The Nipah Virus Fusion Protein Is Cleaved within the Endosomal Compartment*

Nipah virus (NiV) is a recently emerged and highly pathogenic paramyxovirus that causes a systemic infection in animals and humans and can infect a wide range of cultured cells. Interestingly, the NiV fusion (F) protein has a single arginine at the cleavage site similar to paramyxoviruses that are activated by exogenous trypsin-like enzymes only present in specific cells and tissues and therefore only cause localized infections. We show here that NiV F activation is not mediated by an exogenous serum protease but by an endogenous ubiquitous cellular protease after endocytosis of the protein. In addition to endocytosis, acidification of the endosome is a prerequisite for F cleavage. These results show that activation of the NiV F protein depends on a type of proteolytic cleavage that is clearly different from what is known for other paramyxoviral and orthomyxoviral fusion proteins. To our knowledge, this is the first example of a viral class I fusion protein whose activation depends on clathrin-mediated constitutive endocytosis.

Nipah virus (NiV) is a recently emerged and highly pathogenic paramyxovirus that causes a systemic infection in animals and humans and can infect a wide range of cultured cells. Interestingly, the NiV fusion (F) protein has a single arginine at the cleavage site similar to paramyxoviruses that are activated by exogenous trypsin-like enzymes only present in specific cells and tissues and therefore only cause localized infections. We show here that NiV F activation is not mediated by an exogenous serum protease but by an endogenous ubiquitous cellular protease after endocytosis of the protein.
In addition to endocytosis, acidification of the endosome is a prerequisite for F cleavage. These results show that activation of the NiV F protein depends on a type of proteolytic cleavage that is clearly different from what is known for other paramyxoviral and orthomyxoviral fusion proteins. To our knowledge, this is the first example of a viral class I fusion protein whose activation depends on clathrin-mediated constitutive endocytosis.
Nipah virus (NiV), 1 a highly pathogenic paramyxovirus, was isolated in 1999 after an outbreak of fatal encephalitis among pig farmers in Malaysia and Singapore (1). In 1998, the virus was transmitted from bats, which are the most likely natural hosts (2), to pigs and then to humans, suggesting that the pig was required as an amplifying host. However, in more recent outbreaks in Bangladesh, NiV was also transmitted directly from bats to humans, and even human-to-human transmission cannot be ruled out (3,4). Because of the broad host range and the high mortality rates associated with infection, NiV has been classified as a biosafety level 4 agent. Because of their unique genetic and biological characteristics NiV and the closely related Hendra virus form a new genus, Henipavirus, within the Paramyxoviridae family (5,6).
NiV encodes for two surface glycoproteins, the receptor-binding G protein and the fusion (F) protein. The G protein mediates attachment to a receptor on host cells that has not yet been identified. The F protein is responsible for virus entry into host cells by initiating pH-independent fusion of the viral and cellular membranes. If coexpressed together with the G protein on the surface of infected cells or on cells expressing plasmidencoded NiV glycoproteins, the F protein mediates fusion with adjacent cells, resulting in multinucleated syncytia (7,8). It is known that cleavage of paramyxoviral F proteins at basic amino acid residues is a prerequisite for fusion activity and, thus, for virus infectivity (9,10). The F proteins are synthesized as inactive precursors (F 0 ) and have to be activated by proteolytic cleavage into the disulfide-linked subunits F 1 and F 2 , thereby releasing the hydrophobic fusion peptide at the amino terminus of F 1 (Fig. 1). So far, two mechanisms of proteolytic F activation are known. Paramyxoviruses that cause systemic infections in vivo generally possess F proteins with multiple basic amino acids at the cleavage site. These F proteins are ubiquitously activated during the transport through the secretory pathway by the Golgi protease furin. Because cleavage occurs intracellularly, F proteins with multibasic cleavage sites reach the cell surface as fusion-active molecules. In contrast, F proteins with only one basic residue at the cleavage site usually cannot be activated intracellularly but have to be activated by extracellular trypsin-like proteases after they have reached the cell surface. As a consequence, the in vitro growth of paramyxoviruses encoding F proteins with monobasic cleavage sites depends on the addition of trypsin to the cell culture medium. In vivo, replication of these viruses is restricted to the respiratory tract where suitable trypsin-like proteases are known to be expressed (11,12). Although the NiV F protein has a single arginine at the cleavage site ( Fig. 1), NiV infection is not restricted to the respiratory tract and does not require exogenous trypsin for its growth in cell culture (5,7,13). This suggests that activation of the NiV F protein differs from what is known for other paramyxoviral and orthomyxoviral fusion proteins. Supporting this view, we have shown recently that the NiV F protein is cleaved in all cell lines tested. Furthermore, this ubiquitous activation does not require a basic residue at the cleavage site and is, therefore, not mediated by a member of the trypsin-or furin-like protease families known to activate other paramyxoviruses (8).

EXPERIMENTAL PROCEDURES
Cell Culture and Virus Infection-MDCK (Madin-Darby canine kidney) cells were maintained in EagleЈs minimal essential medium (Invitrogen) supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Vero (African green monkey kidney) cells and HeLa cells were grown in Dulbecco's modified minimal essential medium (DMEM) containing 10% FCS, penicillin, and streptomycin. To adapt Vero cells to serum-free conditions, the cells were cultivated in virus production serum-free medium (VP-SFM; Invitrogen). Infections with NiV were performed under biosafety level 4 conditions as described previously (8).
Plasmid Vectors-cDNA fragments spanning the F gene and the G gene of the NiV genome (GenBank TM accession number AF212302) were cloned into the pczCFG5 vector as described earlier (14). Generation of the endocytosis-negative mutant NiV F YA (Fig. 1) has been described previously (15). The F gene of the measles virus (MV) Edmonston strain was cloned into the pCG vector (16).
Metabolic Labeling-Cultures of Vero or MDCK cells in 35-mm diameter dishes were transfected with plasmid DNA encoding NiV F or MV F using the cationic lipid transfection reagent Lipofectamine 2000 (Invitrogen). At 24 h posttransfection, cells were incubated for 40 min with medium lacking cysteine and methionine followed by an incubation with medium containing [ 35 S]cysteine and [ 35 S]methionine (Promix; Amersham Biosciences) at a final concentration of 100 Ci/ml for 10 min. Subsequently, labeling medium was replaced by non-radioactive chase medium for 2 h at 37°C. After radiolabeling, cells were lysed in 0.5 ml of radioimmunoprecipitation assay buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 10 mM EDTA, 20 mM Tris-HCl, pH 8.5, protease inhibitors) and centrifuged for 20 min at 100,000 ϫ g. NiV F protein was immunoprecipitated from cell lysates with a polyclonal antiserum derived from infected guinea pigs (a kind gift from M. Czub and H. Weingartl) at a final dilution of 1:500. After addition of a suspension of protein A-Sepharose CL-4B (Sigma), immune complexes were washed three times with radioimmunoprecipitation assay buffer, suspended in reducing sample buffer for SDS-PAGE, and separated on a 12% polyacrylamide gel. Dried gels were exposed to Kodak BIOMAX films.
Inhibitor Studies-To block NiV F protein transport along the secretory pathway, cells were metabolically labeled in the absence or presence of monensin (10 M; Sigma), brefeldin A (15 g/ml; Sigma), and a mixture of 30 mM NaF and 50 M AlCl 3 (Sigma). Inhibition of clathrinmediated endocytosis was examined in the presence of 50 M chlorpromazine (Sigma).
Surface Biotinylation Analysis-Surface labeling with biotin was performed as described previously (17). Briefly, MDCK cells were transfected with plasmid DNA encoding either the wild type NiV F or mutant NiV F YA gene. At 24 h posttransfection, cells were washed and incubated twice for 20 min at 4°C with 2 mg/ml sulfo-N-hydroxysuccinimidobiotin (Calbiochem). Following cell lysis, F proteins were immunoprecipitated as described above and subjected to SDS-PAGE. Proteins were blotted onto nitrocellulose, and biotinylated F proteins were detected by incubation with a streptavidin-biotinylated horseradish peroxidase complex (dilution 1:2000 in phosphate-buffered saline; Amersham Biosciences) and chemiluminescence (West Dura extended; Pierce).
Fusion Assays-Cells were infected with NiV or cotransfected with plasmids encoding the NiV G gene and either the wild type NiV F or the mutant NiV F YA gene. 24 h later, cell-to-cell fusion was visualized after fixation with ethanol for 10 min and staining with a 1:10 diluted Giemsa staining solution for 30 min. Representative microscopic fields were photographed. To analyze fusion after an external pH 5 shift, MDCK cells were cotransfected with the NiV F YA and the NiV G genes. At 24 h posttransfection, cells were washed with phosphate-buffered saline and treated with normal DMEM or with DMEM adjusted to pH 5 at 37°C for 10 min. Subsequently, cells were washed twice with DMEM containing 2% FCS and then incubated with DMEM containing 2% FCS at 37°C for 7 h.
Colocalization Studies-For colocalization studies, antibody uptake assays were performed essentially as described previously (15). Briefly, at 24 h posttransfection NiV F-expressing HeLa cells were incubated with the polyclonal anti-NiV serum (dilution 1:1000) for 1 h at 4°C and then washed and shifted to 37°C for 5 or 30 min to allow endocytosis to occur. Surface-bound primary antibodies were blocked by incubation with a peroxidase-conjugated secondary antibody (dilution 1:5; DAKO). After fixation with 2% paraformaldehyde for 30 min and permeabilization with phosphate-buffered saline containing 0.2% Triton X-100, internalized primary antibodies were stained with a fluorescein isothio-cyanate-conjugated secondary antibody. To analyze the colocalization of NiV F with transferrin, 50 g/ml transferrin-tetramethylrhodamine (Tfn-TR; Molecular Probes) was added during the incubation at 37°C for either 5 or 30 min. For lysosomal costaining, an antibody against the lysosomal-associated membrane protein 1 (Southern Biotech) was added after fixation and permeabilization for 1 h at 4°C. Bound antibodies were detected with rhodamine-conjugated secondary antibodies. Confocal fluorescence images were recorded using a Zeiss Axioplan 2 LSM 510.

Proteolytic Activation of the NiV F Protein Is Not Mediated by
Soluble Serum Proteases-We have shown recently that the NiV F protein is ubiquitously cleaved in cell culture by a protease that cannot be a member of the trypsin-or furin-like protease families (8). To determine whether the ubiquitous activation is mediated by a host cell protease or by an exogenous serum protease, cleavage and fusion activity of the NiV F protein was analyzed in the absence of FCS. For this, Vero cells were cultivated for several weeks under serum-free conditions and either infected with NiV or cotransfected with the NiV F and G genes. Only if the F protein is proteolytically processed can fusogenic G/F 1,2 complexes on the surface of infected or transfected cells form and mediate fusion with adjacent receptor-bearing cells (8,13). To analyze cleavage of the NiV F protein under serum-free conditions, transfected cells were metabolically labeled with [ 35 S]methionine and [ 35 S]cysteine for 10 min and incubated for 2 h to allow the protein to be processed. After cell lysis, F proteins were immunoprecipitated, separated on a 12% SDS gel, and subjected to autoradiography. The results shown in Fig. 2A revealed that the F protein is cleaved with similar efficiency in the absence or presence of FCS. In agreement with these data, the formation of multinucleated syncytia was also observed in NiV-infected and in F ϩ G-cotransfected cells in the absence or presence of FCS (Fig. 2B). These results clearly demonstrate that proteolytic activation of NiV is not mediated by an exogenous serum protease but rather by an endogenous host cell protease.
NiV F Protein Cleavage Does Not Occur during Transport along the Secretory Pathway-To determine where NiV F cleavage takes place, F activation was tested in cells treated with inhibitors of the secretory pathway. As a control, processing of the MV F protein was analyzed. In contrast to the NiV F protein, MV F has a multibasic cleavage site and is, therefore, activated by furin in the trans-Golgi network (TGN) (9,10,18). To prevent transport of the F proteins from the endoplasmic reticulum to the Golgi apparatus, F-expressing cells were pulse labeled and then chased for 2 h at 37°C in the presence of brefeldin A (19). To block the transport from the medial to trans-Golgi, the ionophore monensin was added to the chase medium (20). As shown in Fig. 3A, retention in the endoplasmic reticulum or the medial Golgi completely prevented cleavage of both MV F and NiV F, showing that NiV F activation does not occur before reaching the TGN. By using a mixture of NaF and AlCl 3 , trafficking of secretory vesicles from the TGN to the cell surface was blocked (21). As expected, furin-mediated cleavage of the MV F protein in the TGN was not affected by this treatment. In contrast, cleavage of the NiV F protein could no longer be detected (Fig. 3B), showing that the NiV F protein is not activated during the transport along the secretory pathway but needs to reach the plasma membrane before it can be cleaved.
Endocytosis of the NiV F Protein Is Required for Proteolytic Cleavage-Very recently, we have shown that both NiV glycoproteins are internalized from the surface of infected and transfected cells. Whereas the G protein appeared to be internalized with the bulk flow during membrane turnover, constitutive endocytosis of the F protein was found to be signal mediated (15). To test whether F uptake is required for proteolytic activation, F cleavage was analyzed in the presence of chlorpromazine, an inhibitor of clathrin-mediated endocytosis (22). Fig.  3C shows that NiV F cleavage was completely prevented in the presence of 50 M chlorpromazine whereas furin cleavage of the MV F protein was not affected. This clearly indicates that not only transport to the cell surface but also internalization via clathrin-coated pits is required for proteolytic activation of the NiV F protein.
To demonstrate the intracellular localization of the NiV F protein after endocytosis, an antibody uptake experiment with F-expressing HeLa cells was performed. At 24 h posttransfec-tion, surface-expressed F protein was labeled on ice with a NiV-specific antiserum. Then the cells were shifted to 37°C for either 5 or 30 min to allow endocytosis to occur. As transferrin is known to be a specific marker for the recycling pathway (23), rhodamine-labeled transferrin (Tfn-TR) was added to the Ftransfected cells during incubation at 37°C. After endocytosis of NiV F and Tfn-TR, surface-bound F-specific primary antibodies were blocked on ice with a peroxidase-conjugated secondary antibody. The cells were permeabilized, and internalized F protein was visualized using a fluorescein isothiocyanate-conjugated secondary antibody. After 5 min of endocytosis, a punctuate staining pattern with an almost complete colocalization of NiV F and Tfn-TR was found, demonstrating that both proteins had been co-internalized and entered the early or sorting endosomal compartment (Fig. 4A). In agreement with this, both Tfn-TR and NiV F colocalized with the early endosomal antigen 1 marker protein, EEA-1 (not shown). After endocytosis for 30 min at 37°C, both Tfn-TR and NiV F colocalized in a perinuclear area, indicating that both proteins had reached the recycling endosomal compartment (Fig. 4B). The marginal colocalization with lysosomal-associated membrane protein 1, lamp-1 (Fig. 4C) confirmed that the F protein is not significantly targeted to the lysosomal compartment.
NiV F Cleavage Requires Acidification of the Endosome-In contrast to the extracellular milieu and the cytoplasm, the pH in the endocytic-lysosomal compartment ranges from 6.5 in the early endosome to 4 in the lysosome. Weakly basic amines such as ammonium chloride (NH 4 Cl) inhibit acidification of the endosomes (24). To test whether raising of the endosomal pH affects NiV F activation, 20 mM NH 4 Cl was added to NiV F-expressing cells at 8 h posttransfection. 16 h later, cells were surface biotinylated and the F protein was immunoprecipitated . Following endocytosis, surface-bound primary anti-NiV F antibodies were blocked by incubation with a peroxidaseconjugated secondary antibody. After fixation and permeabilization, internalized anti-NiV F antibodies were stained with a fluorescein isothiocyanate-conjugated secondary antibody. C, after antibody uptake for 30 min and blocking of surface-bound anti-NiV F antibodies, cells were fixed and permeabilized. Endocytosed anti-NiV F antbodies were again detected by fluorescein isothiocyanate-conjugated secondary antibodies. Lysosomes were stained with an anti-lamp-1 antibody and a rhodamineconjugated secondary antibody. Colocalization of the proteins was analyzed by confocal microscopy. and separated by SDS-PAGE under reducing conditions. Surface-labeled F protein was detected by peroxidase-conjugated streptavidin and chemiluminescence. Fig. 5A shows that in the presence of NH 4 Cl almost no cleaved F protein was expressed on the cell surface (NiV F ϩ NH 4 Cl). As a consequence, syncytia formation in NH 4 Cl-treated cells coexpressing NiV G and F was barely detectable (data not shown). This result shows that not only endocytosis but also acidification of the endosome is required for NiV F activation.
A Decrease in the Extracellular pH Is Not Sufficient for Cleavage-As mentioned above, recent studies have shown that the NiV F protein possesses intrinsic internalization signals that ensure rapid endocytosis of the protein. The YSRL sequence in the F cytoplasmic domain (position 525-528) perfectly fits the YXXØ-type endocytosis signal consensus motif (where Y is a tyrosine, X can be any amino acid, and Ø is a large hydrophobic amino acid). These motifs are recognized by the cytosolic adaptor complex 2, which selectively concentrates proteins within clathrin-coated vesicles (25). In addition, the NiV F cytoplasmic domain contains a YY motif, which further increases the YRSL motif-mediated endocytosis rate to 2%/min (15). By exchanging the three tyrosine residues in the F cytoplasmic domain against alanines (Fig. 1, F YA ), we generated a mutant that was internalized with a rate of Ͻ0.1%/min (15). To confirm that endocytosis is required for F cleavage, proteolytic activation of the endocytosis-negative F YA mutant was analyzed. The surface biotinylation of F YA -expressing cells showed that the mutant protein was abundantly expressed on the cell surface but was not cleaved at a detectable level (Fig. 5A, NiV F YA ). Consequently, F YA was barely able to induce cell-to-cell fusion (Fig. 5B, control). Cleavage deficiency of the endocytosisnegative mutant is likely due to the fact that the F protein no longer encounters the activating protease in the endosomal compartment. Nevertheless, it could not be ruled out that the protease is a cell surface resident protease that cannot cleave non-endocytosed F protein because the protein must pass through the endosomal-recycling pathway to undergo a low pH-dependent conformational change before it can be activated on the cell surface. To test whether an acidic step can restore the fusion activity, F YA -transfected cells were subjected to an external pH 5 shift (26). At 24 h posttransfection, medium adjusted to pH 5 was added for 10 min at 37°C. Then the medium was again exchanged against normal culture medium (pH 7). When fusion was analyzed at 31 h posttransfection, cells expressing the NiV F YA protein together with the NiV G protein did not show increased syncytia formation (Fig. 5B, ϩ  pH 5 shift). This indicates that the deficient F YA cleavage on the cell surface is due not to the lack of a low pH-induced conformational change but to the lack of active protease on the cell surface. DISCUSSION Class I fusion proteins, which are present in paramyxoviruses, orthomyxoviruses, filoviruses, and retroviruses, are proteolytically processed at basic cleavage sites either in the TGN by ubiquitous furin-like proteases or after arrival at the cell surface by exogenous trypsin-like proteases (9). In contrast to what is reported for other class I fusion proteins, activation of the NiV F protein is neither mediated during transport along the secretory pathway by a ubiquitous Golgi protease nor accomplished by an exogenous protease at the cell surface. Using intracellular transport inhibitors and the endocytosis-negative F YA mutant, we clearly demonstrated that activation of the NiV F protein occurs after internalization of the protein by clathrin-mediated endocytosis, showing for the first time that proteolytic activation of a paramyxoviral F protein can occur within the endosomal compartment. In addition to endocytosis, acidification of the endosome is required for NiV proteolytic activation. Fusion activity of the F YA mutant could not be restored by decreasing the extracellular pH, indicating that the activating protease is a low pH-dependent protease that is not present on the cell surface in an active form.
In full agreement with the finding that endocytosis is an essential prerequisite for proteolytic activation, the surprising observation that endocytosis-deficient F mutants showed a decreased fusion activity despite an increased surface expression (15) can now be conclusively explained by the defective proteolytic processing of the surface-expressed mutant F proteins. The idea that proteolytic activation of the NiV F protein is completely different from what is known for other paramyxoviruses was already suggested by our earlier findings that cleavage of the NiV F monobasic cleavage site occurs ubiquitously and does not even require a basic residue at the cleavage site (8). Thus, activation cannot be mediated by a trypsin-or furin-like enzyme such as those known to process other paramyxoviruses. We now know not only that activation of the NiV F protein is exceptional with respect to protease usage but also that the subcellular localization where proteolytic cleavage takes place is strikingly different. Similar to other F proteins with monobasic cleavage sites, the NiV F protein is not activated by the ubiquitous Golgi protease furin during transport and, thus, reaches the cell surface as fusion-inactive protein.
However, whereas other paramyxoviral F proteins depend on exogenous trypsin-like proteases, making their activation on the cell surface restricted, the NiV F protein is ubiquitously internalized, activated within the endosomal compartment, and subsequently recycled to the cell surface as fusion-active protein. As recycled NiV F protein has not entered late endosomes or lysosomes, it must be concluded that activation occurs within early or recycling endosomes. The minor colocalization of endocytosed F protein with lamp-1 further supported the idea that only a small amount of the protein is transported to the late endosomal/lysosomal compartment. As proteins that are recycled to the cell surface and proteins that are targeted to lysosomes have similar sorting signals, it must be assumed that the relative position of the signal within the NiV F cytoplasmic tail mediates internalization and recycling rather than lysosomal targeting (27,28).
Although endosomes were originally thought to only function FIG. 5. Influence of NH 4 Cl and pH 5 treatment on cleavage and fusion activity of wild type NiV F and mutant NiV F YA protein. A, MDCK cells were transfected with either wild type F (NiV F), wild type F in the presence of 20 mM NH 4 Cl (NiV F ϩ NH 4 Cl), or the mutant NiV F YA gene encoding for an endocytosis-negative F protein, respectively. At 24 h posttransfection, cells were surface labeled with biotin and lysed. Following immunoprecipitation, samples were subjected to SDS-PAGE under reducing conditions and blotted onto nitrocellulose. Surface-labeled F proteins were visualized with streptavidin-peroxidase and chemiluminescence. B, at 24 h posttransfection, NiV G and F YA -expressing MDCK cells were either kept untreated (control) or treated with medium adjusted to pH 5 for 10 min and then neutralized by incubation in fresh medium containing 2% FCS (ϩ pH 5 shift). 7 h later, syncytium formation was monitored by Giemsa staining. Magnification, ϫ200.
as a major sorting station of the endocytic pathway and receive endocytosed materials from the cell surface for recycling or transport to late endosomes and lysosomes, it is now well accepted that endosomes also represent a biologically important processing compartment within cells (29,30). Endosomal cathepsins, metalloproteases, and secretases mediate cleavage or limited proteolysis of a variety of cellular proteins, leading either to degradation or activation of the respective proteins (31)(32)(33)(34)(35)(36)(37)(38). Furthermore, proteases of the endosomal compartment are known to be involved in the proteolytic processing of a few viral proteins such as the outer capsid proteins of reoviruses (39). However, the NiV F protein is the first viral surface glycoprotein for which it is now shown that site-specific cleavage within endosomes takes place and is required for biological activation. This raises the interesting possibility that pharmacological drugs that block clathrin-mediated endocytosis might be potential tools for therapeutic treatment in the early phase of a NiV-induced encephalitis.
In agreement with our findings, Pager et al. (40) recently reported that cleavage of the Hendra virus F protein is also sensitive to increases in intracellular pH levels. Because the Hendra virus F protein also contains a YSRL endocytosis motif in its cytoplasmic tail (6), our results strongly suggest that the closely related Hendra virus F protein is also cleaved by a pH-dependent endosomal protease. Thus, activation in the endosomal compartment is likely to be a common property of the highly pathogenic henipaviruses, making these viruses unique not only within the Paramyxoviridae but also when compared with all enveloped viruses encoding for class I fusion proteins.