Human Influenza a Viruses Are Proteolytically Activated and Do Not

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Virology 313 (2003) 198Ð212 www.elsevier.com/locate/yviro

Human inﬂuenza A viruses are proteolytically activated and do not induce apoptosis in CACO-2 cells

Oleg Zhirnova,* and Hans-Dieter Klenkb

a D.I. Ivanovsky Institute of Virology, Gamaleya 16, Moscow 123098, Russia b 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 intestinal epithelial differentiated cells. Hemagglutinin (HA) was cleaved in these cells by an endogenous protease. Thus, infectious virus was produced that underwent multiple cycle replication and plaque formation in the absence of trypsin added to the media. Cleavage of de novo-synthesized HA occurred at a late stage of the exocytic pathway as indicated by pulse-chase labeling and by experiments employing endoglycosidase H and brefeldin A treatment. However, surface-labeling experiments employing biotinylation suggested that there is no cleavage at the plasma membrane. Unlike HA of serotypes H5 and H7 cleaved at multibasic cleavage sites by furin, the HAs with monobasic cleavage 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 culture medium. These observations indicate that the activating enzyme of CACO-2 cells is a trypsinlike protease functioning in the trans-Golgi network and presumably endosomes. In support of this concept immune staining with antibodies specific to human and bovine trypsin revealed the presence of a trypsinlike protease in CACO-2 cells. Unlike MDCK and CV-1 cells undergoing rapid apoptosis after influenza virus 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 site is exposed in a peptide loop at the surface of the HA spike (Chen et al., 1998), and its accessibility to the cleav- Hemagglutinin (HA), the major envelope glycoprotein of age enzyme may be modulated by an adjacent carbohydrate influenza A virus, mediates attachment to cell receptors and side chain (Kawaoka et al., 1984; Ohuchi et al., 1989). The virus entry into target cells. HA is composed of two sub- multibasic site is found only with some H5 and H7 strains, units, HA1 and HA2, that are generated from the precursor whereas all other influenza A viruses as well as influenza B HA0 by proteolytic cleavage. Cleavage that is exerted by and C viruses contain HAs with monobasic sites (Klenk and host proteases activates virus infectivity and has been Garten, 1994). As a rule, influenza viruses having multiba- shown to be an important determinant of influenza virus sic sites are highly virulent and induce systemic infection in pathogenicity (Klenk and Garten, 1994; Steinhauer, 1999). their avian hosts, whereas viruses with monobasic sites are The prime characteristic of HA that determines its sen- less virulent and provoke local inflammation in the respira- sitivity to host proteases (influenza virus activating pro- tory tract (Klenk and Garten, 1994; Steinhauer, 1999). teases, IAP) is the cleavage site, which consists of either a HAs with multibasic cleavage sites are activated in the single arginine (monobasic site) or an R-X-K/R-R motif trans-Golgi network by furin or related proproteinconverta- (multibasic site) (Klenk and Garten, 1994). The cleavage ses (Stieneke-Gro¬ber et al., 1992; Morsy et al., 1994). HAs containing monobasic cleavage sites are activated by trypsin * Corresponding author. Fax ϩ007-095-190-3058. or trypsinlike proteases (Klenk et al., 1975; Lazarowitz and E-mail address: [email protected] (O. Zhirnov). Choppin, 1975), such as plasmin (Lazarowitz et al., 1973);

0042-6822/03/$ Ð see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0042-6822(03)00264-2 O. Zhirnov, H.-D. Klenk / Virology 313 (2003) 198–212 199 kallikrein; urokinase; thrombin (Scheiblauer et al., 1992); are an efficient host system for productive growth of human blood clotting factor Xa (Gotoh et al., 1990); acrosin (Gar- influenza A viruses. These cells are interesting in two re- ten et al., 1981); tryptase Clara (Kido et al., 1992, 1999); spects. First, they are able to cleave and, thus, to maintain miniplasmin from rat lungs (Murakami et al., 2001); pro- multicycle replication of human influenza A viruses. Cleav- teases from human respiratory lavage (Barbey-Morel et al., age of HA was sensitive to aprotinin, indicating that the 1987); and proteases secreted from bacteria, such as Staphy- endogenous CACO-2 enzyme is a trypsinlike serine pro- lococcus aureus (Tashiro et al., 1987) and Pseudomonas tease. Second, infected CACO-2 cells showed no apoptosis, aeruginosa (Callan et al., 1997). Cleavage by these pro- and necrotic markers were dominant in CACO-2 cells at late teases is thought to occur either at the plasma membrane or stages of infection. These data show that cell killing by in extracellular fluids. Furthermore, evidence has been ob- influenza virus varies in different cell types and may de- tained that the hemagglutinin of the A/WSN/33 (H1N1) velop through apoptosis or necrosis. strain is cleaved in MDBK cells after virus internalization by an unknown, presumably endosomal, protease (Boycott et al., 1994). Results A fair amount of information is available on the effects of protease inhibitors on HA activation. Peptidyl-chloro- Proteolytic activation of hemagglutinin with monobasic methylketones mimicking furin-specific cleavage sites, such cleavage site in CACO-2 cells as decREKR-CMK, have been shown to suppress HA cleavage by furin in cultured cells (Garten et al., 1989; Stieneke- First we compared multicycle replication of human in- Gro¬ber et al., 1992). Several inhibitors of trypsinlike pro- fluenza A/Aichi/68 and A/PR/8/34 viruses in CACO-2 and ⑀ teases, such as -aminocaproic acid, an inhibitor of MDCK cells. The results with Aichi virus are shown in Fig. plasminogen activation (Goto and Kawaoka, 1998; Zhirnov 1A and B, and similar data were obtained with PR8 virus et al., 1982b); aprotinin (Zhirnov et al. 1984); leupeptin (not shown). Multicycle replication and accumulation of (Tashiro et al., 1987); pulmonary surfactant (Kido et al. significant amounts of virus in culture fluid were found to 1993); and human mucus protease inhibitor (Beppu et al., develop in CACO-2 cells, whereas virus replication in 1997), were found to possess antiviral potential by interfer- MDCK cells was observed only after the addition of trypsin ing with cleavage at monobasic sites in cultured cells sup- to the culture medium. Under single-cycle growth condi- plemented with blood serum, in chicken embryos, and in tions, CACO-2 cells produced virus to similar titers as lungs of infected mice. MDCK cells, but growth rates were slower, and CACO-2 Influenza infection triggers apoptosis in infected cells cells retained viability for a longer period then MDCK cells (Hinshaw et al., 1994; Takizawa et al., 1993). There is (Fig. 1A). In the next set of experiments formation of viral evidence that influenza apoptosis is a multifactorial process, foci under agarose overlayer was studied in both cells. Viral involving the FAS-dependent caspase 8 pathway (Balachan- foci were formed in CACO-2 cells, whereas they developed dran et al., 2000), the PKR/NF-kappaB/INF cascade (Zhir- in MDCK cells only when trypsin was present in the aga- nov et al., 2002a), transcription factor c-Jun/AP-1, and cy- rose layer (Fig. 1B). These observations suggest that tokine TGF-␤ (Lin et al., 2001). Several viral proteins have CACO-2 cells are able to activate HA by proteolytic cleav- been shown to be involved, too. The nonstructural protein age and thus permit multicycle virus replication. Polypep- NS1 down-regulates apoptosis in infected cells in an IFN- tide patterns of influenza viruses synthesized in CACO-2 dependent manner (Zhirnov et al., 2002a). The ion channel cells were therefore analyzed by PAGE, and virus infectiv- protein M2 and the nucleocapsid protein NP were observed ity was measured by double plaque assay. The results show to be specifically cleaved during apoptosis by host cell that virus grown in CACO-2 cells has 80Ð90% of its HA in caspases (Zhirnov et al., 1999, 2002a). It was also suggested cleaved form and that the real infectivity of this virus is that proteins NA (Morris et al., 1999) and M1 (Zhirnov et almost as high as the potential one (Fig. 1C and Table 1 ). al., 2002b) may participate in regulation of apoptosis. Re- In contrast, virus produced in MDCK cells contained ex- cently, the interesting observation has been made that in an clusively uncleaved HA and had low real infectivity that epithelial cell line derived from porcine lung (SJPL) (Seo et was readily activated by trypsin cleavage of HA (Fig. 1C al., 2001) and some human bronchiolar cell lines (Arndt et and Table 1). These data show that CACO-2 cells contain a al., 2002) influenza virus infection does not lead to apopto- protease able to activate HA with a monobasic cleavage site. sis but probably to necrosis. These data suggest that death Cultured CACO-2 cells are known to differentiate during mechanisms in cells infected with influenza virus depend on proliferation. First growing as islets, they differentiate after the host. However, host and viral factors that determine confluence by acquiring functional properties of entero- death mechanisms remain so far unknown. cytes, including secretory activity (Pinto et al., 1983; Ju- We report here that CACO-2 cells that develop in culture marie and Malo, 1991). To find out whether the ability to into differentiated enterocytes resembling a natural intesti- cleave HA depends on differentiation, influenza viruses nal epithelium (Pinto et al., 1983; Jumarie and Malo, 1991) were grown in young CACO-2 cultures (ϳ70% confluency) Fig. 1. Characteristics of influenza virus replication and viral polypeptides patterns in CACO-2 and MDCK cells. Cultures of CACO-2 and MDCK cells were ⑀ infected with Aichi and PR8 viruses at a MOI of 0.005 (A1),2(BÐE), or about 5 (A2) PFU/cell and then incubated in medium with or without 0.1 g/ml of trypsin. After different intervals virus yields in culture medium were evaluated by hemagglutination titration. Samples of purified virus were prepared from culture 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 ability of 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 were covered with 1% agarose either lacking trypsin or containing trypsin (ϩTr). Viral foci were stained at 38 h.p.i. by antivirus antibodies, HRP conjugates and insoluble 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 fluid and cell pellets were analyzed by PAGE-WB. WBs were scanned, and the HA0/HA1 ratios were calculated by the TINA program, version 2.09. Cytopathic effects (c.p.e.) in cell monolayer were estimated visually as the amount of detached cells (“ϩ” shows one-third of the monolayer). O. Zhirnov, H.-D. Klenk / Virology 313 (2003) 198–212 201 and in aged cultures (1 week after confluency), and the viral Table 1 a polypeptides patterns were analyzed (Fig. 1D and E). Al- Real and potential infectivity of influenza viruses grown in CACO-2 and MDCK cells though the amount of cleaved HA was higher in virions than in cells, the age and thus the differentiation of the cells had Host cells Aichi/68 PR/8/34 no marked effect on HA cleavage. Real Potential Real Potential We then looked at the cleavage process in more detail. CACO-2 1.2 ϫ 107 2.2 ϫ 107 0.7 ϫ 107 1.0 ϫ 107 When analyzed by pulse-chase labeling, first only the un- MDCK 1.6 ϫ 104 3.0 ϫ 107 0.8 ϫ 104 1.7 ϫ 107 cleaved glycoprotein HA0 was detectable in pulse-labeled a CACO-2 and MDCK cells were infected with Aichi/68 and PR/8/34 infected cells (Fig. 2A). The cleavage products HA1 and viruses at m.o.i. about 1 PFU/cell. Infectivity titers of culture fluids (PFU/ HA2 were identified after 70 min and increased after 2.5 h ml) were measured at 24 h.p.i. by two plaque assays, modified and stan- chase. Subunit HA1 of Aichi/2/68 virus was not clearly seen dard, showing real and potential virus infectivity, as described (Zhirnov et on this gel since it comigrated with NP. To obtain more al., 1982a). information on the cleavage enzyme we tested the effect of different protease inhibitors, such as ⑀-aminocaproic acid, Cleavage occurs in exocytotic and endocytotic an inhibitor of plasminogen activation (Brockway and Cas- compartments of CACO-2 cells tellino, 1971); aprotinin (6 kDa), a natural inhibitor of ␣ trypsinlike proteases (Fritz and Wunderer, 1983); 2-mac- We have then investigated the subcellular compartmen- ␣ roglobulin (725 kDa); 1-antitrypsin (53 kDa); protease talization of HA cleavage. For this purpose two parameters inhibitors from human plasma (Travis and Salvesen, 1983); were analyzed. First, cleavage was investigated in CACO-2 and ovomucoid (28 kDa), a trypsin inhibitor from hen egg cells incubated in the presence of brefeldin A (BFA), known white (Tomimatsu et al., 1966) (not shown). Cycloheximide to cause disassembly of cis and medial Golgi compartments was also used as an inhibitor of protein synthesis. Infected and to inhibit the anterograde transport of proteins along the cells were pulse-labeled and then incubated with the inhib- secretory pathway (Lippinscott-Schwartz et al., 1989). Sec- itors during a 2.5-h chase period. Only aprotinin (0.1 mg/ ond, the glycosylation patterns of HA0, HA1, and HA2 ml) was found to suppress HA0 cleavage in infected were tested by endoglycosidase-H (endoH) treatment. Be- CACO-2 cells (Fig. 2A), whereas cycloheximide and other cause N-glycosylated proteins acquire endoH resistance in ⑀ ␣ antiproteases, such as -aminocaproic acid, 1-antitrypsin, the medial Golgi, endoH sensitivity is a characteristic for ␣ 2-macroglobulin, and ovomucoid at concentrations as high glycoproteins that have not yet reached this compartment. as 0.2, 0.35, 0.5, and 0.2 mg/ml, respectively, were not Both parameters were investigated in pulse-chase experi- effective (not shown). Since low-molecular-weight aproti- ments with Aichi and PR8 virus-infected CACO-2 cells. As nin inhibits cleavage, whereas the high-molecular-weight shown in Fig. 2A, BFA prevented cleavage indicating that ␣ ␣ this process occurred in a trans-Golgi compartment or at the inhibitors 1-antitrypsin and 2-macroglobulin, which have a similar specificity, do not, it appears that cleavage occurs plasma membrane. Next we incubated labeled cells with inside cells. We have also analyzed whether 0.45 M sucrose, peptidyl-N-acetylglucosaminidase F (PNAGase-F) to iden- which is an effective suppressor of clathrin-dependent en- tify the electrophoretic positions of the fully deglycosylated docytosis (Heuser and Anderson, 1989), interferes with the products HA0f, HA1f, and HA2f (Fig. 2D). PNAGase treat- inhibitory activity of aprotinin (Fig. 2B). When CACO-2 ment did not affect electrophoretic mobility of Aichi HA2 cells were incubated with sucrose, aprotinin retained the because HA2 of this mouse-adapted variant has lost its glycosylation site by the substitution of Asn at position 154 ability to inhibit cleavage, implying that it entered infected for Thr as indicated by nucleotide sequence analysis (data cells in a clathrin-independent manner. Taken together, not shown). It has to be pointed out that most of uncleaved these data support the view that HA generated in CACO-2 HA labeled by a 40-min pulse was fully sensitive to endoH cells is cleaved within cells by a trypsinlike protease. The as indicated by the major band HA0h, representing the inability of cycloheximide to inhibit cleavage suggests a deglycosylated polypeptide. The minor band HA0h* prob- preexisting pool of this protease in CACO-2 cells. ably represents HA containing both mannose-rich (endoH- To test the effect of aprotinin on virus replication, sensitive) and complex (endoH-insensitive) carbohydrates. CACO-2 cell cultures were infected with different dilutions After 70- and 150-min chase periods, when HA1 and HA2 of Aichi virus and then overlayed with agarose containing appeared, only minor fractions of HA0 and HA1 were or not containing aprotinin. Viral foci were analyzed at endoH sensitive. The results show that HA is cleaved during 46 h.p.i. (Fig. 2C). It can be seen, that normal foci about 1Ð2 or after its passage through the trans-Golgi region when mm in diameter developed in control wells, whereas in cells complex glycosylation is completed. Similar data were ob- covered with aprotinin agarose foci were significantly retained with PR8 virus (data not shown). duced in size and number. Similar observations were made Further experiments have been done to find out if clea- with PR8 virus (not shown). These data support the concept vage occurs before or after exposure of HA at the plasma that aprotinin interferes with multicycle virus replication. membrane of infected CACO-2 cells. In the latter case the 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-2 cells 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 and 150 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 ␮M cycloheximide (CHX). In separate experiment (B) infected cells were pulse-labeled (pul40) and chased in medium alone (none) or in medium containing 0.1 mg/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 after infection 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 fixed and immunostained with True Blue dye, as described under Materials and methods. O. Zhirnov, H.-D. Klenk / Virology 313 (2003) 198–212 203

The ability to cleave at a single arginine suggested that IAP of CACO-2 cells is a trypsinlike protease. We have therefore tested CACO-2 cells for the presence of such an enzyme by immunostaining with antibodies developed against human and bovine trypsin. The results obtained with mock-infected CACO-2 cells and antihuman trypsin antibodies are shown in Fig. 4B. Similar results were obtained in influenza-infected CACO-2 cells (not shown). It can be clearly seen that CACO-2 cells contain a trypsinlike protein. The most intensive staining was observed in polarized cells forming domes (Fig. 3B; arrow), whereas other cells were less stained, suggesting that expression of the protease de- pended on the differentiation of cells. Permeabilization with the nonionic detergent Nonidet P-40 increased staining in- tensity suggesting intracellular accumulation of the trypsinlike protease. Similar results were obtained in CACO-2 cells with antibovine trypsin antibodies. However, there was no immunostaining of non-HA-activting human epitelial Hep-2 cells and monkey CV-1 cells with either antihuman or antibovine trypsin antibodies (not shown). Thus, there is a good correlation between HA cleavage and the presence of the trypsinlike protein, suggesting that this is the activation enzyme of CACO-2 cells. The above experiments have shown that HA cleavage in the CACO-2 cell line is associated with cells but they did not exclude cleavage in the culture medium. To test if extracellular cleavage occurs, the following experiments were done. Aichi virus was grown in MDCK cells without Fig. 3. Cell surface biotinylation labeling (A) and immune detection of trypsin-like protease (B) in CACO-2 cells infected with influenza A viruses. (A) CACO-2 cells were infected with Aichi and PR8 viruses at a m.o.i. of 4 PFU per cell and incubated for 8.5 h. Aichi and PR8 viruses grown in chicken eggs and infected CACO-2 cells were labeled with nonpermeable biotinÐNHSÐsulfo agent for 35 min at 4¡C. After labeling, virus (E virus) and cells (cell-Bt) were desintegrated, and viral proteins were precipitated with antiviral antibodies and protein GÐSepharose. Im- mune complexes were analyzed by PAGE-WB, using streptavidinÐHRP conjugate and ECL. WBs were scanned, and the HA0/HA1 ratios were calculated by the TINA program, version 2.09. (B) CACO-2 cells were fixed with paraformaldehyde, incubated either without (Ϫ) or with (ϩ) antihuman trypsin antibodies, followed by visualization of proteaseÐanti- body complexes with secondary species-specific HRP conjugate and insoluble TMB substrate (True Blue), and observed in the light microscope. The “dome” structure is shown by an arrow. proportion of cleaved to uncleaved HA should be higher on the surface than in whole cells. Viral glycoproteins localized on the cell surface were therefore labeled with nonpermeable Biotin-NHS-sulfo cross-linking reagent, and the HA0/ HA1 ratios at the cell surface, in whole cells, and in virions Fig. 4. CACO-2 cells are able to cleave HA upon virus entry. Aichi virus were compared (Fig. 3A and Fig. 1D and E). The ratios grown in MDCK cells was used as reference virus containing HA0. (A) were similar at the plasma membrane and in whole cells, This virus was incubated for3hat37¡C with growing CACO-2 cells. After indicating the lack of marked cleavage at the plasma mem- incubation a virus pelleted from culture medium (medium) and cells brane of infected CACO-2 cells. Furthermore, the ratio of removed from matrix (cell) were analyzed by PAGE-WB. (B) Reference biotinylated HA0/HA1 in plasma membranes was higher virus was incubated with homogenates of CACO-2 cells and with concentrated culture medium of mock-infected CACO-2 cells. Virus incubated than that in virions. This result may be explained either by with cell homogenate (cell.hom.) or medium concentrate (med.conc.) was a more effective incorporation of cleaved HA into virions or applied directly to PAGE-WB. Lanes: none and trypsin-initial reference additional cleavage in extracellular virus. virus nontreated and treated in vitro with trypsin, respectively. 204 O. Zhirnov, H.-D. Klenk / Virology 313 (2003) 198Ð212 trypsin. As expected it contained predominantly uncleaved 1999); (3) annexin-V binding to cell surface-exposed phos- HA and had low real infectivity (Fig. 1C and Table 1). phatidylserine (PS), which is specific for early apoptosis CACO-2 cultures were incubated with this virus for 3 h. lacking necrosis, whereas after necrotic plasma membrane Thereafter, virus in the culture medium and in pelleted cells permeabilization annexin-V binds also to intracellular PS was analyzed by PAGE to study HA cleavage. Virus re- (Martin et al., 1995); (4) proteolytic activation of caspases maining in culture medium did not display an increased 3, 8, and 9; and (5) caspase-dependent cleavage of nucleo- amount of cleaved HA (Fig. 4A; medium). However, there capsid protein NP occurring during apoptosis (Zhirnov et was a clear cleavage increase on cell bound virus after the al., 1999). Additionally, PI staining of infected cells has 3-h incubation period (Fig. 4A; cell). These results suggest been used to estimate the level of cell plasma membrane that (1) IAP is not released from CACO-2 cells to cleave leakage as one of the main hallmarks differentiating necro- HA on free extracellular virions and (2) IAP is a cell- sis from apoptosis (McGahon et al., 1995). associated enzyme that can cleave HA on input virus after In accordance with our earlier observations (Zhirnov et adsorption. al., 1999, 2002a), MDCK and CV-1 cells infected with Next, we have analyzed whether a homogenate prepared Aichi or PR8 viruses were killed at 20Ð24 h.p.i., showing from CACO-2 cells will cleave HA and activate virus in- DNA fragmentation (Fig. 5C); accumulation of the apopto- fectivity. Nonactivated Aichi virus grown in MDCK cells tic form of NP (aNP) (Fig. 5D); and cleavage of caspases 3, was again used as reference virus. Homogenates were pre- 8, and 9 (Fig. 5E). After PI staining, only a few CV-1 cells pared from mock-infected and A/WSN/33-infected were found to be stained at 20 h.p.i., showing the absence of CACO-2 cells at 7.5 h.p.i. In addition to homogenates of necrosis and supporting the notion that apoptosis is the main cells, culture medium concentrate was also tested in the death mechanism of these cells (Fig. 5F). cleavage assay. Virus grown in MDCK cells was incubated In all of these aspects CACO-2 cells were markedly with homogenates for3hat37¡C and then analyzed by different from MDCK and CV-1 cells. First, CACO-2 cells PAGE (Fig. 4B). It was found that neither the cell homog- were more resistant to influenza-mediated killing, and enates nor the medium concentrate were able to cleave HA prominent cell death developed only at 60Ð70 h.p.i. (Fig. (Fig. 3A and B) or to increase virus infectivity (not shown). 5A). Second, there were no marked apoptotic signs in in- The failure to detect IAP in cell homogenates suggests that fluenza-infected CACO-2 cells, such as DNA laddering structural integrity of the cellular compartment containing (Fig. 5C); aNP accumulation (Fig. 5D); and apoptosis-spe- the enzyme is essential for its activity. cific staining with M30-cytodeath antibodies (Fig. 5H) at the late stage of infection, when a prominent cytopathic Influenza virus infection causes necrosis of CACO-2 cells effect was seen. Additionally, cleavage of caspases 3, 8, and 9 was not detected in infected CACO-2 cells (Figs. 5CÐE). We have observed here that influenza-infected CACO-2 Host cell caspases are known to be activated by limited cells retained viability for at least 3 days, whereas other proteolysis, and the following precursors and cleavage frag- cells, such as MDCK and CV-1 cells, were killed already 1 ments have been identified: p323p20ϩp11 for caspase 3, day after infection. This prompted us to compare the mech- p553p42/343p20ϩp10 for caspase 8, and p483p35ϩp10 anisms of cell death in these cell lines. Apoptotic and for caspase 9 (Boesen et al., 1998; Sun et al., 1999). The necrotic markers were therefore investigated at different cleavage products p20, p42, and p35 of caspases 3, 8, and 9, intervals after virus infection. Cytopathic effects were as- respectively, were clearly seen in CV-1 cells and were not sessed by microscopic monitoring of rounding and detach- detectable in CACO-2 cells as late as 60 h.p.i., when the ment of cells from support matrix and by Trypan blue cytopathic effect developed. It was also noted that activation staining of dead cells. Visible cytopathic effects developed cleavage of caspases 3, 8, and 9 was detectable only at the in CACO-2 cells at 42Ð45 h.p.i. (about 10Ð30% of cells terminal stage of cell death when almost all infected were detached and dead at this stage), and about 60Ð80% of CACO-2 cells were dead at around 70 h.p.i. (not shown). cells were dead and detached from the supporting matrix by These data suggest that signaling for caspase cleavage was 70 h.p.i. In contrast, in MDCK (not shown) and CV-1 cells delayed in infected CACO-2 cells, and cell death preceded almost total death and cell detachment were observed at such activation cleavage of host caspases. Third, unlike about 25 h.p.i. (Fig. 5A). Staining with Trypan blue showed influenza virus infection, treatment of CACO-2 cells with that 80Ð90% of cells still attached on the matrix were dead staurosporine, a well-known inducer of apoptosis (Bertrand at 20Ð22 h.p.i. in CV-1 and MDCK cells and at around et al., 1994), rapidly provoked apoptosis as indicated by the 70 h.p.i. in CACO-2 cells (Fig. 5B). appearance of DNA laddering (Fig. 5F) and significant Apoptosis was analyzed by five different approaches: (1) apoptosis staining of cells with M30 cytodeath antibodies monitoring chromatin DNA fragmentation (DNA laddering) (Fig. 5H). This experiment shows that the apoptotic system as a major indicator of apoptosis; (2) immuno staining of is functional in CACO-2 cells but is not activated during cells with M30 monoclonal antibody, which selectively influenza infection. Fourth, in contrast to MDCK and CV-1 recognizes apoptotic cytokeratin 18, a specific marker of cells, influenza-infected CACO-2 cells were intensively apoptosis, but does not stain necrotic cells (Leers et al., stained with PI late after infection, indicative of necrotic O. Zhirnov, H.-D. Klenk / Virology 313 (2003) 198Ð212 205

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 were examined by light microscopy (LM) for Trypan blue uptake (TB) (A and B); DNA laddering (C and F); aNP accumulation (D); the cleavage of caspases 3, 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; 206 O. Zhirnov, H.-D. Klenk / Virology 313 (2003) 198Ð212

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 of positive cells as a percentage of total cells are shown at the bottom. O. Zhirnov, H.-D. Klenk / Virology 313 (2003) 198Ð212 207 death (Fig. 5G). Fifth, increased annexin-V binding was influenza virus. Allowing effective HA cleavage and mul- observed only late after infection (50Ð70 h.p.i.), whereas at ticycle virus replication, CACO-2 cells may become a use- earlier stages (25Ð40 h.p.i.) annexin-V staining was found ful tool for isolating virus from clinical specimens, for to be comparable with the background of uninfected control selection of virus mutants generated by reverse genetics, cells (Fig. 5I). However, when apoptosis was induced by and for other applications. For instance, we have success- staurosporine, extensive annexin binding was observed in fully used CACO-2 cells in reverse genetics experiments to CACO-2 cells (Fig. 5I; ST). The results obtained with prepare influenza virus mutants containing NP with a mod- annexin-V indicate that there is no apoptosis in CACO-2 ified caspase cleavage site (data not published). Further- cells early after infection, whereas later cells become ne- more, intestinal replication of many influenza A viruses, crotic, allowing annexin-V binding to intracellular PS. including human epidemic viruses containing monobasic Taken together, all of these observations indicate that most HA, has been described in the intestine of pigs, ferrets, and of the cells in CACO-2 cultures are resistant to influenza- birds (Kawaoka et al., 1987; Slemons and Easterday, 1978; mediated apoptosis and die through necrosis only late after Webster et al., 1978; Glathe et al., 1984). These observa- infection. tions are consistent with our present finding that enterocytes have the ability to maintain influenza replication. Exogenous aprotinin inhibits HA cleavage in infected Discussion CACO-2 cells. This finding is interesting for several rea- sons. First, the activity of aprotinin, an inhibitor of trypsin- The data presented in this article have shown that human like serine proteases, indicates that human mucosal IAP influenza A viruses containing hemagglutinin with a mono- belongs to this group of proteases. Second, our data suggest basic cleavage site are activated in CACO-2 cells. Virus that aprotinin is internalized by clathrin-independent endo- yields and replication rates in CACO-2 cells were similar to cytosis and transported to the intracellular cleavage com- those obtained in MDCK cells supplemented with trypsin. partment. Third, aprotinin may be a useful tool for therapy. CACO-2 cells are epithelial cells that differentiate sponta- Previously we have shown that aprotinin suppresses HA neously in culture during proliferation, and they exhibit cleavage and virus activation in cultured cells (Zhirnov et many functional properties of enterocytes (Pinto et al., al., 1982b), chicken embryonated eggs (Zhirnov et al., 1983; Jumarie and Malo, 1991). Before confluency, there 1985), and mouse lungs (Zhirnov et al., 1984) and protects are islets containing mainly undifferentiated dividing cells, against lethal influenza infection of mice (Ovcharenko and whereas cells become polarized and differentiated when Zhirnov, 1994; Zhirnov, 1987). Furthermore, therapeutic cultures are confluent. Virus grown in young and aged cell efficacy against influenza and parainfluenza infections with- cultures contained predominantly cleaved HA. Thus, IAP out side effects (Zhirnov et al., 1994a, 1994b) has been expression occurred already in young CACO-2 cells and demonstrated in humans (Zhirnov et al., 1996). These ob- was not linked to cell differentiation and concomitant se- servations are in agreement with earlier data showing that cretory activity. In fact, there was no secretion of IAP into aprotinin is able to inhibit respiratory proteases, such as the culture fluid of CACO-2 cells. Tryptase Clara (Kido et al., 1992), miniplasmin (Murakami Cleavage of monobasic HA in CACO-2 cells takes place et al., 2001), and plasmin (Zhirnov et al., 1982b), which at a late stage of the exocytic pathway. In this respect it were considered to be involved in influenza virus activation resembles cleavage of multibasic HA, which occurs in the in mucosal membranes of the respiratory tract. Taken to- trans-Golgi network (Stieneke-Gro¬ber et al., 1992; Walker gether these results additionally support our previous con- et al., 1992). However, we show here that cleavage of cept that aprotinin is a potential anti-influenza drug (Zhir- monobasic HA is probably accomplished by cell-associated nov, 1983). trypsinlike protease(s), whereas multibasic HA is cleaved Influenza virus has been reported to provoke apoptosis in by the proproteinconvertase furin recirculating between the many types of cells, such as epithelial MadinÐDarby canine trans-Golgi network and plasma membrane (Stroh et al., kidney (MDCK) cells, epithelial mink lung cell (Mv1Lu) 1999). Thus, both HA types may be cleaved in the same cells, HeLa cells, chicken fibroblasts, monkey kidney intracellular compartment of CACO-2 cells, yet by different (VERO) cells, lymphocytes, and macrophages (Takizawa et proteases. Cell-associated cleavage of human influenza A al., 1993; Hinshaw et al., 1994; Lowy and Dimitrov, 1997; virus hemagglutinin has also been found in cultures of Morris et al., 1999; Zhirnov et al., 2002a). In contrast, human respiratory epithelium prepared from adenoid tissue several epithelial cell lines originated from human (Arndt et (Zhirnov et al., 2002c). Taken together these data suggest al., 2002) and porcine (Seo et al., 2001) lung have been that epithelial cells of human mucosa may contain intracel- reported recently to die after influenza infection not through lular trypsinlike IAP(s) that can cleave monobasic hemag- apoptosis but probably through necrosis. Here we present glutinin intracellularly and produce proteolytically activated another cell line of intestinal origin that maintained effec- virions. tive virus replication but was resistant to rapid influenza- Our data show that CACO-2 cells are a suitable cell mediated killing. When these cells were infected with in- culture system for production of high yields of infectious fluenza virus apoptotic markers were minimal and necrotic 208 O. Zhirnov, H.-D. Klenk / Virology 313 (2003) 198Ð212 signs were dominant. Thus, death in influenza virus-infected Propidium iodide (PI) staining of cells cells may develop not only via apoptosis but also through necrosis. Interestingly, both apoptotic and necrotic changes Uninfected and influenza virus-infected cells grown on 1 have been detected in cells infected with other viruses, such cm2 coverslips were incubated in the darkness with DMEM as Semliki Forest virus, poliovirus, human herpesvirus type containing 2 ␮g/ml of PI for 15 min and fixed with 2% 7, and murine polyomavirus (Agol et al., 1998; An et al., paraformaldehyde prepared in PBS at 4¡C for 20 min. Fixed 2000; Glasgow et al., 1997; Secchiero et al., 1997; Nargi- cells were then washed twice with PBS, covered with Aizenman et al., 2001). These data show that in virus- Moviol mount medium, and observed in a fluorescent mi- infected cells apoptotic and necrotic changes may develop croscope with a 600 EFLP filter. in parallel and, depending on unknown factors, one of them may become dominant. It is known that necrosis and apo- Immune staining of cells with anticytokeratin 18 (M30 CytoDEATH) antibodies ptosis may have common intracellular pathways in the initial signaling phase, such as ␣-TNF receptor signaling and Uninfected, influenza virus-infected and staurosporine- FAS-mediated oligomerization of FADD by the DED do- treated CACO-2 cells grown on 4-well plates (Nunclon, main. However, necrosis does not need caspase activities Denmark) were fixed with 2% paraformaldehyde prepared (Matsumata et al., 2000). It is assumed that cell death in PBS for overnight incubation at 4¡C and permeabilized develops slowly in cells with caspase deficiency and that with PBS containing 0.1% of Nonidet P-40 for 15 min at such cells show necrosis rather than apoptosis (Lemaire et room temperature. Permeabilized cells were washed with al., 1998; Matsumata et al., 2000). Necrosis in influenza- PBS and incubated for 1 h with a mouse monoclonal anti- infected CACO-2 cells may therefore reflect insufficient or body (clone M30-CytoDEATH) (Roche) specific for an delayed signaling for caspase activation. Our data showing epitope of the intracellular apoptotic form of cytokeratin 18 the lack of caspase activation cleavage in influenza-infected (Leers et al., 1999) at a dilution of 1:200 in PBS containing CACO-2 cells support this concept. However, the exact 0.5% of bovine serum albumin. Cells were then incubated mechanisms leading to necrosis in influenza-infected with horseradish peroxidase conjugated secondary antispe- CACO-2 cells are not yet clear and remain to be determined. cies antibodies (Dako), followed by visualization of keratin- positive cells with TMB insoluble substrate “True Blue” (KPL). Stained slides were photographed in a light microscope (magnification, ϫ100), and the percentage of positive Materials and methods cells was counted.

Cells and viruses Staining of cells with biotinylated annexin-V

Influenza A/PR/8/34 (H1N1) virus (PR8) and a mouse Uninfected, influenza virus-infected (25, 36, and lung-adapted strain of A/Aichi/2/68 (H3N2) virus (Aichi) 58 h.p.i.), and staurosporine-treated (7.5 ␮M for 20 h) were grown in 10-day-old embryonated chicken eggs (Zhir- CACO-2 cells grown in 4-well plates were washed with nov et al., 1985). MadinÐDarby canine kidney (MDCK) binding buffer (BB; 2.5 mM Hepes-NaOH, pH 7.4; 140 mM cells (Roche Products Ltd.), monkey kidney (CV-1) cells, NaCl; 5 mM CaCl2), incubated for 15 min with recombinant and a human epithelial colon carcinoma cell line (CACO-2) biotinylated annexin-V (Biosource International; USA) di- (European Collection of Cell Cultures; ECACC) were pas- luted in BB 1:50, and then washed with BB. After that, cells saged in Dulbeco’s minimal essential medium (DMEM) were fixed for 15 min with 2% paraformaldehyde prepared containing 10% bovine fetal calf serum (FCS) (Gibco/ ex-temporo in BB, washed with BB, and incubated for 30 BRL). CACO-2 cells were passaged at dilutions of 1:4 to min with horseradish peroxidase-conjugated streptavidin 1:6. Monolayers became confluent after about 1 week and (Amersham-Biotech, England), followed by visualization of could be maintained for several weeks. annexin-V-positive cells with TMB water-insoluble substrate “True Blue” (KPL). Stained cells were observed in a light microscope (magnification, ϫ250). Trypan blue exclusion assay Immune staining of cells with antitrypsin antibodies The number of dead cells was estimated by the Trypan blue exclusion assay where viable cells exclude the dye, Uninfected and influenza virus-infected CACO-2 cells whereas dead cells take up the dye and are stained. Infected and human epithelial Hep-2 cells grown on 4-well plates and mock-infected cell monolayers were incubated with (Nunclon, Denmark) were fixed with 2% paraformaldehyde 0.1% Trypan blue prepared in phophate-buffered saline prepared in PBS for overnight incubation at 4¡C. Fixed cells (PBS) for 30 min at 37¡C. Then cells were washed with were washed with PBS and incubated for 1 h with IgG PBS and photographed in a light microscope. fractions either of antibodies developed in rabbits against O. Zhirnov, H.-D. Klenk / Virology 313 (2003) 198Ð212 209 human trypsin (Calbiochem, USA) or of antibodies specific with streptavidinÐHRP conjugate (Amersham-Biotech) and for purified bovine trypsin (Sigma, USA) raised in guinea enhanced chemiluminescence (ECL) (Pierce). pigs at the D. I. Ivanovsky Institute of Virology (Moscow, Russia). Antibodies were diluted in PBS containing 0.5% of Polyacrylamide gel electrophoresis (PAGE) bovine serum albumin from 1:100 to 1:400 in different experiments. After washing with PBS cells were incubated with horseradish peroxidase-conjugated secondary anti-IgG Polypeptides were electrophoresed in 12% polyacryl- rabbit or guinea pig species-specific antibodies (Dako). amide gels using TrisÐglycineÐSDS buffer followed by Trypsinlike protease-positive cells were identified with autoradiography as previously described (Zhirnov et al., TMB-insoluble substrate “True Blue” (KPL) in a light mi- 1999). For gel staining with Coomassie brilliant blue R-350, croscope (magnification, ϫ100). the protocol of Pharmacia was applied. For electrophoresis, cellular polypeptides were treated in dissociation buffer (2% Pulse-chase labeling and immune precipitation of cellular SDS, 0.01 M dithiotreitol, 0.02 M TrisÐHCl, pH 6.8) for 5 polypeptides min at 96¡C.

CACO-2 cells were infected with influenza Aichi or PR8 Western blot analysis (WB) viruses 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 cold After SDS-PAGE the polypeptides were transferred from methionine/cysteine and containing S35-labeled methionine the gel onto Protran-nitrocellulose membranes (pore size ϭ and cysteine (Amersham) at a concentration of 75 ␮Ci/ml 0.45 ␮m) (Schleicher & Schuell) by semidry electroblotting (pulse). Then about 106 cells were washed twice with MEM with a TrisÐglycine buffer (pH ϳ9.8) containing 15% eth- containing the 10-fold amount of cold methionine and cys- anol. Membranes were washed with 150 mM PBS and teine and were incubated in this medium for additional 1.5 incubated overnight at 4¡C in 5% dried milk in PBS. After and 3.0 h (chase). After labeling, the cells were mechani- cally removed from the plate and pelleted. Cell pellets were washing with PBS, membranes were incubated for2hat dissolved in 200 ␮l of RIPA buffer containing 250 mM room temperature in PBS containing 1% BSA, and rabbit NaCl, 50 mM TrisÐHCl (pH 7.8), 0.4% nonionic detergent antisera specific for H3 hemagglutinin, PR8 virus, caspase 3 NP-40, 250 ␮M benzamidine, and 50 TIU/ml aprotinin and (Pharmingen), caspase 9 (Pharmingen), and caspase 8 centrifuged at 12,000g to remove undissolved debris. (kindly given by Dr. Xiao-Ming Sun from the MRC Toxi- Twenty- to forty-microliter aliquots of supernatant were cology Unit, University of Leicester, UK; Sun et al., 1999). diluted in 250 ␮1 PBS and mixed with antibodies raised in Mouse monoclonal antibodies against influenza virus NP rabbits against A/X31/82 (H3N2) hemagglutinin or A/PR/ (clones A1 and A3; CDC, Atlanta) were also used. After 8/34 virus. Samples were incubated for1hatroom tem- incubation with antibodies, membranes were exposed to perature and then mixed with 25 ␮l of a 40% suspension of horseradish peroxidase-(HRP) conjugated secondary anti- protein GÐSepharose (Pharmacia) and incubated for addi- species antibodies (Dako), followed by visualization of pos- tional 45 min. Immune complexes were washed 5 times itive bands by ECL on Kodak BioMax film. with PBS and dissolved in SDS-containing dissociation buffer. Samples were analyzed by PAGE followed by au- Focus assay in MDCK cells toradiography on Kodak BioMax film. MDCK cells grown in 24-well plates were incubated Biotinylation of viral glycoproteins at the cell surface with 0.4 ml/well of 10-fold virus dilutions in PBS. After a 60-min incubation at room temperature, the virus inoculum Influenza virus-infected CACO-2 cells grown on 6-cm dishes were washed 2 times to remove contaminating pro- was removed, and cells were washed and covered with 1.5 teins and then incubated twice at 20¡C for 20 min each time ml of agarose (culture quality; Sigma) at a final concentra- with 3 ml PBS containing 1 mg/ml nonpermeable biotin- tion of 1% in DMEM containing 0.1% of BSA. In some experiments, the agarose overlay contained 0.1 ␮g/ml of NHS-sulfo (Calbiochem), 5 mM CaCl2, and 2.5 mM acetylated trypsin (Sigma) to permit virus activation and MgCl2. Cells were then incubated with 50 mM glycine prepared in PBS to saturate the remaining biotinylation secondary rounds of virus replication. Thirty-five to fifty reagent. Cells (ϳ106) were collected, suspended in 200 ␮1 hours after infection, cells were fixed with 4% paraformal- of PBS, and homogenized by ultrasonication. Fifty micro- dehyde, and agarose layers were removed. Fixed cells were liters of cell homogenates were mixed with 350 ␮1 of RIPA incubated for 1 h with anti-influenza virus antibodies and buffer and clarified at 12,000g for 10 min. Viral glycopro- after that with HRP-conjugated secondary antispecies anti- teins were precipitated by rabbit antivirus antibodies bound bodies (Dako), followed by visualization of virus foci with to protein GÐSepharose and analyzed by PAGE-WB fol- TMB insoluble substrate “True Blue” (KPL). Stained foci lowed by identification of biotinylated viral glycoproteins were counted. 210 O. Zhirnov, H.-D. Klenk / Virology 313 (2003) 198Ð212

DNA fragmentation analysis with uncleaved HAs, was measured by the standard plaque method (Zhirnov et al., 1982a). Briefly, infected cells were For the isolation of cellular low-molecular-weight DNA, covered with 1 ml of 1% agarose (culture quality; Sigma) in the SDSÐhigh salt extraction method has been used (Zhir- DMEM containing 0.3 ␮g/ml of acetylated trypsin (Sigma) nov et al., 1999). Mock-infected and influenza-infected cells in the case of standard plaque assay and no trypsin in the were suspended in 80 ␮1 PBS and gently mixed with 300 modified plaque assay. According to the modified assay, ␮1 of buffer containing 10 mM TrisÐHCl (pH 7.6), 10 mM after 24 h of incubation at 37¡C, the cells were overlaid EDTA, and 0.6% SDS. In some experiments, cells were additionally with 0.7% agarose (0.5 ml per well) containing incubated for 15Ð20 h with staurosporine (Sigma) at a 0.8 ␮g/ml of acetylated trypsin. In both plaque assays, cells concentation of 7.5 ␮M. Cell lysates were incubated over- were stained 3 days after infection with hematoxilin-eosin, night at 4¡Cin100␮1 of 5 M NaCl and centrifuged at and plaques were counted. 14,000 rpm for 20 min at 4¡C. Supernatants were treated sequentially with RNAase A (1 mg/ml) and proteinase K (0.2 mg/ml) for 20 min at 37¡C, mixed with two volumes of Acknowledgments ethanol, and left overnight at Ϫ20¡C. Precipitated DNA was resuspended in TE buffer (10 mM TrisÐHCl, 1 mM EDTA, The authors acknowledge Drs. Francoise Debierre- pH 7.6), electrophoresed in 1.5% agaroseÐTBE buffer, and Grockiego, and Mikhail Matrosovich for valuable discus- stained with ethidium bromide. sions and help. We thank D. Alexey Zhigulin for assistance with microscopy and digital pictures. This work was sup- HA cleavage in CACO-2 cell homogenates ported by RFBR Grants 04-48442 and 04-04000, DFG- RFBR Grant 436 RUS 113/587, NATO linkage Grant HT- ϳ 6 CACO-2 cells ( 10 ) were removed from dishes with a 974619, Howard Hughes Medical Institute (USA) Grant rubber policeman, pelleted at 1000g for 15 min, and sus- 75195546302, and Sonderforschungsbereich 286. pended in 50Ð70 ␮1 of PBS. Cell suspensions were homogenized either by 20 frictions in a 1-ml syringe/24GA-1 needle or by ultrasonic disintegration for 2 min at 350 Wt (Branson Sonifier 450). All manipulations were performed References at 4¡C. To perform the HA0 cleavage test, influenza Aichi Agol, V.I., Belov, G.A., Bienz, K., Egger, M.S., Kolesnikova, N.T., Raikh- or PR8 viruses were grown in MDCK cells, culture fluids lin, I.I., Romanova, E.A., Smirnova, E.A., Tolskaya, E.A., 1998. Two were clarified at 4,000g for 20 min, and viruses were pel- types of death of polivirus-infected cells: caspase involvement in the leted through 5.0 ml of 20% (Wt/Wt) sucrose in PBS at apoptosis but not cytopathic effect. Virology 252, 343Ð353. 22,000 rpm for 2.5 h in a SW 27.1 rotor (Beckman). Virus An, K., Fattacy, H.K., Panisco, A.O., Consigli, R.A., 2000. Murine poly- pellets were suspended in PBS, mixed with CACO-2 cell oma virus infection of 3T6 mouse cells shows evidence of predominant necrosis as well as limited apoptosis. Virus. Res. 67, 81Ð90. homogenates, and incubated at 37¡C for 3Ð4 h. After incu- Arndt, U., Wennemuth, G., Barth, P., Nain, M., Al-Abed, Y., Meinhardt, bation, the mixtures were analyzed by PAGE-WB with A., Gemsa, D., Bacher, M., 2002. Release of macrophage migration virus-specific antibodies. inhibitory factor and CXCL8/interleukin-8 from lung epithelial cells rendered necrotic by influenza A virus infection. J. Virol. 76, 9298Ð CACO-2 culture medium concentrate 9306. Balachandran, S., Roberts, P.C., Kipperman, T., Bhalla, K.N., Compans, R.W., Archer, D.R., Barber, G.N., 2000. Alpha/beta interferons poten- Confluent CACO-2 cell cultures in 6-cm dishes were tiate virus-induced apoptosis through activation of theFADD/caspase-8 incubated for 1 week at 37¡C in 8 ml of DMEM lacking death signaling pathway. J. Virol. 74, 1513Ð1523. FCS containing streptomycin and penicillin. Culture me- Barbey-Morel, C.L., Oeltmann, T.N., Edwards, K.M., Wright, P.F., 1987. dium was clarified at 3000g for 10 min and concentrated in Role of respiratory tract proteases in infectivity of influenza A virus. J. Infect. Dis. 155, 667Ð672. Centriplus 10 tubes (Amicon) with a cutoff value of 10 kDa. Beppu, Y., Imamura, Y., Tashiro, M., Towatari, T., Ariga, H., Kido, H., The final volume of medium concentrate was about 80 ␮1. 1997. Human mucus protease inhibitor in airway fluids is a potential In cleavage experiments, 20 ␮1 of concentrate (5Ð10 ␮gof defensive compound against infection with influenza A and Sendai protein) were mixed with 3 ␮1 of purified virus (ϳ 0.5 ␮g virus. J. Biochem. 121, 309Ð316. of protein), incubated for 3Ð4hat37¡C and then analyzed Bertrand, R., Solary, E., O’Connor, P., Kohn, K.W., Pommier, Y., 1994. Induction of a common pathway of apoptosis by staurosporine. Exp. by PAGE-WB and plaque assay. Cell Res. 211, 314Ð321. Boesen-de, C., Jeanine, G.R., Tepper, A.D., Evert de, V., van Blitterswijk, Double plaque assay W.J., Borst, J., 1999. Common regulation of apoptosis signaling in- diced by CD95 and the DNA-damaging stimuli etoposide and ␥-irra- Plaque assays were performed in MDCK cells prepared diation downstream of caspse-8 activation. J. Biol. Sci. 274, 14255Ð 14261. in 24-well plates. Real infectivity, i.e., of virions with Boycott, R., Klenk, H.-D., Ohuchi, M., 1994. 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