human inﬂuenza a viruses are proteolytically activated and ... · endogenous caco-2 enzyme is a...

Human influenza A viruses are proteolytically activated and do notinduce apoptosis in CACO-2 cells

Oleg Zhirnova,* and Hans-Dieter Klenkb

a D.I. Ivanovsky Institute of Virology, Gamaleya 16, Moscow 123098, Russiab Institute of Virology, Philipps University, Marburg 35037, Germany

Received 2 December 2002; returned to author for revision 13 January 2003; accepted 5 March 2003

Abstract

Replication of human influenza A/H3N2 and A/H1N1 viruses was studied in human CACO-2 cells, a continuous line of intestinalepithelial differentiated cells. Hemagglutinin (HA) was cleaved in these cells by an endogenous protease. Thus, infectious virus wasproduced that underwent multiple cycle replication and plaque formation in the absence of trypsin added to the media. Cleavage of denovo-synthesized HA occurred at a late stage of the exocytic pathway as indicated by pulse-chase labeling and by experiments employingendoglycosidase H and brefeldin A treatment. However, surface-labeling experiments employing biotinylation suggested that there is nocleavage at the plasma membrane. Unlike HA of serotypes H5 and H7 cleaved at multibasic cleavage sites by furin, the HAs with monobasiccleavage sites analyzed here were not cleaved in CACO-2 cells in the presence of aprotinin, a natural inhibitor of trypsinlike proteases.Growing CACO-2 cells were able to cleave HA of incoming virus, although influenza virus activating protease was not detected in culturemedium. These observations indicate that the activating enzyme of CACO-2 cells is a trypsinlike protease functioning in the trans-Golginetwork and presumably endosomes. In support of this concept immune staining with antibodies specific to human and bovine trypsinrevealed the presence of a trypsinlike protease in CACO-2 cells. Unlike MDCK and CV-1 cells undergoing rapid apoptosis after influenzavirus infection, CACO-2 cells showed no apoptosis but displayed cytopathic effects with necrotic signs significantly later after infection.It follows from these data that, depending on the cell type, influenza virus may kill cells either by apoptosis or by necrosis.© 2003 Elsevier Science (USA). All rights reserved.

Introduction

Hemagglutinin (HA), the major envelope glycoprotein ofinfluenza A virus, mediates attachment to cell receptors andvirus entry into target cells. HA is composed of two sub-units, HA1 and HA2, that are generated from the precursorHA0 by proteolytic cleavage. Cleavage that is exerted byhost proteases activates virus infectivity and has beenshown to be an important determinant of influenza viruspathogenicity (Klenk and Garten, 1994; Steinhauer, 1999).

The prime characteristic of HA that determines its sen-sitivity to host proteases (influenza virus activating pro-teases, IAP) is the cleavage site, which consists of either asingle arginine (monobasic site) or an R-X-K/R-R motif(multibasic site) (Klenk and Garten, 1994). The cleavage

site is exposed in a peptide loop at the surface of the HAspike (Chen et al., 1998), and its accessibility to the cleav-age enzyme may be modulated by an adjacent carbohydrateside chain (Kawaoka et al., 1984; Ohuchi et al., 1989). Themultibasic site is found only with some H5 and H7 strains,whereas all other influenza A viruses as well as influenza Band C viruses contain HAs with monobasic sites (Klenk andGarten, 1994). As a rule, influenza viruses having multiba-sic sites are highly virulent and induce systemic infection intheir avian hosts, whereas viruses with monobasic sites areless virulent and provoke local inflammation in the respira-tory tract (Klenk and Garten, 1994; Steinhauer, 1999).

HAs with multibasic cleavage sites are activated in thetrans-Golgi network by furin or related proproteinconverta-ses (Stieneke-Grober et al., 1992; Morsy et al., 1994). HAscontaining monobasic cleavage sites are activated by trypsinor trypsinlike proteases (Klenk et al., 1975; Lazarowitz andChoppin, 1975), such as plasmin (Lazarowitz et al., 1973);

* Corresponding author. Fax �007-095-190-3058.E-mail address: [email protected] (O. Zhirnov).

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kallikrein; urokinase; thrombin (Scheiblauer et al., 1992);blood clotting factor Xa (Gotoh et al., 1990); acrosin (Gar-ten et al., 1981); tryptase Clara (Kido et al., 1992, 1999);miniplasmin from rat lungs (Murakami et al., 2001); pro-teases from human respiratory lavage (Barbey-Morel et al.,1987); and proteases secreted from bacteria, such as Staphy-lococcus aureus (Tashiro et al., 1987) and Pseudomonasaeruginosa (Callan et al., 1997). Cleavage by these pro-teases is thought to occur either at the plasma membrane orin extracellular fluids. Furthermore, evidence has been ob-tained that the hemagglutinin of the A/WSN/33 (H1N1)strain is cleaved in MDBK cells after virus internalizationby an unknown, presumably endosomal, protease (Boycottet al., 1994).

A fair amount of information is available on the effectsof protease inhibitors on HA activation. Peptidyl-chloro-methylketones mimicking furin-specific cleavage sites, suchas decREKR-CMK, have been shown to suppress HA cleav-age by furin in cultured cells (Garten et al., 1989; Stieneke-Grober et al., 1992). Several inhibitors of trypsinlike pro-teases, such as �-aminocaproic acid, an inhibitor ofplasminogen activation (Goto and Kawaoka, 1998; Zhirnovet al., 1982b); aprotinin (Zhirnov et al. 1984); leupeptin(Tashiro et al., 1987); pulmonary surfactant (Kido et al.1993); and human mucus protease inhibitor (Beppu et al.,1997), were found to possess antiviral potential by interfer-ing with cleavage at monobasic sites in cultured cells sup-plemented with blood serum, in chicken embryos, and inlungs of infected mice.

Influenza infection triggers apoptosis in infected cells(Hinshaw et al., 1994; Takizawa et al., 1993). There isevidence that influenza apoptosis is a multifactorial process,involving the FAS-dependent caspase 8 pathway (Balachan-dran et al., 2000), the PKR/NF-kappaB/INF cascade (Zhir-nov et al., 2002a), transcription factor c-Jun/AP-1, and cy-tokine TGF-� (Lin et al., 2001). Several viral proteins havebeen shown to be involved, too. The nonstructural proteinNS1 down-regulates apoptosis in infected cells in an IFN-dependent manner (Zhirnov et al., 2002a). The ion channelprotein M2 and the nucleocapsid protein NP were observedto be specifically cleaved during apoptosis by host cellcaspases (Zhirnov et al., 1999, 2002a). It was also suggestedthat proteins NA (Morris et al., 1999) and M1 (Zhirnov etal., 2002b) may participate in regulation of apoptosis. Re-cently, the interesting observation has been made that in anepithelial cell line derived from porcine lung (SJPL) (Seo etal., 2001) and some human bronchiolar cell lines (Arndt etal., 2002) influenza virus infection does not lead to apopto-sis but probably to necrosis. These data suggest that deathmechanisms in cells infected with influenza virus depend onthe host. However, host and viral factors that determinedeath mechanisms remain so far unknown.

We report here that CACO-2 cells that develop in cultureinto differentiated enterocytes resembling a natural intesti-nal epithelium (Pinto et al., 1983; Jumarie and Malo, 1991)

are an efficient host system for productive growth of humaninfluenza A viruses. These cells are interesting in two re-spects. First, they are able to cleave and, thus, to maintainmulticycle replication of human influenza A viruses. Cleav-age of HA was sensitive to aprotinin, indicating that theendogenous CACO-2 enzyme is a trypsinlike serine pro-tease. Second, infected CACO-2 cells showed no apoptosis,and necrotic markers were dominant in CACO-2 cells at latestages of infection. These data show that cell killing byinfluenza virus varies in different cell types and may de-velop through apoptosis or necrosis.

Results

Proteolytic activation of hemagglutinin with monobasiccleavage site in CACO-2 cells

First we compared multicycle replication of human in-fluenza A/Aichi/68 and A/PR/8/34 viruses in CACO-2 andMDCK cells. The results with Aichi virus are shown in Fig.1A and B, and similar data were obtained with PR8 virus(not shown). Multicycle replication and accumulation ofsignificant amounts of virus in culture fluid were found todevelop in CACO-2 cells, whereas virus replication inMDCK cells was observed only after the addition of trypsinto the culture medium. Under single-cycle growth condi-tions, CACO-2 cells produced virus to similar titers asMDCK cells, but growth rates were slower, and CACO-2cells retained viability for a longer period then MDCK cells(Fig. 1A). In the next set of experiments formation of viralfoci under agarose overlayer was studied in both cells. Viralfoci were formed in CACO-2 cells, whereas they developedin MDCK cells only when trypsin was present in the aga-rose layer (Fig. 1B). These observations suggest thatCACO-2 cells are able to activate HA by proteolytic cleav-age and thus permit multicycle virus replication. Polypep-tide patterns of influenza viruses synthesized in CACO-2cells were therefore analyzed by PAGE, and virus infectiv-ity was measured by double plaque assay. The results showthat virus grown in CACO-2 cells has 80–90% of its HA incleaved form and that the real infectivity of this virus isalmost as high as the potential one (Fig. 1C and Table 1 ).In contrast, virus produced in MDCK cells contained ex-clusively uncleaved HA and had low real infectivity thatwas readily activated by trypsin cleavage of HA (Fig. 1Cand Table 1). These data show that CACO-2 cells contain aprotease able to activate HA with a monobasic cleavage site.

Cultured CACO-2 cells are known to differentiate duringproliferation. First growing as islets, they differentiate afterconfluence by acquiring functional properties of entero-cytes, including secretory activity (Pinto et al., 1983; Ju-marie and Malo, 1991). To find out whether the ability tocleave HA depends on differentiation, influenza viruseswere grown in young CACO-2 cultures (�70% confluency)

199O. Zhirnov, H.-D. Klenk / Virology 313 (2003) 198–212

Fig. 1. Characteristics of influenza virus replication and viral polypeptides patterns in CACO-2 and MDCK cells. Cultures of CACO-2 and MDCK cells wereinfected with Aichi and PR8 viruses at a MOI of 0.005 (A1), 2 (B–E), or about 5 (A2) PFU/cell and then incubated in medium with or without 0.1 �g/mlof trypsin. After different intervals virus yields in culture medium were evaluated by hemagglutination titration. Samples of purified virus were prepared fromculture fluids at 20 (MDCK) and 30 h.p.i. (CACO-2) and analyzed by PAGE followed by gel staining with Coomassie Blue R-350 (C). To study the abilityof viruses to produce foci, CACO-2 and MDCK cells were grown in 24-well plates and infected with different dilutions of Aichi virus. Infected cells werecovered with 1% agarose either lacking trypsin or containing trypsin (�Tr). Viral foci were stained at 38 h.p.i. by antivirus antibodies, HRP conjugates andinsoluble TMB reagent. Young (Y) and aged (A) CACO-2 cells were infected with Aichi (D) and PR8 (E) viruses. At 28 h.p.i. viruses from culture fluidand cell pellets were analyzed by PAGE-WB. WBs were scanned, and the HA0/HA1 ratios were calculated by the TINA program, version 2.09. Cytopathiceffects (c.p.e.) in cell monolayer were estimated visually as the amount of detached cells (“�” shows one-third of the monolayer).

and in aged cultures (1 week after confluency), and the viralpolypeptides patterns were analyzed (Fig. 1D and E). Al-though the amount of cleaved HA was higher in virions thanin cells, the age and thus the differentiation of the cells hadno marked effect on HA cleavage.

We then looked at the cleavage process in more detail.When analyzed by pulse-chase labeling, first only the un-cleaved glycoprotein HA0 was detectable in pulse-labeledinfected cells (Fig. 2A). The cleavage products HA1 andHA2 were identified after 70 min and increased after 2.5 hchase. Subunit HA1 of Aichi/2/68 virus was not clearly seenon this gel since it comigrated with NP. To obtain moreinformation on the cleavage enzyme we tested the effect ofdifferent protease inhibitors, such as �-aminocaproic acid,an inhibitor of plasminogen activation (Brockway and Cas-tellino, 1971); aprotinin (6 kDa), a natural inhibitor oftrypsinlike proteases (Fritz and Wunderer, 1983); �2-mac-roglobulin (725 kDa); �1-antitrypsin (53 kDa); proteaseinhibitors from human plasma (Travis and Salvesen, 1983);and ovomucoid (28 kDa), a trypsin inhibitor from hen eggwhite (Tomimatsu et al., 1966) (not shown). Cycloheximidewas also used as an inhibitor of protein synthesis. Infectedcells were pulse-labeled and then incubated with the inhib-itors during a 2.5-h chase period. Only aprotinin (0.1 mg/ml) was found to suppress HA0 cleavage in infectedCACO-2 cells (Fig. 2A), whereas cycloheximide and otherantiproteases, such as �-aminocaproic acid, �1-antitrypsin,�2-macroglobulin, and ovomucoid at concentrations as highas 0.2, 0.35, 0.5, and 0.2 mg/ml, respectively, were noteffective (not shown). Since low-molecular-weight aproti-nin inhibits cleavage, whereas the high-molecular-weightinhibitors �1-antitrypsin and �2-macroglobulin, which havea similar specificity, do not, it appears that cleavage occursinside cells. We have also analyzed whether 0.45 M sucrose,which is an effective suppressor of clathrin-dependent en-docytosis (Heuser and Anderson, 1989), interferes with theinhibitory activity of aprotinin (Fig. 2B). When CACO-2cells were incubated with sucrose, aprotinin retained theability to inhibit cleavage, implying that it entered infectedcells in a clathrin-independent manner. Taken together,these data support the view that HA generated in CACO-2cells is cleaved within cells by a trypsinlike protease. Theinability of cycloheximide to inhibit cleavage suggests apreexisting pool of this protease in CACO-2 cells.

To test the effect of aprotinin on virus replication,CACO-2 cell cultures were infected with different dilutionsof Aichi virus and then overlayed with agarose containingor not containing aprotinin. Viral foci were analyzed at46 h.p.i. (Fig. 2C). It can be seen, that normal foci about 1–2mm in diameter developed in control wells, whereas in cellscovered with aprotinin agarose foci were significantly re-duced in size and number. Similar observations were madewith PR8 virus (not shown). These data support the conceptthat aprotinin interferes with multicycle virus replication.

Cleavage occurs in exocytotic and endocytoticcompartments of CACO-2 cells

We have then investigated the subcellular compartmen-talization of HA cleavage. For this purpose two parameterswere analyzed. First, cleavage was investigated in CACO-2cells incubated in the presence of brefeldin A (BFA), knownto cause disassembly of cis and medial Golgi compartmentsand to inhibit the anterograde transport of proteins along thesecretory pathway (Lippinscott-Schwartz et al., 1989). Sec-ond, the glycosylation patterns of HA0, HA1, and HA2were tested by endoglycosidase-H (endoH) treatment. Be-cause N-glycosylated proteins acquire endoH resistance inthe medial Golgi, endoH sensitivity is a characteristic forglycoproteins that have not yet reached this compartment.Both parameters were investigated in pulse-chase experi-ments with Aichi and PR8 virus-infected CACO-2 cells. Asshown in Fig. 2A, BFA prevented cleavage indicating thatthis process occurred in a trans-Golgi compartment or at theplasma membrane. Next we incubated labeled cells withpeptidyl-N-acetylglucosaminidase F (PNAGase-F) to iden-tify the electrophoretic positions of the fully deglycosylatedproducts HA0f, HA1f, and HA2f (Fig. 2D). PNAGase treat-ment did not affect electrophoretic mobility of Aichi HA2because HA2 of this mouse-adapted variant has lost itsglycosylation site by the substitution of Asn at position 154for Thr as indicated by nucleotide sequence analysis (datanot shown). It has to be pointed out that most of uncleavedHA labeled by a 40-min pulse was fully sensitive to endoHas indicated by the major band HA0h, representing thedeglycosylated polypeptide. The minor band HA0h* prob-ably represents HA containing both mannose-rich (endoH-sensitive) and complex (endoH-insensitive) carbohydrates.After 70- and 150-min chase periods, when HA1 and HA2appeared, only minor fractions of HA0 and HA1 wereendoH sensitive. The results show that HA is cleaved duringor after its passage through the trans-Golgi region whencomplex glycosylation is completed. Similar data were ob-tained with PR8 virus (data not shown).

Further experiments have been done to find out if clea-vage occurs before or after exposure of HA at the plasmamembrane of infected CACO-2 cells. In the latter case the

Table 1Real and potential infectivitya of influenza viruses grown in CACO-2and MDCK cells

Host cells Aichi/68 PR/8/34

Real Potential Real Potential

CACO-2 1.2 � 107 2.2 � 107 0.7 � 107 1.0 � 107

MDCK 1.6 � 104 3.0 � 107 0.8 � 104 1.7 � 107

a CACO-2 and MDCK cells were infected with Aichi/68 and PR/8/34viruses at m.o.i. about 1 PFU/cell. Infectivity titers of culture fluids (PFU/ml) were measured at 24 h.p.i. by two plaque assays, modified and stan-dard, showing real and potential virus infectivity, as described (Zhirnov etal., 1982a).


Fig. 2. Effect of protease inhibitors and brefeldin A on intracellular HA0 cleavage and sensitivity of viral glycoproteins to glycosidases. (A,B, and D) CACO-2cells were infected with Aichi virus at a MOI of 2 PFU per cell. At 6.5 h.p.i. infected cells were labeled for 40 min (pulse) and then incubated for 70 and150 min in DMEM containing on excess of cold amino acids (chase) and the following inhibitors: 0.1 mg/ml of aprotinin, 10 �g/ml BFA, and 75 �Mcycloheximide (CHX). In separate experiment (B) infected cells were pulse-labeled (pul40) and chased in medium alone (none) or in medium containing 0.1mg/ml of aprotinin (APR) or 0.45 M of sucrose (Suc). Viral polypeptides were immunoprecipitated and analyzed by PAGE followed by autoradiograpgy.Some samples were pretreated with endo-H and PNGase-F before PAGE (D). (C) CACO-2 cells were infected with different dilutions of Aichi virus afterinfection cells were covered either with standard agarose layer (none) or with agarose containing 0.1 mg/ml of aprotinin (apr). At 45 h.p.i. cells were fixedand immunostained with True Blue dye, as described under Materials and methods.

proportion of cleaved to uncleaved HA should be higher onthe surface than in whole cells. Viral glycoproteins localizedon the cell surface were therefore labeled with nonperme-able Biotin-NHS-sulfo cross-linking reagent, and the HA0/HA1 ratios at the cell surface, in whole cells, and in virionswere compared (Fig. 3A and Fig. 1D and E). The ratioswere similar at the plasma membrane and in whole cells,indicating the lack of marked cleavage at the plasma mem-brane of infected CACO-2 cells. Furthermore, the ratio ofbiotinylated HA0/HA1 in plasma membranes was higherthan that in virions. This result may be explained either bya more effective incorporation of cleaved HA into virions oradditional cleavage in extracellular virus.

The ability to cleave at a single arginine suggested thatIAP of CACO-2 cells is a trypsinlike protease. We havetherefore tested CACO-2 cells for the presence of such anenzyme by immunostaining with antibodies developedagainst human and bovine trypsin. The results obtained withmock-infected CACO-2 cells and antihuman trypsin anti-bodies are shown in Fig. 4B. Similar results were obtainedin influenza-infected CACO-2 cells (not shown). It can beclearly seen that CACO-2 cells contain a trypsinlike protein.The most intensive staining was observed in polarized cellsforming domes (Fig. 3B; arrow), whereas other cells wereless stained, suggesting that expression of the protease de-pended on the differentiation of cells. Permeabilization withthe nonionic detergent Nonidet P-40 increased staining in-tensity suggesting intracellular accumulation of the trypsin-like protease. Similar results were obtained in CACO-2 cellswith antibovine trypsin antibodies. However, there was noimmunostaining of non-HA-activting human epitelial Hep-2cells and monkey CV-1 cells with either antihuman orantibovine trypsin antibodies (not shown). Thus, there is agood correlation between HA cleavage and the presence ofthe trypsinlike protein, suggesting that this is the activationenzyme of CACO-2 cells.

The above experiments have shown that HA cleavage inthe CACO-2 cell line is associated with cells but they didnot exclude cleavage in the culture medium. To test ifextracellular cleavage occurs, the following experimentswere done. Aichi virus was grown in MDCK cells without

Fig. 4. CACO-2 cells are able to cleave HA upon virus entry. Aichi virusgrown in MDCK cells was used as reference virus containing HA0. (A)This virus was incubated for 3 h at 37°C with growing CACO-2 cells. Afterincubation a virus pelleted from culture medium (medium) and cellsremoved from matrix (cell) were analyzed by PAGE-WB. (B) Referencevirus was incubated with homogenates of CACO-2 cells and with concen-trated culture medium of mock-infected CACO-2 cells. Virus incubatedwith cell homogenate (cell.hom.) or medium concentrate (med.conc.) wasapplied directly to PAGE-WB. Lanes: none and trypsin-initial referencevirus nontreated and treated in vitro with trypsin, respectively.

Fig. 3. Cell surface biotinylation labeling (A) and immune detection oftrypsin-like protease (B) in CACO-2 cells infected with influenza A vi-ruses. (A) CACO-2 cells were infected with Aichi and PR8 viruses at am.o.i. of 4 PFU per cell and incubated for 8.5 h. Aichi and PR8 virusesgrown in chicken eggs and infected CACO-2 cells were labeled withnonpermeable biotin–NHS–sulfo agent for 35 min at 4°C. After labeling,virus (E virus) and cells (cell-Bt) were desintegrated, and viral proteinswere precipitated with antiviral antibodies and protein G–Sepharose. Im-mune complexes were analyzed by PAGE-WB, using streptavidin–HRPconjugate and ECL. WBs were scanned, and the HA0/HA1 ratios werecalculated by the TINA program, version 2.09. (B) CACO-2 cells werefixed with paraformaldehyde, incubated either without (�) or with (�)antihuman trypsin antibodies, followed by visualization of protease–anti-body complexes with secondary species-specific HRP conjugate and in-soluble TMB substrate (True Blue), and observed in the light microscope.The “dome” structure is shown by an arrow.


trypsin. As expected it contained predominantly uncleavedHA and had low real infectivity (Fig. 1C and Table 1).CACO-2 cultures were incubated with this virus for 3 h.Thereafter, virus in the culture medium and in pelleted cellswas analyzed by PAGE to study HA cleavage. Virus re-maining in culture medium did not display an increasedamount of cleaved HA (Fig. 4A; medium). However, therewas a clear cleavage increase on cell bound virus after the3-h incubation period (Fig. 4A; cell). These results suggestthat (1) IAP is not released from CACO-2 cells to cleaveHA on free extracellular virions and (2) IAP is a cell-associated enzyme that can cleave HA on input virus afteradsorption.

Next, we have analyzed whether a homogenate preparedfrom CACO-2 cells will cleave HA and activate virus in-fectivity. Nonactivated Aichi virus grown in MDCK cellswas again used as reference virus. Homogenates were pre-pared from mock-infected and A/WSN/33-infectedCACO-2 cells at 7.5 h.p.i. In addition to homogenates ofcells, culture medium concentrate was also tested in thecleavage assay. Virus grown in MDCK cells was incubatedwith homogenates for 3 h at 37°C and then analyzed byPAGE (Fig. 4B). It was found that neither the cell homog-enates nor the medium concentrate were able to cleave HA(Fig. 3A and B) or to increase virus infectivity (not shown).The failure to detect IAP in cell homogenates suggests thatstructural integrity of the cellular compartment containingthe enzyme is essential for its activity.

Influenza virus infection causes necrosis of CACO-2 cells

We have observed here that influenza-infected CACO-2cells retained viability for at least 3 days, whereas othercells, such as MDCK and CV-1 cells, were killed already 1day after infection. This prompted us to compare the mech-anisms of cell death in these cell lines. Apoptotic andnecrotic markers were therefore investigated at differentintervals after virus infection. Cytopathic effects were as-sessed by microscopic monitoring of rounding and detach-ment of cells from support matrix and by Trypan bluestaining of dead cells. Visible cytopathic effects developedin CACO-2 cells at 42–45 h.p.i. (about 10–30% of cellswere detached and dead at this stage), and about 60–80% ofcells were dead and detached from the supporting matrix by70 h.p.i. In contrast, in MDCK (not shown) and CV-1 cellsalmost total death and cell detachment were observed atabout 25 h.p.i. (Fig. 5A). Staining with Trypan blue showedthat 80–90% of cells still attached on the matrix were deadat 20–22 h.p.i. in CV-1 and MDCK cells and at around70 h.p.i. in CACO-2 cells (Fig. 5B).

Apoptosis was analyzed by five different approaches: (1)monitoring chromatin DNA fragmentation (DNA laddering)as a major indicator of apoptosis; (2) immuno staining ofcells with M30 monoclonal antibody, which selectivelyrecognizes apoptotic cytokeratin 18, a specific marker ofapoptosis, but does not stain necrotic cells (Leers et al.,

1999); (3) annexin-V binding to cell surface-exposed phos-phatidylserine (PS), which is specific for early apoptosislacking necrosis, whereas after necrotic plasma membranepermeabilization annexin-V binds also to intracellular PS(Martin et al., 1995); (4) proteolytic activation of caspases3, 8, and 9; and (5) caspase-dependent cleavage of nucleo-capsid protein NP occurring during apoptosis (Zhirnov etal., 1999). Additionally, PI staining of infected cells hasbeen used to estimate the level of cell plasma membraneleakage as one of the main hallmarks differentiating necro-sis from apoptosis (McGahon et al., 1995).

In accordance with our earlier observations (Zhirnov etal., 1999, 2002a), MDCK and CV-1 cells infected withAichi or PR8 viruses were killed at 20–24 h.p.i., showingDNA fragmentation (Fig. 5C); accumulation of the apopto-tic form of NP (aNP) (Fig. 5D); and cleavage of caspases 3,8, and 9 (Fig. 5E). After PI staining, only a few CV-1 cellswere found to be stained at 20 h.p.i., showing the absence ofnecrosis and supporting the notion that apoptosis is the maindeath mechanism of these cells (Fig. 5F).

In all of these aspects CACO-2 cells were markedlydifferent from MDCK and CV-1 cells. First, CACO-2 cellswere more resistant to influenza-mediated killing, andprominent cell death developed only at 60–70 h.p.i. (Fig.5A). Second, there were no marked apoptotic signs in in-fluenza-infected CACO-2 cells, such as DNA laddering(Fig. 5C); aNP accumulation (Fig. 5D); and apoptosis-spe-cific staining with M30-cytodeath antibodies (Fig. 5H) atthe late stage of infection, when a prominent cytopathiceffect was seen. Additionally, cleavage of caspases 3, 8, and9 was not detected in infected CACO-2 cells (Figs. 5C–E).Host cell caspases are known to be activated by limitedproteolysis, and the following precursors and cleavage frag-ments have been identified: p323p20�p11 for caspase 3,p553p42/343p20�p10 for caspase 8, and p483p35�p10for caspase 9 (Boesen et al., 1998; Sun et al., 1999). Thecleavage products p20, p42, and p35 of caspases 3, 8, and 9,respectively, were clearly seen in CV-1 cells and were notdetectable in CACO-2 cells as late as 60 h.p.i., when thecytopathic effect developed. It was also noted that activationcleavage of caspases 3, 8, and 9 was detectable only at theterminal stage of cell death when almost all infectedCACO-2 cells were dead at around 70 h.p.i. (not shown).These data suggest that signaling for caspase cleavage wasdelayed in infected CACO-2 cells, and cell death precededsuch activation cleavage of host caspases. Third, unlikeinfluenza virus infection, treatment of CACO-2 cells withstaurosporine, a well-known inducer of apoptosis (Bertrandet al., 1994), rapidly provoked apoptosis as indicated by theappearance of DNA laddering (Fig. 5F) and significantapoptosis staining of cells with M30 cytodeath antibodies(Fig. 5H). This experiment shows that the apoptotic systemis functional in CACO-2 cells but is not activated duringinfluenza infection. Fourth, in contrast to MDCK and CV-1cells, influenza-infected CACO-2 cells were intensivelystained with PI late after infection, indicative of necrotic

204 O. Zhirnov, H.-D. Klenk / Virology 313 (2003) 198–212

Fig. 5. Death parameters in CACO-2, MDCK, and CV-1 cells infected with influenza A virus. CACO-2, MDCK, and CV-1 cells were infected with Aichi(Ai) or PR8 (PR) viruses at a m.o.i. of about 2 PFU per cell. At 22 (MDCK and CV-1 cells) and 24, 46, and 72 h.p.i. (CACO-2 cells) infected cells wereexamined by light microscopy (LM) for Trypan blue uptake (TB) (A and B); DNA laddering (C and F); aNP accumulation (D); the cleavage of caspases3, 8, and 9 (E); and propidium iodide staining (G) as described under Materials and Methods. (C) U, uninfected cells; M, MW markers. (F) M, MW markers;


U, uninfected cells; ST, uninfected cells incubated with staurosporine for 18 h, Ai, cells infected with Aichi virus for 60 h. (H and I) Mock-infected,Aichi-infected (32, 48, and 58 h.p.i), and staurosporine-treated for 20 h (ST-20) CACO-2 cells were tested either with M30-cytodeath monoclonal antibodies(H) or with annexin-V (I) procedures, respectively, and were visualized with “True Blue” substrate followed by light microscope observation. Amounts ofpositive cells as a percentage of total cells are shown at the bottom.


death (Fig. 5G). Fifth, increased annexin-V binding wasobserved only late after infection (50–70 h.p.i.), whereas atearlier stages (25–40 h.p.i.) annexin-V staining was foundto be comparable with the background of uninfected controlcells (Fig. 5I). However, when apoptosis was induced bystaurosporine, extensive annexin binding was observed inCACO-2 cells (Fig. 5I; ST). The results obtained withannexin-V indicate that there is no apoptosis in CACO-2cells early after infection, whereas later cells become ne-crotic, allowing annexin-V binding to intracellular PS.Taken together, all of these observations indicate that mostof the cells in CACO-2 cultures are resistant to influenza-mediated apoptosis and die through necrosis only late afterinfection.

Discussion

The data presented in this article have shown that humaninfluenza A viruses containing hemagglutinin with a mono-basic cleavage site are activated in CACO-2 cells. Virusyields and replication rates in CACO-2 cells were similar tothose obtained in MDCK cells supplemented with trypsin.CACO-2 cells are epithelial cells that differentiate sponta-neously in culture during proliferation, and they exhibitmany functional properties of enterocytes (Pinto et al.,1983; Jumarie and Malo, 1991). Before confluency, thereare islets containing mainly undifferentiated dividing cells,whereas cells become polarized and differentiated whencultures are confluent. Virus grown in young and aged cellcultures contained predominantly cleaved HA. Thus, IAPexpression occurred already in young CACO-2 cells andwas not linked to cell differentiation and concomitant se-cretory activity. In fact, there was no secretion of IAP intothe culture fluid of CACO-2 cells.

Cleavage of monobasic HA in CACO-2 cells takes placeat a late stage of the exocytic pathway. In this respect itresembles cleavage of multibasic HA, which occurs in thetrans-Golgi network (Stieneke-Grober et al., 1992; Walkeret al., 1992). However, we show here that cleavage ofmonobasic HA is probably accomplished by cell-associatedtrypsinlike protease(s), whereas multibasic HA is cleavedby the proproteinconvertase furin recirculating between thetrans-Golgi network and plasma membrane (Stroh et al.,1999). Thus, both HA types may be cleaved in the sameintracellular compartment of CACO-2 cells, yet by differentproteases. Cell-associated cleavage of human influenza Avirus hemagglutinin has also been found in cultures ofhuman respiratory epithelium prepared from adenoid tissue(Zhirnov et al., 2002c). Taken together these data suggestthat epithelial cells of human mucosa may contain intracel-lular trypsinlike IAP(s) that can cleave monobasic hemag-glutinin intracellularly and produce proteolytically activatedvirions.

Our data show that CACO-2 cells are a suitable cellculture system for production of high yields of infectious

influenza virus. Allowing effective HA cleavage and mul-ticycle virus replication, CACO-2 cells may become a use-ful tool for isolating virus from clinical specimens, forselection of virus mutants generated by reverse genetics,and for other applications. For instance, we have success-fully used CACO-2 cells in reverse genetics experiments toprepare influenza virus mutants containing NP with a mod-ified caspase cleavage site (data not published). Further-more, intestinal replication of many influenza A viruses,including human epidemic viruses containing monobasicHA, has been described in the intestine of pigs, ferrets, andbirds (Kawaoka et al., 1987; Slemons and Easterday, 1978;Webster et al., 1978; Glathe et al., 1984). These observa-tions are consistent with our present finding that enterocyteshave the ability to maintain influenza replication.

Exogenous aprotinin inhibits HA cleavage in infectedCACO-2 cells. This finding is interesting for several rea-sons. First, the activity of aprotinin, an inhibitor of trypsin-like serine proteases, indicates that human mucosal IAPbelongs to this group of proteases. Second, our data suggestthat aprotinin is internalized by clathrin-independent endo-cytosis and transported to the intracellular cleavage com-partment. Third, aprotinin may be a useful tool for therapy.Previously we have shown that aprotinin suppresses HAcleavage and virus activation in cultured cells (Zhirnov etal., 1982b), chicken embryonated eggs (Zhirnov et al.,1985), and mouse lungs (Zhirnov et al., 1984) and protectsagainst lethal influenza infection of mice (Ovcharenko andZhirnov, 1994; Zhirnov, 1987). Furthermore, therapeuticefficacy against influenza and parainfluenza infections with-out side effects (Zhirnov et al., 1994a, 1994b) has beendemonstrated in humans (Zhirnov et al., 1996). These ob-servations are in agreement with earlier data showing thataprotinin is able to inhibit respiratory proteases, such asTryptase Clara (Kido et al., 1992), miniplasmin (Murakamiet al., 2001), and plasmin (Zhirnov et al., 1982b), whichwere considered to be involved in influenza virus activationin mucosal membranes of the respiratory tract. Taken to-gether these results additionally support our previous con-cept that aprotinin is a potential anti-influenza drug (Zhir-nov, 1983).

Influenza virus has been reported to provoke apoptosis inmany types of cells, such as epithelial Madin–Darby caninekidney (MDCK) cells, epithelial mink lung cell (Mv1Lu)cells, HeLa cells, chicken fibroblasts, monkey kidney(VERO) cells, lymphocytes, and macrophages (Takizawa etal., 1993; Hinshaw et al., 1994; Lowy and Dimitrov, 1997;Morris et al., 1999; Zhirnov et al., 2002a). In contrast,several epithelial cell lines originated from human (Arndt etal., 2002) and porcine (Seo et al., 2001) lung have beenreported recently to die after influenza infection not throughapoptosis but probably through necrosis. Here we presentanother cell line of intestinal origin that maintained effec-tive virus replication but was resistant to rapid influenza-mediated killing. When these cells were infected with in-fluenza virus apoptotic markers were minimal and necrotic


signs were dominant. Thus, death in influenza virus-infectedcells may develop not only via apoptosis but also throughnecrosis. Interestingly, both apoptotic and necrotic changeshave been detected in cells infected with other viruses, suchas Semliki Forest virus, poliovirus, human herpesvirus type7, and murine polyomavirus (Agol et al., 1998; An et al.,2000; Glasgow et al., 1997; Secchiero et al., 1997; Nargi-Aizenman et al., 2001). These data show that in virus-infected cells apoptotic and necrotic changes may developin parallel and, depending on unknown factors, one of themmay become dominant. It is known that necrosis and apo-ptosis may have common intracellular pathways in the ini-tial signaling phase, such as �-TNF receptor signaling andFAS-mediated oligomerization of FADD by the DED do-main. However, necrosis does not need caspase activities(Matsumata et al., 2000). It is assumed that cell deathdevelops slowly in cells with caspase deficiency and thatsuch cells show necrosis rather than apoptosis (Lemaire etal., 1998; Matsumata et al., 2000). Necrosis in influenza-infected CACO-2 cells may therefore reflect insufficient ordelayed signaling for caspase activation. Our data showingthe lack of caspase activation cleavage in influenza-infectedCACO-2 cells support this concept. However, the exactmechanisms leading to necrosis in influenza-infectedCACO-2 cells are not yet clear and remain to be determined.

Materials and methods

Cells and viruses

Influenza A/PR/8/34 (H1N1) virus (PR8) and a mouselung-adapted strain of A/Aichi/2/68 (H3N2) virus (Aichi)were grown in 10-day-old embryonated chicken eggs (Zhir-nov et al., 1985). Madin–Darby canine kidney (MDCK)cells (Roche Products Ltd.), monkey kidney (CV-1) cells,and a human epithelial colon carcinoma cell line (CACO-2)(European Collection of Cell Cultures; ECACC) were pas-saged in Dulbeco’s minimal essential medium (DMEM)containing 10% bovine fetal calf serum (FCS) (Gibco/BRL). CACO-2 cells were passaged at dilutions of 1:4 to1:6. Monolayers became confluent after about 1 week andcould be maintained for several weeks.

Trypan blue exclusion assay

The number of dead cells was estimated by the Trypanblue exclusion assay where viable cells exclude the dye,whereas dead cells take up the dye and are stained. Infectedand mock-infected cell monolayers were incubated with0.1% Trypan blue prepared in phophate-buffered saline(PBS) for 30 min at 37°C. Then cells were washed withPBS and photographed in a light microscope.

Propidium iodide (PI) staining of cells

Uninfected and influenza virus-infected cells grown on 1cm2 coverslips were incubated in the darkness with DMEMcontaining 2 �g/ml of PI for 15 min and fixed with 2%paraformaldehyde prepared in PBS at 4°C for 20 min. Fixedcells were then washed twice with PBS, covered withMoviol mount medium, and observed in a fluorescent mi-croscope with a 600 EFLP filter.

Immune staining of cells with anticytokeratin 18(M30 CytoDEATH) antibodies

Uninfected, influenza virus-infected and staurosporine-treated CACO-2 cells grown on 4-well plates (Nunclon,Denmark) were fixed with 2% paraformaldehyde preparedin PBS for overnight incubation at 4°C and permeabilizedwith PBS containing 0.1% of Nonidet P-40 for 15 min atroom temperature. Permeabilized cells were washed withPBS and incubated for 1 h with a mouse monoclonal anti-body (clone M30-CytoDEATH) (Roche) specific for anepitope of the intracellular apoptotic form of cytokeratin 18(Leers et al., 1999) at a dilution of 1:200 in PBS containing0.5% of bovine serum albumin. Cells were then incubatedwith horseradish peroxidase conjugated secondary antispe-cies antibodies (Dako), followed by visualization of keratin-positive cells with TMB insoluble substrate “True Blue”(KPL). Stained slides were photographed in a light micro-scope (magnification, �100), and the percentage of positivecells was counted.

Staining of cells with biotinylated annexin-V

Uninfected, influenza virus-infected (25, 36, and58 h.p.i.), and staurosporine-treated (7.5 �M for 20 h)CACO-2 cells grown in 4-well plates were washed withbinding buffer (BB; 2.5 mM Hepes-NaOH, pH 7.4; 140 mMNaCl; 5 mM CaCl2), incubated for 15 min with recombinantbiotinylated annexin-V (Biosource International; USA) di-luted in BB 1:50, and then washed with BB. After that, cellswere fixed for 15 min with 2% paraformaldehyde preparedex-temporo in BB, washed with BB, and incubated for 30min with horseradish peroxidase-conjugated streptavidin(Amersham-Biotech, England), followed by visualization ofannexin-V-positive cells with TMB water-insoluble sub-strate “True Blue” (KPL). Stained cells were observed in alight microscope (magnification, �250).

Immune staining of cells with antitrypsin antibodies

Uninfected and influenza virus-infected CACO-2 cellsand human epithelial Hep-2 cells grown on 4-well plates(Nunclon, Denmark) were fixed with 2% paraformaldehydeprepared in PBS for overnight incubation at 4°C. Fixed cellswere washed with PBS and incubated for 1 h with IgGfractions either of antibodies developed in rabbits against


human trypsin (Calbiochem, USA) or of antibodies specificfor purified bovine trypsin (Sigma, USA) raised in guineapigs at the D. I. Ivanovsky Institute of Virology (Moscow,Russia). Antibodies were diluted in PBS containing 0.5% ofbovine serum albumin from 1:100 to 1:400 in differentexperiments. After washing with PBS cells were incubatedwith horseradish peroxidase-conjugated secondary anti-IgGrabbit or guinea pig species-specific antibodies (Dako).Trypsinlike protease-positive cells were identified withTMB-insoluble substrate “True Blue” (KPL) in a light mi-croscope (magnification, �100).

Pulse-chase labeling and immune precipitation of cellularpolypeptides

CACO-2 cells were infected with influenza Aichi or PR8viruses at a m.o.i. of about 4 PFU per cell. After 8.5 h.p.i.cells were incubated for 40 min in MEM lacking coldmethionine/cysteine and containing S35-labeled methionineand cysteine (Amersham) at a concentration of 75 �Ci/ml(pulse). Then about 106 cells were washed twice with MEMcontaining the 10-fold amount of cold methionine and cys-teine and were incubated in this medium for additional 1.5and 3.0 h (chase). After labeling, the cells were mechani-cally removed from the plate and pelleted. Cell pellets weredissolved in 200 �l of RIPA buffer containing 250 mMNaCl, 50 mM Tris–HCl (pH 7.8), 0.4% nonionic detergentNP-40, 250 �M benzamidine, and 50 TIU/ml aprotinin andcentrifuged at 12,000g to remove undissolved debris.Twenty- to forty-microliter aliquots of supernatant werediluted in 250 �1 PBS and mixed with antibodies raised inrabbits against A/X31/82 (H3N2) hemagglutinin or A/PR/8/34 virus. Samples were incubated for 1 h at room tem-perature and then mixed with 25 �l of a 40% suspension ofprotein G–Sepharose (Pharmacia) and incubated for addi-tional 45 min. Immune complexes were washed 5 timeswith PBS and dissolved in SDS-containing dissociationbuffer. Samples were analyzed by PAGE followed by au-toradiography on Kodak BioMax film.

Biotinylation of viral glycoproteins at the cell surface

Influenza virus-infected CACO-2 cells grown on 6-cmdishes were washed 2 times to remove contaminating pro-teins and then incubated twice at 20°C for 20 min each timewith 3 ml PBS containing 1 mg/ml nonpermeable biotin-NHS-sulfo (Calbiochem), 5 mM CaCl2, and 2.5 mMMgCl2. Cells were then incubated with 50 mM glycineprepared in PBS to saturate the remaining biotinylationreagent. Cells (�106) were collected, suspended in 200 �1of PBS, and homogenized by ultrasonication. Fifty micro-liters of cell homogenates were mixed with 350 �1 of RIPAbuffer and clarified at 12,000g for 10 min. Viral glycopro-teins were precipitated by rabbit antivirus antibodies boundto protein G–Sepharose and analyzed by PAGE-WB fol-lowed by identification of biotinylated viral glycoproteins

with streptavidin–HRP conjugate (Amersham-Biotech) andenhanced chemiluminescence (ECL) (Pierce).

Polyacrylamide gel electrophoresis (PAGE)

Polypeptides were electrophoresed in 12% polyacryl-amide gels using Tris–glycine–SDS buffer followed byautoradiography as previously described (Zhirnov et al.,1999). For gel staining with Coomassie brilliant blue R-350,the protocol of Pharmacia was applied. For electrophoresis,cellular polypeptides were treated in dissociation buffer (2%SDS, 0.01 M dithiotreitol, 0.02 M Tris–HCl, pH 6.8) for 5min at 96°C.

Western blot analysis (WB)

After SDS-PAGE the polypeptides were transferred fromthe gel onto Protran-nitrocellulose membranes (pore size �0.45 �m) (Schleicher & Schuell) by semidry electroblottingwith a Tris–glycine buffer (pH �9.8) containing 15% eth-anol. Membranes were washed with 150 mM PBS andincubated overnight at 4°C in 5% dried milk in PBS. Afterwashing with PBS, membranes were incubated for 2 h atroom temperature in PBS containing 1% BSA, and rabbitantisera specific for H3 hemagglutinin, PR8 virus, caspase 3(Pharmingen), caspase 9 (Pharmingen), and caspase 8(kindly given by Dr. Xiao-Ming Sun from the MRC Toxi-cology Unit, University of Leicester, UK; Sun et al., 1999).Mouse monoclonal antibodies against influenza virus NP(clones A1 and A3; CDC, Atlanta) were also used. Afterincubation with antibodies, membranes were exposed tohorseradish peroxidase-(HRP) conjugated secondary anti-species antibodies (Dako), followed by visualization of pos-itive bands by ECL on Kodak BioMax film.

Focus assay in MDCK cells

MDCK cells grown in 24-well plates were incubatedwith 0.4 ml/well of 10-fold virus dilutions in PBS. After a60-min incubation at room temperature, the virus inoculumwas removed, and cells were washed and covered with 1.5ml of agarose (culture quality; Sigma) at a final concentra-tion of 1% in DMEM containing 0.1% of BSA. In someexperiments, the agarose overlay contained 0.1 �g/ml ofacetylated trypsin (Sigma) to permit virus activation andsecondary rounds of virus replication. Thirty-five to fiftyhours after infection, cells were fixed with 4% paraformal-dehyde, and agarose layers were removed. Fixed cells wereincubated for 1 h with anti-influenza virus antibodies andafter that with HRP-conjugated secondary antispecies anti-bodies (Dako), followed by visualization of virus foci withTMB insoluble substrate “True Blue” (KPL). Stained fociwere counted.


DNA fragmentation analysis

For the isolation of cellular low-molecular-weight DNA,the SDS–high salt extraction method has been used (Zhir-nov et al., 1999). Mock-infected and influenza-infected cellswere suspended in 80 �1 PBS and gently mixed with 300�1 of buffer containing 10 mM Tris–HCl (pH 7.6), 10 mMEDTA, and 0.6% SDS. In some experiments, cells wereincubated for 15–20 h with staurosporine (Sigma) at aconcentation of 7.5 �M. Cell lysates were incubated over-night at 4°C in 100 �1 of 5 M NaCl and centrifuged at14,000 rpm for 20 min at 4°C. Supernatants were treatedsequentially with RNAase A (1 mg/ml) and proteinase K(0.2 mg/ml) for 20 min at 37°C, mixed with two volumes ofethanol, and left overnight at �20°C. Precipitated DNA wasresuspended in TE buffer (10 mM Tris–HCl, 1 mM EDTA,pH 7.6), electrophoresed in 1.5% agarose–TBE buffer, andstained with ethidium bromide.

HA cleavage in CACO-2 cell homogenates

CACO-2 cells (�106) were removed from dishes with arubber policeman, pelleted at 1000g for 15 min, and sus-pended in 50–70 �1 of PBS. Cell suspensions were homog-enized either by 20 frictions in a 1-ml syringe/24GA-1needle or by ultrasonic disintegration for 2 min at 350 Wt(Branson Sonifier 450). All manipulations were performedat 4°C. To perform the HA0 cleavage test, influenza Aichior PR8 viruses were grown in MDCK cells, culture fluidswere clarified at 4,000g for 20 min, and viruses were pel-leted through 5.0 ml of 20% (Wt/Wt) sucrose in PBS at22,000 rpm for 2.5 h in a SW 27.1 rotor (Beckman). Viruspellets were suspended in PBS, mixed with CACO-2 cellhomogenates, and incubated at 37°C for 3–4 h. After incu-bation, the mixtures were analyzed by PAGE-WB withvirus-specific antibodies.

CACO-2 culture medium concentrate

Confluent CACO-2 cell cultures in 6-cm dishes wereincubated for 1 week at 37°C in 8 ml of DMEM lackingFCS containing streptomycin and penicillin. Culture me-dium was clarified at 3000g for 10 min and concentrated inCentriplus 10 tubes (Amicon) with a cutoff value of 10 kDa.The final volume of medium concentrate was about 80 �1.In cleavage experiments, 20 �1 of concentrate (5–10 �g ofprotein) were mixed with 3 �1 of purified virus (� 0.5 �gof protein), incubated for 3–4 h at 37°C and then analyzedby PAGE-WB and plaque assay.

Double plaque assay

Plaque assays were performed in MDCK cells preparedin 24-well plates. Real infectivity, i.e., of virions withcleaved HA, was measured by a modified plaque assay,whereas potential infectivity, which includes also virions

with uncleaved HAs, was measured by the standard plaquemethod (Zhirnov et al., 1982a). Briefly, infected cells werecovered with 1 ml of 1% agarose (culture quality; Sigma) inDMEM containing 0.3 �g/ml of acetylated trypsin (Sigma)in the case of standard plaque assay and no trypsin in themodified plaque assay. According to the modified assay,after 24 h of incubation at 37°C, the cells were overlaidadditionally with 0.7% agarose (0.5 ml per well) containing0.8 �g/ml of acetylated trypsin. In both plaque assays, cellswere stained 3 days after infection with hematoxilin-eosin,and plaques were counted.

Acknowledgments

The authors acknowledge Drs. Francoise Debierre-Grockiego, and Mikhail Matrosovich for valuable discus-sions and help. We thank D. Alexey Zhigulin for assistancewith microscopy and digital pictures. This work was sup-ported by RFBR Grants 04-48442 and 04-04000, DFG-RFBR Grant 436 RUS 113/587, NATO linkage Grant HT-974619, Howard Hughes Medical Institute (USA) Grant75195546302, and Sonderforschungsbereich 286.

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