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Bovine Viral Diarrhoea Virus is Internalized by Clathrin-dependent Receptor-mediated Endocytosis

Bovine Viral Diarrhoea Virus is Internalized by Clathrin-dependent Receptor-mediated Endocytosis
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  Department of Infectious Diseases, Institute for Virology, University of Veterinary Medicine Hannover, Hannover, Germany Bovine Viral Diarrhoea Virus is Internalized by Clathrin-dependentReceptor-mediated Endocytosis B. Grummer 1,2 , S.  Grotha 1 and I.  Greiser-Wilke 1 Addresses of authors:  1 Department of Infectious Diseases, Institute for Virology, University of Veterinary Medicine Hannover,Foundation Buenteweg 17, 30559 Hannover, Germany;  2 Corresponding author: Tel.: (+49) 511 953 8845; fax: (+49) 511 9538898; E-mail: beatrice.grummer@tiho-hannover.deWith 2 figures and 1 table Received for publication July 27, 2004 Summary Bovine viral diarrhoea virus (BVDV) is a pestivirus within thefamily Flaviviridae. In contrast to the members of the genusflavivirus, nothing is known about the viral entry route forpestiviruses. In this study, the process of BVDV infectionfollowing attachment to the cell surface was examined. BVDVclearly co-localizes with clathrin, with early endosome antigen-1 (EEA-1), an early endosome marker, and also with lyso-somal-associated membrane protein-2 (LAMP-2), a lysosomalmarker. BVDV internalization is inhibited by compounds thatblock clathrin- but not caveolae-dependent endocytosis. Thesefindings demonstrate that BVDV enters the cells via theclathrin-coated pit pathway. Introduction Bovine viral diarrhoea virus (BVDV) belongs to the familyFlaviviridae, genus pestivirus (Wengler et al., 1995) and is asmall, enveloped, positive-stranded RNA virus. It is serolog-ically closely related to the other members of the genuspestivirus, classical swine fever virus and border disease virusof sheep. The genome is approximately 12.5 kb long andcontains one large open reading frame which is translated intoa polyprotein of about 4000 amino acids. The BVDV envelopehas a complex structure comprising three viral glycoproteins.The envelope glycoproteins Erns and E2 form disulphide-linked homodimers, whereas E1 and E2 are additionallypresent as heterodimers (Weiland et al., 1990; Ruemenapf et al., 1993). The function of the different envelope proteins isnot well understood. Erns exhibits a ribonuclease activitywhich is unique for pestiviruses. In addition, antibodies whichreduce the enzymatic activity of Erns also inhibit infectivity of the virus. This implies an essential role of Erns in the viral lifecycle (Schneider et al., 1993; Windisch et al., 1996). E2 is themain target molecule for neutralizing antibodies (Bolin et al.,1988; Donis et al., 1988) and is probably involved in the initialbinding of virus particles to the surface of permissive cells(Donis et al., 1988; Xue and Minocha, 1993). Entry of BVDVis believed to occur by receptor-mediated endocytosis. Thecellular proteins responsible for attachment and penetration of BVDV are not unequivocally characterized. Xue and Minocha(1993) identified a 50-kDa protein which is probably involvedin binding of E2. However, this protein seems to be present onboth permissive and non-permissive cell types (Xue andMinocha, 1996; Xue et al., 1997). In another study, it wasshown that various monoclonal antibodies (MAbs) thatrecognize cell surface proteins on bovine cells efficiently blockinfection with BVDV (Schelp et al., 1995). With one of theseMAbs, a 50–58-kDa membrane protein was identified. Furtheranalysis provided strong evidence that this protein is thebovine CD46 which probably serves as a cellular receptor forBVDV (Maurer et al., 2004). CD46 or membrane cofactorprotein (MCP) is a 50–60-kDa glycoprotein, expressed on awide variety of cells and is a complement regulatory factor.Interestingly, there are inconsistent informations about its rolein receptor-mediated endocytosis. Maisner et al. (1997)showed that CD46 is not endocytosed. In contrast, Crimeen-Irwin et al. (2003) demonstrated that it depends on the ligandif CD46 is constitutively internalized via clathrin-coated pits,traffics to multivesicular bodies, and is recycled to the cellsurface or not.The aim of our study was to clarify the route of internal-ization of BVDV and to figure out if BVDV enters cells viaclathrin-coated pits or the clathrin-independent caveolaesystem which produces uncoated vesicles. Material and Methods Cell cultures and viruses Foetal bovine kidney (FBK) cells were seeded in 96-well platesin a concentration of 4  ·  10 5 cells/ml medium. The cells weregrown in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 5% bovine serum tested for the absenceof BVDV and antibodies directed against BVDV. All cells weremaintained at 37  C in a 5% CO 2  incubator. The cells wereinfected with a multiplicity of infection (moi) of 1 with thecytopathogenic (cp) BVDV strain NADL or with the non-cytopathogenic (ncp) BVDV strain 7443, as follows: virus wasallowed to adsorb to the cells for 60 min at 4  C. Subsequently,the cells were washed with ice-cold phosphate-buffered saline(PBS) to remove unbound particles. Then, infection wasinitiated by shifting the cells to 37  C with warm medium. As anegative control, mock-infected cells were included. Co-localization with markers of endocytic uptake To study the internalization pathway followed by cp or ncpBVDV, its possible co-localization with established markers of endocytic pathways was examined under a fluorescencemicroscope with 450–490 and 550–570 nm band-pass filters.The markers studied included the following: clathrin (goatanti-bovine polyclonal antibody; working dilution 1:20) U. S. Copyright Clearance Center Code Statement: 0931–1793/2004/5110–0427$15.00/0 J. Vet. Med. B  51,  427–432 (2004)   2004 Blackwell Verlag, BerlinISSN 0931–1793  (abcam; Cambridge, UK), early endosome antigen-1(EEA-1) [fluorescein isothiocyanate (FITC)-conjugated anti-human monoclonal antibody; working dilution 1:200] andlysosomal-associated membrane protein-2 (LAMP-2)(FITC-conjugated anti-human monoclonal antibody; work-ing dilution 1:10) (both BD Bioscience, Heidelberg,Germany).For all antibodies, control images were evaluated to ensurenon-overlapping binding of secondary antibodies and specificdetection for each excitation channel. Cells were allowed tointernalize BVDV for defined time periods (for 1, 2, 3, 4, 5, or6 h after shifting the cells from 4 to 37  C; herein after referredto as: h.p.i.). They were then fixed with ice-cold methanol/acetone (1:1) for 1 min and stained with the various antibodiesdirected against the marker proteins and the BVDV/CA3antibody against the viral envelope protein E2 (Bolin et al.,1988), or BVDV/C42 antibody against the viral envelopeprotein Erns (Cay et al., 1989). In case of staining with theclathrin antibody, a second anti-species antibody conjugatedwith FITC was used (mouse anti-goat) (DAKO Cytomation,Hamburg, Germany). To visualize the viral antigen, a secondanti-species antibody conjugated with tetramethylrhodamineisothiocyanate (TRITC)2 was used (goat anti-mouse). Inhibition of BVDV infection ToelucidatetheBVDVinternalizationpathway,theinfluenceof a variety of chemical compounds known to inhibit distinctcellular processes was analysed. These compounds were bafilo-mycin A, chlorpromazine, filipin III, and nystatin. Filipin IIIwas purchased from Sigma-Aldrich (Munich, Germany).Chlorpromazine, bafilomycin A, and nystatin were obtainedfrom CN Calbiochem Novabiochem (Bad Soden, Germany).Filipin III was used in an end concentration of 400 n m ,bafilomycin A was applied in an end concentration of 100 n m (stocks prepared in3 dimethylsulfoxide, DMSO). Nystatin wasapplied in a 27- l m  end concentration (stocks prepared in PBS)and chlorpromazine was used in a 25- l m  end concentration(stocks prepared in4 ddH 2 O). Cell viability assay Dulbecco’s modified Eagle’s medium containing 25  l m  chlor-promazine, 100 n m  bafilomycin A, 400 n m  filipin III, or 27  l m nystatin was added to the cell cultures 24 h after seeding. As acontrol, cells were incubated with DMEM only. After 12 and24 h of incubation, the cells were stained with 0.1% trypanblue for monitoring viable and dead cells under a phase-contrast microscope (Phillips, 1973). Influence of the inhibitors on virus entry and replication Foetal bovine kidney cells were infected with 7443 or NADL(moi: 1) at 4  C. After 1 h, the cells were washed with ice-coldPBS to remove non-attached virions.In a preliminary experiment, the different inhibitors wereadded to the infected cells with fresh medium directly for 24 h.As a control, medium without inhibitors was added to infectedand non-infected cells.The cells were then heat-fixed at 80  C for 4 h. After fixation,the cells were incubated with a polyclonal pig anti-BVDVserum conjugated with horse radish peroxidase for 1 h in ahumidified chamber. Viral antigen was detected by peroxidase-linked antibody assay using 3 ¢ -amino-9 ¢ -ethyl-carbazole (AEC)and H 2 O 2  as a substrate for the peroxidase.To determine when each of the inhibitory drugs exerted itsinfluence on infection, cells were infected as before and thecompounds were added for 1, 2, 3, 4, 5, or 6 h. After eachdefined period, the inhibitor-containing medium was replacedby fresh medium. Overall, the cells were incubated for 24 h toallow viral replication. As a control, medium without inhibitorwas used. Virus titration Virus titration was performed by standard methods in micro-titre plates as described previously (Grummer et al., 2002). Allexperiments were performed twice in duplicates. Results Co-localization with markers of endocytic uptake To investigate whether BVDV enters the host cell via theclathrin endocytic pathway, we used immunofluorescencelabelling with MAbs against the virus envelope proteins Ernsand E2 (Fig. 1b,e,h) and markers for the entry pathways: anantibody directed against bovine clathrin (Fig. 1a) and anti-bodies directed against human EEA-1 and human LAMP-2which both cross-reacted specifically with bovine cells(Fig. 2d,g).Double labelling with anti-BVDV-E2 and anti-clathrinantibodies at 1 and 2 h.p.i. showed co-localization, suggestingthat the virus enters via clathrin-coated pits (Fig. 1c). At 2 and3 h.p.i. a co-localization with the EEA-1 was shown. At3 h.p.i. no co-localization was seen with clathrin, indicatingthat the virus has already been taken up into the earlyendosomes (Fig. 1e). BVDV E2 clearly co-localized withLAMP-2, a marker for lysosomes at 4 h.p.i. (Fig. 1i). Stainingwith the antibody BVD/C42 against BVDV Erns revealed thesame results for clathrin as well as for EEA-1 co-localization.In contrast, we were not able to show co-localization of BVDVErns with LAMP-2 at any time point (data not shown). Inhibition of BVDV infection To support the co-localization results, we examined thesensitivity of BVDV infection to a variety of compoundsknown to inhibit distinct cellular processes. To identifysuccessful virus entry and replication, viral antigen wasdetected via peroxidase linked antibody (PLA)5 assay and thecell culture supernatant was titrated. Before, the influence of bafilomycin A, chlorpromazine, filipin, and nystatin on the cellviability was tested to exclude cytotoxic side-effects.Twelve and 24 h after incubation with the chosen concen-tration for each of the four substances, cell viability did notseem to be affected. As in the control, only few cells (about5–10%) were positive for trypan blue staining, indicating thatthe concentrations used were not toxic for the cells.Our experiments revealed that chlorpromazine and bafilo-mycin A were able to prevent infection. Neither nystatin norfilipin influenced BVDV infection and replication as demon-strated by antigen detection and virus quantification (Fig. 2;Table 1). 428 B. Grummer  et al.  To determine when each of the inhibitory drugs exerted itsinfluence on infection, FBK cell cultures were infected withBVDV in the presence of the drug for 1–6 h. Added directly orduring the first 3 h after infection to the cells, chlorpromazineand bafilomycin A inhibited the infection nearly completely(Fig. 2c,e). When added later than 3 h.p.i., the inhibitory effectof both compounds failed (Fig. 2d,f). In contrast, at any timepoint, neither filipin III nor nystatin were able to preventinfection (Fig. 2g–k). Discussion The early events of viral infection include virion attachment tocellular receptors, penetration, and uncoating followed by viralgene transcription. For BVDV, this early phase of infection isnot fully elucidated. Several cell surface molecules have beenproposed as receptor candidates for BVDV, e.g. the low-density lipoprotein (LDL) receptor (Agnello et al., 1999) andthe bovCD46 (Maurer et al., 2004). It is likely that BVDVrequires a set of co-receptors for efficient cell entry as describedfor the related hepatitis C virus (Bartosch et al., 2003). Incontrast, BVDV attachment proteins are unequivocally iden-tified. The viral envelope protein E2 is needed for successfulcell entry (Donis et al., 1988; Xue and Minocha, 1993) andErns seems to be attached to the virus envelope via a directinteraction with E2 (Lazar et al., 2003). Previous studiessuggested that both envelope proteins interact with differentcell surface receptors (Hulst and Moormann, 1997).To analyse the subsequent steps of BVDV entry, weperformed several experiments that concentrate on the routeof internalization. In general, it is believed that BVDV entersthe cell via receptor-mediated endocytosis and we were able toshow, by subcellular localization, that BVDV co-localized withmarkers of the clathrin-dependent pathway.Coated pits contain two major structural proteins, clathrinand the adapter protein AP-2 (Pearse, 1989). Clathrin is a180-kDa protein that coats the cytoplasmic face of coated pitsinvolved in receptor-mediated endocytosis of a variety of ligands. The observed co-localization of BVDV Erns and E2with clathrin in early events of infection strongly implicates theclathrin-dependent pathway in viral penetration. This conclu-sion was further supported by co-localization of BVDV Ernsand E2 with the EEA-1, which is a 180-kDa hydrophilicperipheral membrane protein. Immunofluorescence studiesshowed that EEA-1 co-localizes to early endosomes withtransferrin receptor and Rab5, but not with the late endosome- Fig. 1. Co-localization8 of BVDV with endocytic markers. Ncp BVDV strain 7443 was internalized in FBK cells for 1 (a, b, and c), 2 (d, e, and f),or 4 h (g, h, and i). Cells were fixed and double-stained for endocytic markers and BVDV. Staining against BVDV E2 is shown in (b), (e), and (h).Monoclonal antibody BVD/CA3 was detected with goat anti-mouse IgG-TRITC. Anti-clathrin staining, shown in (a), was detected with goatanti-mouse IgG-FITC. The merge of the two pictures is shown in (c). Localization of the early endosome antigen (EEA)-1 is shown in D. Themerged image is in (f). Lysosomal-associated membrane protein (LAMP)-2 is detected in (g). The merged image is in (i). For all antibodies,control images were evaluated to ensure nonoverlapping binding of secondary antibodies and specific detection for each excitation channel.(FITC filter: 450–490 nm; rhodamine filter: 550–570 nm). Analogous results were obtained for cp BVDV strain NADL (data not shown). BVDV Uptake by Receptor-mediated Endocytosis1 429  localizing Rab7 (Mu et al., 1995). Although some viruses mayescape from, or undergo uncoating in early endosomes (Greberet al., 1993; Bartlett et al., 2000), many continue to bepassaged to late endosomes and then to lysosomes (Zeichhardtet al., 1985). Using LAMP-2 as a marker for late endosomesand lysosomes (Chen et al., 1985), we found that it is co-localized with BVDV E2. Interestingly, we were not able todetect BVDV Erns in the same way. The negative reactivity tothe MAb BVD/C42 in the lysosomal compartment suggeststhat the Erns has undergone some conformational changes inthe meantime. This is in concert with our former findings thatMAb BVD/C42 does not react with the Erns protein inWestern blot under reducing conditions. In contrast, it couldbe demonstrated previously that E2 dimers are resistant toreducing agents and degradation and possess a pH stability(Branza-Nichita et al., 2002) which supports our findings.Biochemically, BVDV infection of susceptible cells wasinhibited by two drugs that interfere with the clathrin-dependent pathway but not by drugs that inhibit the caveo-lae-dependent pathway. We used the drugs chlorpromazineand bafilomycin A to inhibit clathrin-dependent endocytosisand filipin and nystatin to inhibit caveolae-dependent endocy-tosis. Chlorpromazine is a cationic amphiphilic drug whichspecifically blocks clathrin-mediated endocytosis by causing aredistribution of the adaptor protein AP-2 away from clathrin-containing pits (Wang et al., 1993; Subtil et al., 1994). Bafilo-mycin A has been shown to affect early endosomal trafficking(Clague et al., 1994; Bayer et al., 1998), in addition to its well-established inhibition of vacuolar ATPases (Bowman et al.,1988; Yoshimori et al., 1991). The antibiotic nystatin is aknown sterol-binding agent and acts to remove membranecholesterol, which is important for both the maintenance of caveolae and for the ability of caveolae to seal off from theplasma membrane (Chen and Norkin, 1999; Okamoto et al.,2000). Filipin is an invasive sterol-binding agent, which insertsinto the membrane. It is known to bind cholesterol in theplasma membrane and impair the invagination of caveolae,thereby inhibiting caveolae internalization (Rothberg et al.,1992; Schnitzer et al., 1994). Unambiguously, chlorpromazineand bafilomycin A inhibited BVDV infection during the first3 h of infection, whereas, at later time points, no inhibitingeffect could be observed. Neither filipin III nor nystatin wereable to prevent infection at any time point.The ability of chlorpromazine to inhibit early infectionsuggests that early events in BVDV infection are mediated bya clathrin-dependent process. The inhibition with bafilomycinA implies that BVDV uncoating depends on exposure to lowpH.Our data demonstrate, for the first time, that entry of BVDVleading to virus replication proceeds by clathrin-dependentendocytosis and probably requires low endosomal pH for viraluncoating. However, there are many open questions, especiallyhow the uncoating process takes place in detail. There are hintsthat BVDV E2 could play a pivotal role as proteomicscomputational analyses suggest that it is a truncated class IIfusion protein (Garry and Dash, 2003). Table 1. Inhibition of BVDV infection at different time points and the influence on the virus yield (TCID 50 ) after 24 h.p.i.Cell control Virus control Chlorpromazine Bafilomycin A Fillipin III NystatinNegative 10 4.5 Inhibitor added 1 h.p.i. Negative Negative 10 4.5 10 4.5 Inhibitor added 5 h.p.i. 10 4 10 4.0 10 4.5 10 4.5 These results represent mean values from two assays performed separately (±3 standard variation). a bc de fg hj k  Fig. 2. Inhibition of BVDV infection. FBK cells were infected withncp BVDV strain 7443, incubated with the different compounds, andstained for viral antigen by peroxidase-linked antibody assay. (c, e,g, j): added 1 h.p.i. and (d, f, h, k): added 5 h.p.i. Overall, the cells wereincubated for 24 h to allow viral replication; (a): virus control withoutinhibitors; (b): cell control; (c and d): incubation with chlorpromazine;(e and f): incubation with bafilomycin (a); (g and h): incubation withnystatin; (j and k): incubation with filipin III. 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