scrapie strain glycoform variation[END_REF]). Moreover, PrP C glycosylation may differ in individual cell types or brain regions [START_REF] Beringue | Regional heterogeneity of cellular prion protein isoforms in the mouse brain[END_REF][START_REF] Ermonval | Glycosylation-related genes are variably expressed depending on the differentiation state of a bioaminergic neuronal cell line: implication for the cellular prion protein[END_REF]. The glycoform ratio associated with a defined strain also varies depending on the brain region or organ in which PrP Sc is formed [START_REF] Dearmond | Selective neuronal targeting in prion disease[END_REF][START_REF] Levavasseur | Regulating factors of PrP glycosylation in Creutzfeldt-Jakob disease-implications for the dissemination and the diagnosis of human prion strains[END_REF][START_REF] Rubenstein | Scrapie-infected spleens: analysis of infectivity, scrapieassociated fibrils, and protease-resistant proteins[END_REF][START_REF] Somerville | Transmissible spongiform encephalopathy strain, PrP genotype and brain region all affect the degree of glycosylation of PrPSc[END_REF]. Such observations have led to the speculation that the glycoform ratio of PrP C expressed by neuronal cells might be a determinant of the different brain-targeting selectivity manifested by prion strains [START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF]. Acellular amplification of prions is possible through protein misfolding cyclic amplification (PMCA) [START_REF] Castilla | Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions[END_REF]. In one study where reconstituted PrP C material was used as a substrate, the proportion of each glycotype was shown to affect the efficiency of PrP Sc formation, therefore arguing that the stoichiometry of the various glycotypes within PrP Sc multimers obeys strainspecific conformational constraints [START_REF] Nishina | The stoichiometry of host PrPC glycoforms modulates the efficiency of PrPSc formation in vitro[END_REF]. Also, inoculation of aglycosylated PrP Sc material produced by PMCA into mice provided evidence that the carbohydrate moiety of the input PrP Sc is not required for the maintenance of the strain-specific neurotropism [START_REF] Piro | Prion protein glycosylation is not required for strainspecific neurotropism[END_REF]. Such observations support the view that PrP Sc glycoform variation may be a consequence rather than a cause of the prion strain diversity, with the strain specificity being essentially enciphered by distinct conformations of PrP Sc backbone [START_REF] Jones | Fibril conformation as the basis of species-and strain-dependent seeding specificity of mammalian prion amyloids[END_REF]48).

To what extent host PrP glycosylation influences the outcome of prion infection and disease in a host is an important issue. Studies in transgenic mouse models expressing PrP with no glycan chain at either one or both sites established that the diglycosylated form of PrP is not mandatory for prion infection [START_REF] Dearmond | Selective neuronal targeting in prion disease[END_REF][START_REF] Neuendorf | Glycosylation deficiency at either one of the two glycan attachment sites of cellular prion protein preserves susceptibility to bovine spongiform encephalopathy and scrapie infections[END_REF]. More recent work using gene-targeted transgenic mice expressing glycosylation mutants under the control of the endogenous promoter demonstrated that PrP C glycosylation is not essential for establishing infection within a host by intracerebral inoculation; however, prion replication in mice expressing aglycosylated PrP was fully or strongly impaired, depending on the strain [START_REF] Cancellotti | Altered glycosylated PrP proteins can have different neuronal trafficking in brain but do not acquire scrapie-like properties[END_REF][START_REF] Tuzi | Host PrP glycosylation: a major factor determining the outcome of prion infection[END_REF]. Glycosylation was shown to influence the incubation period before disease onset and not the neuropathological features in the brain [START_REF] Tuzi | Host PrP glycosylation: a major factor determining the outcome of prion infection[END_REF]. Following inoculation by a peripheral route, however, PrP C glycosylation profoundly influenced both the disease timing and the PrP Sc deposition pattern, and mice without PrP glycosylation were resistant [START_REF] Cancellotti | Glycosylation of PrPC determines timing of neuroinvasion and targeting in the brain following transmissible spongiform encephalopathy infection by a peripheral route[END_REF]. This suggested a role for PrP-attached glycans in the spread of prions within the organism. One general limitation of such studies, however, is the limited number of mutant PrPs expressed in mice and hence the possibility that the observed differential strain requirement is linked to the replaced amino acid rather than to the removed glycan chain. Moreover, there is evidence from experiments both in cultured cells [START_REF] Korth | Expression of unglycosylated mutated prion protein facilitates PrP(Sc) formation in neuroblastoma cells infected with different prion strains[END_REF][START_REF] Lehmann | Blockade of glycosylation promotes acquisition of scrapie-like properties by the prion protein in cultured cells[END_REF][START_REF] Rogers | Intracellular accumulation of the cellular prion protein after mutagenesis of its Asn-linked glycosylation sites[END_REF] and in vivo [START_REF] Cancellotti | Altered glycosylated PrP proteins can have different neuronal trafficking in brain but do not acquire scrapie-like properties[END_REF][START_REF] Dearmond | Selective neuronal targeting in prion disease[END_REF]) that glycosylation-abolishing mutations can alter the intracellular PrP trafficking, thus possibly affecting the efficiency of the conversion process.

The strategy generally employed to address how the glycosylation status of PrP influences its ability to sustain prion propagation has consisted of transiently expressing tagged mouse PrP constructs with mutated glycosylation sites in steadily infected ScN2a mouse cells and monitoring acute conversion into PrP Sc [START_REF] Ikeda | Thr but not Asn of the N-glycosylation sites of PrP is indispensable for its misfolding[END_REF][START_REF] Korth | Expression of unglycosylated mutated prion protein facilitates PrP(Sc) formation in neuroblastoma cells infected with different prion strains[END_REF][START_REF] Neuendorf | Glycosylation deficiency at either one of the two glycan attachment sites of cellular prion protein preserves susceptibility to bovine spongiform encephalopathy and scrapie infections[END_REF]. In the present study, we have taken advantage of the Rov cell model, which is infectible by ovine prions [START_REF] Vilette | Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein[END_REF], to further address this issue. The approach used here differs in several aspects. First, the trafficking properties of mutants and their conversion competence were analyzed in cultures that stably expressed only the glycosylation mutant protein, as the concomitant expression of wild-type molecules within the same cells may create a less than optimal context to do this. Second, the conversion competence was examined in a situation of de novo infection and along subpassages of the cultures, which the robustness of the Rov cell infection system made feasible. We observed dramatic differences depending on the amino acid change introduced and that proper trafficking to the cell surface did not suffice to confer permissiveness to infection. These results are important in the perspective of elucidating the specific roles of PrP Asn-glycan chains in prion biology.

MATERIALS AND METHODS

Cell culture. Rov cells are derived from the epithelial RK13 cell line and express ovine PrP in a doxycycline-inducible manner [START_REF] Vilette | Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein[END_REF]. They were grown in Opti-MEM medium (Invitrogen) supplemented with 10% fetal calf serum (FCS) and antibiotics and were split 1:4 after trypsin dissociation once a week.

Generation of PrP mutants. Mutations were introduced by site-directed mutagenesis (QuikChange II mutagenesis kit; Stratagene) into sheep PrP (allotype V 136 R 154 Q 171 ) cloned into plasmid pTRE (Clontech) using mutagenic primers. To build the N184 mutant series, the same oligonucleotide sequence, 5Ј-GTG CAT GAC TGT GTC AAC ATC ACA GTC AAG CAA-3Ј, was designed by introduction of relevant nucleotide changes in the asparagine codon (bold). Mutant V183I was created by introducing a G-to-A transition in the upstream valine codon (underlined) of the above sequence. To design the N200 mutant series, the oligonucleotide sequence 5Ј-C ACC ACC AAG GGG GAG AAC TTC ACC GAA ACT GAC-3Ј was used to introduce changes of the asparagine codon. F201S and F201V mutants were produced by replacement in the latter sequence of the phenylalanine codon (underlined) by TCC and GTC, respectively. The following primers were used to obtain T-to-X glycosylation mutants: 5Ј-GAC TGT GTC AAC ATC GCA GTC AAG CAA CAC ACA G-3Ј (T186A) and 5Ј-GAC TGT GTC AAC ATC AAC GTC AAG CAA CAC ACA G-3Ј (T186N). G37T and Y52T substitutions, both creating a novel consensus glycosylation site, were obtained using primers 5Ј-GC GGA GGA TGG AAC ACT ACG GGG AGC CGA TAC C-3Ј and 5Ј-CCT GGA GGC AAC CGC ACT CCA CCT CAG GGA GGA G-3Ј, respectively. Anchorless (⌬GPI) PrP mutants were generated by replacing serine 234 by a stop codon using the primer 5Ј-C CAA AGG GGG GCA TGA GTG TAC CTC TTT TC-3Ј and its reverse complement.

Isolation of Rov cells. RK13 cells that stably expressed either wild-type or mutant PrP were obtained by transfection and puromycin selection as described previously [START_REF] Vilette | Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein[END_REF]. When needed, individual clones were isolated from selected cell batches. To assess PrP C expression, cells were cultivated in the continuous presence of 1 g/ml doxycycline (Sigma).

Prion infection of cell cultures. Cells were infected with 127S strain of sheep scrapie as previously described using 1% (wt/vol) of brain pool homogenates of terminally ill tg338 mice [START_REF] Vilette | Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein[END_REF]. Two days after exposure, cells were carefully rinsed and incubated for two additional days before trypsinization and passage of cells at a 1:10 dilution (passage 1). Cells were further incubated for 1 week and then split at a 1:4 dilution at each following passage. Rov cells with ⌬GPI PrP were similarly inoculated with the brain homogenate, incubated for 2 days, and then rinsed and covered with culture medium plus 0.6% agarose. After 1 week of incubation, the agarose layer was mechanically removed using a scalpel blade and replaced by a fresh one, and the cells were further incubated for 1 week prior to harvesting for analysis. The amount of PrPres associated with the removed agarose layer was found to be minimal. To test the infectivity of cultures that propagated mutant N200D PrP Sc , 5 ϫ 10 6 cells were harvested at passage 6 postexposure, pelleted, frozen and thawed, and then sonicated and used as inoculum as for brain homogenate. The infectivity of ⌬GPI PrP Sc was assessed using material prepared from 2-week-old cultures under agarose by disruption in lysis buffer (see the next section) and methanol precipitation. Resuspended dry pellets were used to inoculate Rov9 cells expressing wild-type PrP [START_REF] Vilette | Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein[END_REF].

Cell lysis and PK digestion. The cells were washed twice with cold phosphatebuffered saline (PBS), and whole-cell lysates were prepared at 4°C in TL1 buffer (50 mM Tris-HCl [pH 7.4], 0.5% sodium deoxycholate, 0.5% Triton X-100). Lysates were clarified by centrifugation at 1,000 ϫ g for 2 min, and the protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce). To analyze the PrP C content, 50 g of protein was methanol precipitated. For PrPres, 250 g of protein was digested with proteinase K (PK) under our standard conditions (4 g of PK per 1 mg of protein, 2 h, 37°C) and further centrifuged at 22,000 ϫ g for 30 min, and pellets were dissolved in Laemmli sample buffer.

Detection of PrP C and PrPres by immunoblotting. Samples were loaded on a 12% Bis-Tris polyacrylamide gel (Invitrogen) and run in morpholineethanesulfonic acid (MES) buffer. Proteins were transferred to a nitrocellulose membrane (Schleicher and Schuell) by semidry blotting using a Trans-Blot SD cell (Bio-Rad) as previously described [START_REF] Dron | Endogenous proteolytic cleavage of disease-associated prion protein to produce C2 fragments is strongly cell-and tissue-dependent[END_REF]. For PrP C detection, membranes were further incubated with the mouse monoclonal antiprion antibody 4F2 (antioctarepeat) and horseradish peroxidase-conjugated anti-mouse IgG as a secondary antibody. To detect PrPres, membranes were incubated with the biotinylated mouse monoclonal antiprion antibody Sha31 (epitope from residue 145 to 152 [human numbering]) and then with peroxidase-conjugated streptavidin. Revelation was done using the BM chemiluminescence blotting substrate (Roche). Molecular weight markers were Precision Plus protein standards (Bio-Rad).

Immunofluorescence and confocal microscopy. After fixation with 4% paraformaldehyde, cells were permeabilized with 0.1% Triton X-100 or not as previously described [START_REF] Dron | Proteasome inhibitors promote the sequestration of PrPSc into aggresomes within the cytosol of prion-infected CAD neuronal cells[END_REF]. PrP C was detected using 4F2 antibody and Alexa Fluor 488-conjugated goat anti-mouse antibodies (Molecular Probes). For colocalization experiments, permeabilized cells were further incubated with a rabbit polyclonal antigiantin antibody (Abcam) and Alexa Fluor 568-conjugated goat anti-rabbit as secondary antibody, whereas nuclei were stained with DAPI (4Ј,6Ј-diamidino-2-phenylindole) (Sigma). To assess the presence of PrP C at the outer leaflet of the cell surface, adherent live cells were washed and labeled with 4F2 antibody and Alexa Fluor anti-mouse antibodies at 4°C in medium containing 1% heat-inactivated FCS. Images were acquired with a Zeiss Axiovert 200 M microscope and a charge-coupled device (CCD) cool-snap HQ camera (Roper Scientific) driven by MetaVue imaging system software (Molecular Devices/ MDS Analytical Technologies). For confocal microscopy, a Zeiss LSM510 Meta microscope linked to an Axiocam HRm camera was used, the acquisition and treatment of the image were done with the CLSM 510 software, and multiple optical planes in the axial (Z) direction were collected.

Quantification of PrP cell surface expression by flow cytometry. Cells (5 ϫ 10 6 ) were detached using a protease-free cell dissociation buffer (Sigma). All subsequent steps were performed at 4°C and with FCS. Cells were resuspended in PBS plus 5% FCS and then labeled with 4F2 antibodies (1:500 dilution) and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500; Molecular Probes), with further addition of 5 g/ml of 7-amino-actinomycin D stain. Data were acquired on FACSCalibur (Becton Dickinson), collecting 1,000,000 events, with Cellquest software. Untransfected RK13 and Rov cells labeled with conjugated antibody only were used as negative controls. The mean specific fluorescence was used for quantification of the PrP expression level.

Sucrose density gradient centrifugation. Detergent and flotation procedures were performed as described previously [START_REF] Paquet | Prion infection of epithelial Rov cells is a polarized event[END_REF]. Briefly, Rov cultures expressing wild-type or G37T-N184D-N200D mutant PrP were lysed in TNE/TX100 buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100), and lysates were brought to 40% sucrose. A discontinuous (40%, 35%, and 5%) sucrose gradient was layered onto the lysate, and the flotation gradient was ultracentrifuged for 18 h at 4°C and 150,000 ϫ g in a Beckman SW55Ti rotor. Ten fractions were harvested from the top of the gradient and analyzed for PrP by immunoblotting after methanol precipitation.

RESULTS

Expression and subcellular localization of "monoglycosylated" PrP mutants. We generated an initial series of PrP glycosylation mutants in which the asparagine residue of one each of the consensus sites (N184 and N200 in the ovine sequence) was replaced by one of the five amino acid residues D, H, S, T, and Q. The first four amino acids were selected because they are statistically associated with a lower propensity to modify the structure and/or function of proteins upon substitution (51); Q was used because this amino acid has often been chosen on a steric basis to suppress glycosylation sites [START_REF] Korth | Expression of unglycosylated mutated prion protein facilitates PrP(Sc) formation in neuroblastoma cells infected with different prion strains[END_REF]. Expression vector constructs encoding mutant or wildtype PrP (VRQ allotype) were introduced into RK13 parental cells, and populations of stably transfected cells were selected. PrP expression in the "Rov" cell cultures thus obtained was induced by doxycycline and analyzed by immunoblotting (Fig. 1). The PrP mutants displayed the expected molecular profile, in which the upper, broad band corresponding to the diglycosylated species was lacking whereas the faster-migrating band corresponding to the monoglycosylated species of N200X and N184X mutants was predominant (Fig. 1). The mobility of the glycosylated species appeared overall to be slightly lower for site 1 than that for site 2 mutants, consistent with the lower molecular mass of the glycan chain attached to site 1 [START_REF] Moudjou | Glycan-controlled epitopes of prion protein include a major determinant of susceptibility to sheep scrapie[END_REF]. Additional site 1 glycosylation mutants were produced by replacing T (instead of N) in the consensus sequon by either A or N, the glycoform pattern of which did not differ from that of N184X mutants (data not shown).

The subcellular localization of the mutant PrP proteins was examined by immunocytochemistry on live and paraformaldehyde-fixed cells permeabilized with Triton or not. Both the N184X and N200X mutants were efficiently expressed at the cell surface (Fig. 2 and Table 1). Quantification by fluorescence-activated cell sorting (FACS), performed for nine of these mutants, showed a cell surface expression level of between 55 and 84% of that of wild-type PrP (Fig. 3). However, as observed in permeabilized cultures, the PrP labeling seen in a giantin-positive juxtanuclear organelle was generally more intense with mutant than with wild-type PrP (Fig. 2b and Fig. 4), indicating some retention of glycosylation mutant PrP molecules in the Golgi apparatus. In contrast to the N184X mutants, the T186X mutants did not reach the cell surface (Fig. 4) or only marginally (data not shown).

Prion formation and self-propagation in monoglycosylated PrP cells. Cells stably expressing mutant PrP were exposed to ovine prion strain 127S. All five N200 PrP mutants formed PK-resistant PrP Sc (PrPres) from the first passage postexposure on, as well as on subsequent passages of the cultures (Fig. 5). This result, based on three independent experiments using at least two different cell populations produced through independent transfection and selection, was indicative of sustainable infection, as opposed to simple, acute conversion into PrP Sc . However, the efficiency of conversion, in terms of the level and sustainability of PrPres accumulation, reproducibly differed among the mutants. Thus, while N200D cells accumulated nearly as much PrPres as wild-type Rov cells and were able to propagate the infection steadily for up to 10 consecutive passages (Fig. 5), N200H cells accumulated PrPres at much lower levels (Fig. 5) and not beyond Clearly different results were obtained when Rov cells expressing N184X PrP mutants were exposed to infection. Indeed, N184D was the only one of the site 1 mutants to show evidence of conversion into PrP Sc at a substantial level (Fig. 5 and Table 1). Additional amino acid changes (not listed in Table 1) were tested, including E and G, which were expected to be good substitutes for D. In short, none of the nine mutants produced a detectable accumulation of PrPres, even at the first passage postexposure, except N184D. N184D Rov cell cultures were reproducibly infectible (in five independent experiments) and produced PrPres for at least five passages postexposure (Fig. 5). Cell subclones expressing N184D were isolated and also found to be readily infectible, but again this turned out to be unsuccessful with other mutants.

To confirm that ovine prions propagated in cells expressing monoglycosylated mutant PrP C were truly infectious, cell extracts were prepared from such cultures and then used as the inoculum for de novo infection experiments. As shown in Fig. 6, prions propagated in cell cultures expressing N200D were able to infect cultures expressing the homotypic as well as the wild-type PrP sequence. Similar results were obtained by using infecting prions derived from cells expressing N184D (not shown).

Failure of unglycosylated PrP mutants to form PrP Sc . To produce unglycosylated double PrP mutants, we used constructs that combined the sole amino acid change found to be permissive at position 184 with one each of the changes made at position 200. These mutants will thereafter be designated using a shortened form, for instance, NDND instead of N184D-N200D. Additional mutants were designed, including NQNQ and NTNT, because these mutations had already been used in mouse transgenesis and some combinations of those and substitutions were used in previous studies. Altogether, 13

ϩ ϩ N184H ϩ Ϫ N184Q ϩ Ϫ N184S ϩ Ϫ N184T ϩ Ϫ N184G ϩ Ϫ N184E ϩ Ϫ T186A Ϫ Ϫ T186N Ϫ Ϫ Monoglycosylated site 2 mutants N200D ϩ ϩ N200H ϩ ϩ N200Q ϩ ϩ N200S ϩ ϩ N200T ϩ ϩ Unglycosylated double mutants N184D-N200D Ϫ Ϫ N184D-N200H Ϫ Ϫ N184D-N200Q Ϫ Ϫ N184D-N200S Ϫ Ϫ N184E-N200D Ϫ Ϫ N184G-N200D Ϫ Ϫ N184T-N200T Ϫ Ϫ
a ϩ, Ն 55%; Ϫ, Յ10% (relative to wild-type PrP).

double PrP mutants were generated (Table 1 and data not shown). As a common feature, such mutants were expressed at the cell surface very poorly, despite global expression levels approaching those of monoglycosylated mutants (Fig. 1). PrP immunofluorescent labeling was essentially intracellular and mostly colocalized with a marker of the Golgi complex, which is indicative of a markedly impaired trafficking (Fig. 2a andb). Upon exposure to infectious inoculum, the cultures expressing such mutants consistently failed to accumulate PrPres even transiently, despite repeated attempts.

Restoration of cell surface expression by creation of an artificial glycosylation site.

In the experiments described above, none of the PrP mutants (15 out of [START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF] showing no or minimal cell surface expression was able to form PrP Sc at a detectable level, suggesting that adequate trafficking to the plasma membrane is a key, if not an absolute requirement. Therefore, we asked whether introduction of an ectopic glycosylation site would restore the normal phenotype and, if so, the permissiveness. To this end, an NXT consensus sequon was created in the sequence of the NDND mutant by mutating codon 37 so as to change T to G, downstream from the first asparagine (N35) present in the N-terminal region of the molecule. Immunoblotting analysis showed that the G37T-NDND mutant PrP consisted mostly of glycosylated species, indicating that the newly introduced NTT sequon acted as an efficient sugar attachment site (Fig. 7a). As a control, the same mutation was introduced in the wild-type sequence. The PrP glycoform pattern of the latter mutant (G37T) was modified compared to that of wild-type PrP, with the presence of a larger, presumably triply glycosylated species and also the nearly complete disappearance of the unglycosylated species, further confirming that the ectopic NTT codon was fully functional. The G37T and G37T-NDND mutants had similar subcellular distributions, and both were properly trafficked to the outer cell surface, as revealed by immunofluorescence labeling of live or fixed cells (Fig. 7b; see also Fig. 4M). Moreover, both mutants showed association with flotillin (Fig. 7c), which is considered a raft component and classically used as a marker for lipid microdomains. Upon exposure to prion, however, G37T-NDND turned out to be unable to form PrP Sc , whereas G37T (with three glycan attachment sites) was converted as efficiently as wild-type PrP and allowed robust, sustainable infection (Fig. 7d). Similar results, i.e., efficient glycosylation and expression at the cell surface but lack of conversion into PrP Sc , were obtained with two other mutants, G37T-T186N, and Y52T-NDND, in which the Y52T mutation created a novel consensus sequon (NRT) involving asparagine 50 (Table 2). From these results it was concluded that restoring the trafficking of a mutant PrP to the cell surface was not sufficient to confer on this mutant the ability to convert into PrP Sc .

GPI anchor-deficient PrP can form PrP Sc upon infection. The lack of a GPI anchor has been reported to perturb the process of glycosylation of proteins during their translocation in the endoplasmic reticulum (ER) [START_REF] Hein | Association of a GPIanchored protein with detergent-resistant membranes facilitates its trafficking through the early secretory pathway[END_REF]. Indeed, GPI-deficient (⌬GPI) PrP consists mostly of unglycosylated species in mouse and in cell culture [START_REF] Chesebro | Anchorless prion protein results in infectious amyloid disease without clinical scrapie[END_REF][START_REF] Mcnally | Cells expressing anchorless prion protein are resistant to scrapie infection[END_REF][START_REF] Walmsley | Membrane topology influences N-glycosylation of the prion protein[END_REF]. Production of ⌬GPI PrP was thus a possible means to gain further insight on the role of the glycan chains in prion biology in cultured cells. RK13 cells were transfected using a construct with a stop codon placed at the usual cleavage site of the GPI anchor signal peptide. As expected, ⌬GPI PrP molecules displayed distinctive features compared to wild-type PrP (Fig. 8a and data not shown): (i) higher electrophoretic mobility, (ii) predominance of unglycosylated species and absence of diglycosylated ones, and (iii) lack of expression at the cell surface and release in the supernatant, with a small proportion of molecules remaining intracellular (not shown). In order to augment the concentration of the protein at the monolayer cell surface, prion-exposed cultures were maintained in agarose-containing semisolid medium, which was previously shown to have no adverse effect on prion propagation [START_REF] Paquet | Efficient dissemination of prions through preferential transmission to nearby cells[END_REF]. These cultures were kept without splitting for 2 weeks, at which time the input PrP Sc material was cleared and no longer detectable. Under these conditions, we were able to detect the conversion of the ⌬GPI PrP into a PK-resistant form that was essentially unglycosylated, with a minority species corresponding to a monoglycosylated form (Fig. 8a). These results indicated that unglycosylated PrP with a wild-type core sequence can convert into PK-resistant PrP even when glycosylated species are largely underrepresented. The amount of protein converted was markedly lower than that of wild-type PrP, and PK-resistant ⌬GPI PrP no longer accumulated after a subsequent passage of infected-cell culture overlaid or not with agarose. However, ⌬GPI-PrP Sc was clearly infectious, since it was able to transmit infection to Rov9 cells (58) expressing wild-type PrP C (Fig. 8b). Wild type

Ϫ Ϫ ϩ ϩ ϩ ϩ G37T ϩ Ϫ ϩ ϩ ϩ ϩ N184D-N200D Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ G37T-N184D-N200D ϩ Ϫ Ϫ Ϫ ϩ Ϫ T36I-G37T-N184D-N200D ϩ Ϫ Ϫ Ϫ ϩ Ϫ Y52T-N184D-N200D Ϫ ϩ Ϫ Ϫ ϩ Ϫ G37T-N184Q-N200Q ϩ Ϫ Ϫ Ϫ ϩ Ϫ G37T-N184T-N200T ϩ Ϫ Ϫ Ϫ ϩ Ϫ T186N Ϫ Ϫ Ϫ ϩ Ϫ Ϫ G37T-T186N ϩ Ϫ Ϫ ϩ ϩ Ϫ
a ϩ, Ն 55%; Ϫ, Յ10% (relative to wild-type PrP).

PrP mutations associated with atypical PrP C or PrP Sc glycoform ratios. Several mutations associated with genetic TSE diseases in humans are located within or in the close vicinity of a PrP glycosylation site. These include T183A, which was analyzed here in the ovine sequence context (T186A) (see above), and V180I and F198S, which were reported to modify the glycoform pattern of PrP C or PrP Sc [START_REF] Chasseigneaux | V180I mutation of the prion protein gene associated with atypical PrPSc glycosylation[END_REF][START_REF] Grasbon-Frodl | Loss of glycosylation associated with the T183A mutation in human prion disease[END_REF][START_REF] Zaidi | Characterization of the F198S prion protein mutation: enhanced glycosylation and defective refolding[END_REF]. We sought to examine the effect of the latter mutations on 127S prion propagation when engineered in the ovine PrP sequence. V183I ovine PrP C exhibited a normal glycoform pattern, similar to that of its human counterpart V180I PrP C . This mutation was permissive to infection (Fig. 9), but disappointingly, the PrPres generated did not show an atypical profile such as described in human brain tissue, which is characterized by the total absence of diglycosylated species [START_REF] Chasseigneaux | V180I mutation of the prion protein gene associated with atypical PrPSc glycosylation[END_REF]. The F198S mutation, as well as the recently described F198V mutation [START_REF] Zheng | PRNP mutations in a series of apparently sporadic neurodegenerative dementias in China[END_REF], targets the central amino acid of the consensus sequon (NFT). The equivalent mutations in the ovine sequence, F201S and F201V, respectively, both induced altered glycoform profiles in which unglycosylated PrP C was essentially lacking (Fig. 9), similar to that reported for the human F198S PrP mutation [START_REF] Zaidi | Characterization of the F198S prion protein mutation: enhanced glycosylation and defective refolding[END_REF]. In Rov cells, both PrP mutants were properly expressed at the plasma membrane (not shown). However, upon exposure to infectious prions, these two mutations produced opposite effects, since F201S-engineered cells were refractory to infection while F201V conferred permissiveness.

DISCUSSION

Glycosylation and PrP C trafficking. Asn-linked carbohydrate chains play an important role in the intracellular trafficking and transport to the cell surface of many glycoproteins. Immunofluorescence analysis indeed showed that the subcellular localization of the ovine PrP VRQ glycosylation mutants was altered compared to that of the wild-type protein, although to a variable extent. Despite some intracellular retention, mostly in the Golgi apparatus (which was not seen with wildtype PrP VRQ molecules), all the monoglycosylated N184X and N200X mutants generated were efficiently expressed at the cell surface. Thus, even in the absence of endogenous diglycosylated species, one glycan chain suffices to ensure proper expression of PrP at the cell surface. Two monoglycosylated mutants, however, both produced by replacing T instead of N in the consensus sequon (T186N and T186A, which is homologous to a human PrP mutation [START_REF] Grasbon-Frodl | Loss of glycosylation associated with the T183A mutation in human prion disease[END_REF]), exhibited a dramatically impaired trafficking, although they successfully passed the ER quality control given their massive accumulation in the Golgi apparatus. Similarly, the double nonglycosylated mutants were marginally expressed at the cell surface and localized mostly in the Golgi apparatus. These results were somewhat surprising since in N2A cells, the surface expression levels of T182N and of doubly mutated T182N-T198A mouse PrP were like that of wild-type PrP [START_REF] Neuendorf | Glycosylation deficiency at either one of the two glycan attachment sites of cellular prion protein preserves susceptibility to bovine spongiform encephalopathy and scrapie infections[END_REF]. The different cell type or PrP sequence context, i.e., murine versus ovine, could account for these discrepant observations. Alternatively, wild-type PrP molecules concomitantly expressed in the N2a cell system might exert some rescuing activity in trans. In one study, coexpression of wild-type and mutant prion proteins was found to alter their cellular localization and partitioning into membrane domains [START_REF] Schiff | Coexpression of wild-type and mutant prion proteins alters their cellular localization and partitioning into detergent-resistant membranes[END_REF]. In any case, it should be emphasized that the pattern of subcellular expression observed with nonglycosylated PrP mutants in the Rov cell system resembled that reported in neuronal cells in brain sections from knock-in mice that express the same mutation ( 9), thus arguing that Rov cells are a biologically relevant model for such studies.

A salient observation resulting from this study is that introduction of an Asn glycosylation site in the N-proximal, nonstructured region of the molecule (e.g., mutation G37T) conferred double PrP glycosylation mutants a wild-type-like phenotype in terms of cell surface expression efficiency and membrane subdomain distribution. This implies that attachment of an ectopic glycan chain provided a signal in cis that sufficed to satisfy the Golgi compartment quality control. N glycosylation is regarded as a key determinant in the Golgian quality control of glycoproteins, including GPI-anchored proteins [START_REF] Vagin | Inverse correlation between the extent of N-glycan branching and intercellular adhesion in epithelia. Contribution of the Na,K-ATPase beta1 subunit[END_REF]. Moreover, in Rov cells PrP is mostly expressed apically [START_REF] Paquet | Prion infection of epithelial Rov cells is a polarized event[END_REF], and N-glycans are frequently involved in the apical sorting in polarized cells [START_REF] Pang | N-glycans, not the GPI anchor, mediate the apical targeting of a naturally glycosylated, GPI-anchored protein in polarised epithelial cells[END_REF]. Notwithstanding this, was the lack of a glycan chain the sole cause for the observed strong retention of the PrP double mutants in the Golgi compartment? Three comments can be made in this regard. (i) Intriguingly, the maturation of PrP molecules with mutations not targeting the glycosylation sequon, but also leading to Golgi retention, was recently reported to be favored by a short deletion in the N-terminal sequence (2), close to the ectopic glycan attachment sites of the G37T and Y52T mutants; thus, such a compensating effect cannot be excluded here. (ii) Whether or not wild-type, naturally aglycosylated PrP molecules undergo retention in the Golgi compartment is poorly documented. Mutated PrP molecules lacking a GPI anchor, almost exclusively aglycosylated, exit from the Golgi compartment and are secreted [START_REF] Chesebro | Anchorless prion protein results in infectious amyloid disease without clinical scrapie[END_REF][START_REF] Mcnally | Cells expressing anchorless prion protein are resistant to scrapie infection[END_REF][START_REF] Walmsley | Distance of sequons to the C-terminus influences the cellular N-glycosylation of the prion protein[END_REF], but this may reflect a less stringent quality control of secreted compared to raft-associated proteins in the Golgi complex [START_REF] Fiedler | The role of N-glycans in the secretory pathway[END_REF]. Forward trafficking of PrP was reported to be unimpaired in tunicamycin-treated SH-SY5Y and CHO cells [START_REF] Lehmann | Blockade of glycosylation promotes acquisition of scrapie-like properties by the prion protein in cultured cells[END_REF][START_REF] Walmsley | Membrane topology influences N-glycosylation of the prion protein[END_REF], implying that aglycosylated PrP species can satisfy the Golgi quality control. Lastly, circumstantial data suggest that the glycoform ratio of cell surface-expressed PrP does not greatly differ from that of total PrP [START_REF] Loberto | The membrane environment of endogenous cellular prion protein in primary rat cerebellar neurons[END_REF][START_REF] Nunziante | Essential role of the prion protein N terminus in subcellular trafficking and half-life of cellular prion protein[END_REF][START_REF] Taylor | The low-density lipoprotein receptorrelated protein 1 (LRP1) mediates the endocytosis of the cellular prion protein[END_REF]. (iii) Within cells where glycan synthesis takes place normally, glycosylated molecules may provide in trans a signal that favors the sorting of nonglycosylated molecules. In support of such a "cooperative" scenario, oligomerization has been proposed to be a specific requirement for apical sorting of GPI-anchored proteins [START_REF] Paladino | Oligomerization is a specific requirement for apical sorting of glycosyl-phosphatidylinositol-anchored proteins but not for non-raft-associated apical proteins[END_REF]. Incidentally, it would also provide a simple explanation for the above-mentioned conflicting observations regarding the expression at the cell surface [START_REF] Neuendorf | Glycosylation deficiency at either one of the two glycan attachment sites of cellular prion protein preserves susceptibility to bovine spongiform encephalopathy and scrapie infections[END_REF], or lack of such expression (reference 9 and this study), of aglycosylated PrP mutants, depending on whether wild-type molecules are coexpressed or not in the cell system studied.

Glycosylation and permissiveness to prion infection. Three main findings arose from our experiments. First, all the monoglycosylated site 2 mutants (5/5) allowed de novo infection and prion propagation over several subpassages. In contrast, only one out of the nine mutations on site 1 tested in the infection assay led to sustained prion propagation. This result, while confirming the dispensability of either glycan chain, as also shown in vivo in mouse prion-infected transgenic mice [START_REF] Neuendorf | Glycosylation deficiency at either one of the two glycan attachment sites of cellular prion protein preserves susceptibility to bovine spongiform encephalopathy and scrapie infections[END_REF][START_REF] Tuzi | Host PrP glycosylation: a major factor determining the outcome of prion infection[END_REF], underscores the possibility that any effect observed on prion biology could involve the amino acid substitution itself rather than simply the loss of the glycan chain. Indeed, the greater versatility of site 2 is likely to hold to the attachment of the glycan chain within the helix 2-helix 3 loop, whereas site 1 involves a more constrained domain of the PrP core, helix 2. Of note, two of the site 1 mutants (N184T and T186N) that were found to confer a decreased to transgenic mice, in a strain-dependent manner [START_REF] Neuendorf | Glycosylation deficiency at either one of the two glycan attachment sites of cellular prion protein preserves susceptibility to bovine spongiform encephalopathy and scrapie infections[END_REF][START_REF] Tuzi | Host PrP glycosylation: a major factor determining the outcome of prion infection[END_REF], failed to form PrP Sc in the Rov cell system. Reciprocally, it would be relevant to investigate the effects of the N184D mutation in transgenic mice, because this substitution, which to our knowledge has rarely if ever been used to disrupt an Asn glycosylation sequon in a glycoprotein, was the only one that conferred permissiveness for de novo infection to a site 1 mutant in the present study. A drastic effect of the amino acid change was also observed with two mutations targeting the central residue of the consensus sequon: F201S and F201V mutants showed a similarly altered PrP C glycoform pattern with a marked decrease of unglycosylated species, without alteration of the cell surface expression; however, F201V supported prion infection, whereas F201S did not.

Second, mutations that led to undetectable or hardly detectable expression at the cell surface were invariably associated (15 out of 15 mutants) with failure of the culture to propagate prions. This was notably the case for the monoglycosylated T186X mutants and for all the double mutants, even when the most favorable substitution (N to D) in terms of infectibility was used at both sequons. These results further strengthen the view that prior access of the PrP molecules to the cell surface is mandatory so that the conversion into PrP Sc can take place [START_REF] Borchelt | Evidence for synthesis of scrapie prion proteins in the endocytic pathway[END_REF][START_REF] Caughey | The scrapie-associated form of PrP is made from a cell surface precursor that is both protease-and phospholipase-sensitive[END_REF][START_REF] Enari | Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody[END_REF][START_REF] Gilch | Intracellular re-routing of prion protein prevents propagation of PrP(Sc) and delays onset of prion disease[END_REF].

Third, stable and high-level cell surface expression of the glycosylation PrP mutant turned out not to be sufficient to allow prion propagation. As a matter of the fact, cell surface expression of six of the site 1 mutants found to be fully resistant to conversion (see above) did not greatly differ from that of wild-type molecule. In contrast, five of these same mutations were found to be permissive for transient conversion by mutation scanning in ScN2a [START_REF] Ikeda | Thr but not Asn of the N-glycosylation sites of PrP is indispensable for its misfolding[END_REF]. This suggests that coexpression with wild-type PrP and/or use of preinfected cell cultures is an experimental condition leading to an underestimation of the potential antagonistic effect of a mutation on prion propagation. Also supporting the contention that high-level surface expression was not sufficient, the double mutant NDND, the trafficking of which was restored by an ectopic, truly functional Asn glycosylation site (G37T; see above), did not allow prion multiplication either, whereas PrP molecules bearing both this extra site and the natural glycan attachment sites behaved like wild-type PrP in terms of efficiency and sustainability of the infection.

Strikingly enough, we failed at detecting even transient PrP Sc accumulation in any of the nonpermissive, cell surfaceexpressed mutants, including G37T-NDND, indicating that acute conversion did not take place. This was not unexpected, as there is ample evidence that single-amino-acid differences in the PrP sequence may dramatically affect prion formation for a given strain both in vitro and in vivo [START_REF] Manson | A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy[END_REF][START_REF] Priola | Heterologous PrP molecules interfere with accumulation of protease-resistant PrP in scrapie-infected murine neuroblastoma cells[END_REF]. In contrast, acute conversion (not sustained propagation) was repeatedly observed in Rov cultures expressing PrP lacking a GPI anchor, mostly consisting of aglycosylated species, corroborating a recent study on cells that expressed mouse anchorless PrP [START_REF] Mcnally | Cells expressing anchorless prion protein are resistant to scrapie infection[END_REF]. Various lines of evidence in vivo and in vitro tend to suggest that a glycosylation defect does not necessarily impair, or could even ease, PrP conversion [START_REF] Bradford | Dramatic reduction of PrP C level and glycosylation in peripheral nerves following PrP knock-out from Schwann cells does not prevent transmissible spongiform encephalopathy neuroinvasion[END_REF][START_REF] Chesebro | Anchorless prion protein results in infectious amyloid disease without clinical scrapie[END_REF][START_REF] Korth | Expression of unglycosylated mutated prion protein facilitates PrP(Sc) formation in neuroblastoma cells infected with different prion strains[END_REF][START_REF] Nishina | The stoichiometry of host PrPC glycoforms modulates the efficiency of PrPSc formation in vitro[END_REF][START_REF] Priola | Glycosylation influences cross-species formation of protease-resistant prion protein[END_REF][START_REF] Taraboulos | Acquisition of protease resistance by prion proteins in scrapie-infected cells does not require asparagine-linked glycosylation[END_REF][START_REF] Winklhofer | Inhibition of complex glycosylation increases the formation of PrPsc[END_REF]. Taken together, these observations lend support to the view that the inability of aglycosylated mutants that satisfied various cell sorting quality controls to convert into PrP Sc might involve conformational dynamics of the mutant molecules, in turn affecting the efficiency of conversion process.

In conclusion, the results of this study with the Rov cell culture model established that not only the subcellular trafficking of mutant molecules but also the ability of properly trafficked molecules to form PrP Sc can be variably altered, depending on the amino acid change introduced. The possibility is therefore real, not just theoretical, that any effect observed in vivo on the propagation of the agent in mice expressing PrP with a mutated glycosylation site(s) could actually be unrelated to the glycan chain deficiency per se. This issue deserves greater consideration, all the more because for obvious technical reasons only a few such mutations have been studied by mouse transgenesis. Much deeper investigations will probably be needed to understand the exact role of the Asn-linked carbohydrate moiety of PrP in prion biology.

FIG. 1 .

 1 FIG. 1. Expression of PrP glycosylation mutants in Rov cell cultures. Immunoblotting analysis with 4F2 antibody of Rov cells that expressed PrP mutants with asparagine substitutions at codon 184 or 200 (sheep sequence numbering) or at both codons (double mutants) is shown. The positions of the diglycosylated, monoglycosylated, and unglycosylated forms of PrP are indicated on the left. While diglycosylated species predominate with wild-type (WT) PrP, monoglycosylated species become the major component with single mutants. Double mutants show only one band corresponding to unglycosylated PrP. Molecular weights (in thousands) are indicated on the right. FIG. 2. Subcellular localization of PrP glycosylation mutants. (a) Immunofluorescence staining of Triton X-100-permeabilized and nonpermeabilized cells using 4F2 antibody. Cells expressing monoglycosylated PrP mutants displayed a typical cell surface labeling together with a regionalized intracellular signal, whereas in cells expressing double PrP mutants the labeling was essentially intracellular. (b) Double immunofluorescence staining of PrP (in green) and of a marker of the Golgi apparatus (giantin, in red). Bars, 10 m.

FIG. 3 .

 3 FIG. 3. Comparative cell surface expression of PrP glycosylation mutants as determined by flow cytometry. The expression levels of the mutants are provided as percentage relative to wild-type PrP (means and standard deviations from three experiments; 4F2 antibody). NT, nontransfected RK13 cells. G37T-NDND designates a double mutant with an extra glycosylation site, which is described in the text.

FIG. 5 .

 5 FIG. 5. Prion infection of Rov cells expressing PrP glycosylation mutants. Cultures exposed to an infectious inoculum of the 127S ovine prion strain were tested for the presence of PK-resistant PrP by immunoblotting using Sha31 antibody. Results of a typical experiment are shown, and the number of passages postexposure is indicated. The gels were loaded with 250 g or 25 g of PK-digested proteins per lane for the mutant or wild-type PrP-containing samples, respectively.

FIG. 7 .

 7 FIG. 7. Expression and permissiveness to prion infection of PrP mutants with an extra glycosylation site. (a) Immunoblot showing the PrP C glycoform profiles of mutants bearing either three glycosylation sites (G37T) or the extra glycosylation site only (G37T-NDND). The artificial N-linked glycosylation sequon created by replacement of a glycine by a threonine at position 37 was truly functional, as evidenced by the modification of the glycoform profiles of G37T-NDND and of G37T mutants compared to NDND and wild-type PrP, respectively. (b) Expression of G37T-NDND PrP at the cell surface. Immunofluorescence on living cells using 4F2 antibody is shown. G37T and G37T-NDND PrPs show similar cell surface labeling, while the NDND mutant is essentially negative. Bars, 20 M. (c) G37T-NDND mutant and wild-type PrPs are both associated with lipid rafts. An immunoblot analysis performed on step sucrose gradient fractions using anti-PrP and antiflotillin antibodies is shown. Fractions 3 and 4 correspond to the interface of 5% and 30% sucrose cushions. (d) Permissiveness to de novo prion infection of the G37T PrP mutants. An immunoblot with PK-treated cell lysates from cultures at passage 3 postexposure is shown (Sha31 antibody). No PrPres signal is visible in G37T-NDND mutant-expressing cells, although G37T mutant and wild-type PrPexpressing cells accumulate PrPres at similar levels.

FIG. 9 .

 9 FIG. 9. Permissiveness to prion infection of PrP mutants with an altered glycoform pattern. Human PrP mutations reported to alter the PrP C glycosylation pattern were introduced into the sheep sequence, as indicated (ovine PrP numbering). Immunoblot analysis for PrP C (a) (mockinfected culture, 4F2 antibody) and PrPres (b) (prion-infected cultures at third passage postexposure, Sha31 antibody) is shown. Note that the F210V mutant but not the F210S mutant conferred susceptibility to infection.

TABLE 1 .

 1 Characteristics of the PrP glycosylation mutants

	Glycotype	Genotype	Cell surface expression a	Conversion
	Wild type (VRQ)	Wild type	ϩ	ϩ
	Monoglycosylated site 1	N184D
	mutants

TABLE 2 .

 2 Effect of ectopic glycosylation on PrP cell surface expression and conversion

	Genotype	N35	Glycan attachment sites N50 N184	N200	Cell surface expression a	Conversion

on July 30, 2012 by INRA-MME CORNIC http://jvi.asm.org/ Downloaded from

J. VIROL. on July 30, 2012 by INRA-MME CORNIC http://jvi.asm.org/ Downloaded from

on July 30, 2012 by INRA-MME CORNIC http://jvi.asm.org/ Downloaded from

SALAMAT ET AL. J. VIROL. on July 30, 2012 by INRA-MME CORNIC http://jvi.asm.org/ Downloaded from

ACKNOWLEDGMENTS

We thank Myriam Ermonval (Institut Pasteur, Paris, France) for helpful discussions and Jacques Grassi (C. E. A., Saclay, France) for antibodies. Many thanks go to Ce ´line Urien and to Florian Marquet (Virologie et Immunologie Mole ´culaires, INRA-Jouy) for skilled help with confocal microscopy and FACS analysis, respectively. We acknowledge Vincent Be ´ringue and Rachel Young (INRA-Jouy) for revising the manuscript. M. K. Salamat is a scholar of the Higher Education Commission of Pakistan and is supported by a complementary fellowship from INRA. This work was supported by European Commission FP6 "Neuroprion" Network of Excellence.