Neutrophil elastase, an acid-independent serine protease, facilitates reovirus uncoating and infection in U937 promonocyte cells
© Golden and Schiff; licensee BioMed Central Ltd. 2005
Received: 18 May 2005
Accepted: 31 May 2005
Published: 31 May 2005
Mammalian reoviruses naturally infect their hosts through the enteric and respiratory tracts. During enteric infections, proteolysis of the reovirus outer capsid protein σ3 is mediated by pancreatic serine proteases. In contrast, the proteases critical for reovirus replication in the lung are unknown. Neutrophil elastase (NE) is an acid-independent, inflammatory serine protease predominantly expressed by neutrophils. In addition to its normal role in microbial defense, aberrant expression of NE has been implicated in the pathology of acute respiratory distress syndrome (ARDS). Because reovirus replication in rodent lungs causes ARDS-like symptoms and induces an infiltration of neutrophils, we investigated the capacity of NE to promote reovirus virion uncoating.
The human promonocyte cell line U937 expresses NE. Treatment of U937 cells with the broad-spectrum cysteine-protease inhibitor E64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane] and with agents that increase vesicular pH did not inhibit reovirus replication. Even when these inhibitors were used in combination, reovirus replicated to significant yields, indicating that an acid-independent non-cysteine protease was capable of mediating reovirus uncoating in U937 cell cultures. To identify the protease(s) responsible, U937 cells were treated with phorbol 12-myristate 13-acetate (PMA), an agent that induces cellular differentiation and results in decreased expression of acid-independent serine proteases, including NE and cathepsin (Cat) G. In the presence of E64, reovirus did not replicate efficiently in PMA-treated cells. To directly assess the role of NE in reovirus infection of U937 cells, we examined viral growth in the presence of N-Ala-Ala-Pro-Val chloromethylketone, a NE-specific inhibitor. Reovirus replication in the presence of E64 was significantly reduced by treatment of cells with the NE inhibitor. Incubation of virions with purified NE resulted in the generation of infectious subviron particles that did not require additional intracellular proteolysis.
Our findings reveal that NE can facilitate reovirus infection. The fact that it does so in the presence of agents that raise vesicular pH supports a model in which the requirement for acidic pH during infection reflects the conditions required for optimal protease activity. The capacity of reovirus to exploit NE may impact viral replication in the lung and other tissues during natural infections.
Mammalian reoviruses are the prototypic members of the Reoviridae family, which also includes the pathogenic rotaviruses, coltiviruses, seadornaviruses and orbiviruses. These viruses share elements of their replication cycle as well as structural features, including a non-enveloped multi-layered capsid that surrounds a segmented dsRNA genome. In humans, mammalian reoviruses are typically associated with mild and self-limiting enteric and respiratory infections. However, studies in neonatal mice reveal that reoviruses can spread to distant tissue sites in immunocompromised hosts (reviewed in). The factors that determine reovirus cellular host range are poorly understood. Because reovirus attaches to cells through interactions with broadly expressed receptors, one or more subsequent steps in the viral life cycle must help to regulate host range and pathogenesis. Our recent studies suggest that one such step is proteolysis of the capsid protein σ3 [2, 3].
In cell culture, the first step in infection is attachment to cellular receptors through interactions with the viral protein σ1 [4, 5]. σ1 interacts with two known receptors: sialic acid and junctional adhesion molecule 1 [6–8]. Following binding, virions are internalized by receptor-mediated endocytosis . Endocytosis is an essential step in the viral life cycle under standard infection conditions . Within the endosomal and/or lysosomal compartment, proteases convert virions into particles that resemble in vitro-generated intermediate subvirion particles (ISVPs) [10–14]. These uncoating intermediates, typically prepared using chymotrypsin or trypsin, lack σ3 and have a cleaved form of μ1. Studies using ISVPs and ISVPs recoated with recombinant outer capsid proteins reveal that σ3 plays a key role in regulating reovirus cell entry by interacting with, protecting, and controlling the conformational status of the underlying penetration protein μ1 [15–18]. In cells that cannot efficiently mediate σ3 degradation during uncoating, reovirus infection is slow or blocked; these cells can be productively infected by particles that lack σ3 . In vitro, ISVP-like particles can be generated by a variety of proteases in addition to chymotrypsin and trypsin, including proteinase K, thermolysin, endoproteinase lys-C, Cat L, Cat B and Cat S[3, 19–21].
Recent work has provided insight into the cellular determinants of reovirus uncoating. In murine fibroblasts, where reovirus entry has been best studied, the cysteine proteases Cat L, and to a lesser extent Cat B, are required for σ3 removal, whereas the aspartyl protease Cat D is not [14, 21–25]. Virion disassembly in murine fibroblasts also requires acidic pH[10, 26, 27]. Recently, we demonstrated that reovirus uncoating in the macrophage-like cell line P388D is mediated by the acid-independent lysosomal cysteine protease Cat S. This finding revealed that in different cell types, distinct proteases can facilitate reovirus uncoating. Our results suggested a model in which infection in some cells is acid-dependent because the proteases that mediate σ3 removal in those cells require acidic pH for maximal activity. Thus, in fibroblasts or other cells in which the acid-dependent proteases Cat L and Cat B mediate σ3 removal, infection is acid-dependent [21, 23, 28], whereas in Cat S-expressing cells it is not , because Cat S maintains its activity at neutral pH . Insight from the analysis of reovirus cell entry facilitated the recent discovery that activation of the Ebola virus glycoprotein also depends on the activity of the acid-dependent endosomal proteases Cat B and Cat L .
The role that specific intracellular and extracellular proteases play in regulating reovirus tropism, spread, and disease in animals is largely unknown, except in the murine intestinal tract where pancreatic serine proteases have been shown to mediate σ3 removal [31, 32]. Reovirus also naturally infects hosts via the respiratory tract [33–35]. One protease with well-described effects in the respiratory tract is elastase 2 (GenBank NM_001972), an inflammatory serine protease of the chymotrypsin family, which is predominantly expressed by neutrophils . NE plays a prominent role in wound repair [37–39] and in controlling microbial infections [38–40]. NE expression can also promote pathogenesis; it has been implicated in smoke-induced emphysema , respiratory syncytial viral bronchiolitis  and in the respiratory syndrome ARDS . The fact that reovirus replication in the rodent lung causes an influx of neutrophils [35, 43] and that reovirus infection can recapitulate ARDS , led us to ask whether NE could mediate productive reovirus uncoating. We investigated reovirus infection in the monocyte-like cell line U937, because it is known to express NE . Experiments described in this report demonstrate that reovirus infection in U937 cells does not require cysteine protease activity and is not blocked in the presence of agents that raise vesicular pH. Studies using protease inhibitors suggest that, in the absence of cysteine protease activity, NE is largely responsible for productive infection of U937 cells. NE can directly mediate σ3 removal from reovirus virions; the resultant particles are infectious and do not require additional intracellular proteolysis. Our data raise the possibility that NE is involved in reovirus replication in the respiratory tract.
Reovirus infection of U937 cells does not require cysteine protease activity
Next, we compared reovirus replication in E64-treated U937 and L929 cells. Cells were pre-treated for 3 h and infected with Lang virions or ISVPs at a multiplicity of infection (MOI) of 3. The results of a representative experiment are shown in Fig. 1B. In the absence of E64, both L929 and U937 cells supported reovirus replication, consistent with the fact that these cells express Cat L. As expected, E64 blocked virion infection of L929 cells; however, viral yields in E64-treated U937 cells were only slightly reduced relative to untreated cells. ISVPs, which lack capsid protein σ3, replicated efficiently in treated cells, indicating that 300 μM E64 was not toxic to either cell type. These results demonstrate that productive infection of U937 cells by Lang virions does not require the activity of E64-sensitive, papain-like cysteine proteases.
Infection of U937 cells is acid-independent
Analysis of reovirus replication in U937 cells differentiated by PMA
To examine the effect of U937 cell differentiation on reovirus infection, PMA-treated and untreated U937 cells were left untreated or were treated with E64 for 3 h and infected with Lang virions or ISVPs at an MOI of 3. Yields were measured at 3 d p.i. and the results of a typical experiment are shown in Fig. 3B. In the absence of E64, PMA-treated U937 cells were permissive to infection by virions. PMA treatment only decreased yields by ~0.5 log relative to untreated cells. In contrast, when PMA-differentiated U937 cells were treated with E64 to inhibit cysteine protease activity, they no longer supported productive infection by Lang virions. Because these results could be explained if E64 was toxic to PMA-treated U937 cells, we examined the replication of ISVPs. In the presence of E64, ISVPs replicated to high yields in both undifferentiated and differentiated U937 cells. Since PMA-induced differentiation of U937 cells caused a substantial decrease in NE expression, these results are consistent with the hypothesis that NE or another similarly regulated neutral protease facilitates productive reovirus infection in promonocytic (pre-differentiated) U937 cells.
NE can facilitate reovirus infection in U937 cells
Inhibition of NE activity in U937 cells.a
% NE Activityb
% Cat Activityc
7.5 ± 5.0
84.3 ± 5.8
87.0 ± 9.0
88.3 ± 9.4
8.8 ± 3.5
89.5 ± 9.1
88.8 ± 10.0
86.3 ± 3.6
-4.7 ± 1.6
98.8 ± 3.9
NE-generated subviral particles are infectious and do not require additional proteolytic processing
To determine if NE-generated SVPs required further proteolytic processing of σ3, L929 cells were pre-treated with E64 to block cysteine protease activity and infected at an MOI of 3 with Lang virions, ISVPs or NE-generated subviral particles (NE-SVPs). Viral yields were determined at 1 d p.i. As expected, E64 blocked infection of L929 cells by virions. In contrast, both ISVPs and NE-SVPs replicated efficiently in the presence of the cysteine protease inhibitor (Fig. 5B). Because virion disassembly in L929 cells requires acidic pH , we also examined the capacity of NE-SVPs to infect L929 cells treated with Baf, NH4Cl or monensin, three agents that raise vesicular pH by distinct mechanisms. Cells were treated with these agents and then infected with virions, ISVPs or NE-SVPs at an MOI of 10. At 18 hours post infection (h p.i.), cell lysates were harvested and expression of the reovirus non-structural protein μNS was analyzed by immunoblotting (Fig. 5C). As expected, when treated cells were infected with virions, viral protein expression was blocked. In contrast, μNS expression was evident even in the presence of agents that raise pH when infections were initiated with ISVPs or NE-SVPs (Fig. 5C). Together, these results demonstrate that NE can directly mediate σ3 removal from virions to generate infectious particles that do not require further proteolytic processing by acid-dependent cysteine proteases in L929 cells.
NE, an acid-independent serine protease, can promote productive reovirus infection in U937 promonocytes
Serine proteases are involved in reovirus infection in the mammalian intestinal tract  and in this report we provide evidence that they can mediate uncoating and promote infection in U937 cells. This expands the range of proteases that promote reovirus infection in cell culture to include NE as well as the cysteine proteases Cat L, Cat B, and Cat S. Several lines of evidence now support the notion that protease expression is a cell-specific host factor that can impact reovirus infection. For example, some reovirus strains are inefficiently uncoated by Cat S and thus do not replicate to high yield in P388D macrophages . In this report we demonstrate that PMA-induced differentiation influences the type of protease that mediates reovirus uncoating in U937 cells. In these cells, PMA treatment is reported to increase Cat L expression  and decrease expression of the serine proteases NE and Cat G [56, 57]. Accordingly, when we used PMA to induce U937 cell cultures to differentiate, reovirus infection became sensitive to the cysteine protease inhibitor E64. We suspect that Cat L is largely responsible for uncoating in these PMA-differentiated cells, but the acid-independent protease Cat S may also play a role. We are currently addressing this question by analyzing infection in PMA-differentiated cells treated with either Baf or NH4Cl.
Does the serine protease Cat G also play a role in reovirus infection of U937 cells?
Our data do not completely resolve this question. Cat G is expressed by U937 cells and, like NE, it is down-regulated by PMA treatment. Furthermore, we found that in vitro treatment of reovirus virions with purified Cat G generates SVPs that behave like NE-SVPs in that they are infectious in the absence of further proteolytic processing (data not shown). Results of our experiment with the NE-specific inhibitor suggest that NE is largely responsible for the E64-resistant infection in U937 cells. While this inhibitor is reported not to inhibit Cat G , we have not independently confirmed this. Another approach to assess the role of Cat G in reovirus infection of U937 cells would be to examine the effect of Cat G-specific inhibitors on infection. We tried one such inhibitor, Cathepsin G Inhibitor I (Calbiochem), but found that it was cytotoxic to U937 cell cultures. Given that both NE and Cat G can generate infectious reovirus SVPs, more work needs to be done in order to understand the role that these two proteases play in infection in these cells.
The acid-dependence of reovirus infection is a reflection of the requirements for protease activation
Previously, we reported that virion uncoating mediated by Cat S does not require acidic pH . These results were consistent with the acid-independence of Cat S activity . Together, the results in Fig. 2 and Fig. 4 reveal that, like Cat S, NE-mediates infection in an acid-independent manner. This finding thus provides further support for a model in which the requirement for acidic pH during reovirus infection of some cell types reflects the requirement for acid-dependent protease activity in those cells rather than some other requisite acid-dependent aspect of cell entry. The small effect of Baf and NH4Cl on E64-resistant reovirus growth (Fig. 2) may reflect the participation of one or more acid-dependent proteases (such as Cat D) in the activation of NE.
Does NE mediate uncoating intracellularly or extracellularly in U937 cell cultures?
Elastase is stored in azurophilic granules that are the major source of acid-dependent hydrolases in neutrophils . Although these granules do not contain LAMP-1 or LAMP-2  they contain the lysosomal markers LAMP-3  and CD68  and are accessible to endocytosed fluid-phase markers under conditions of cellular stimulation . NE can be released from neutrophils during degranulation  and its cell surface expression can be induced upon PMA treatment . However, studies in U937 cells have shown that NE is predominantly retained intracellularly and that little if any activity is present in the extracellular medium . Consistent with this, we have been unable to generate ISVP-like particles by treatment of virions with U937 culture supernatants (data not shown). This observation, together with our finding that PMA treatment decreases the capacity of E64-treated U937 cells to support reovirus infection, leads us to favor a model in which NE-mediated virion uncoating in U937 cell cultures occurs intracellularly.
Implications for infection in the host
In vivo, a number of viruses, including dengue and respiratory syncytial virus, induce the release of IL-8, a cytokine that serves as a chemoattractant for neutrophils and promotes their degranulation [66, 67]. Reovirus replication in the rat lung results in neutrophilic invasion [35, 43] and studies in cell culture indicate that reovirus infection can induce IL-8 expression . Thus, the capacity of reovirus to induce IL-8 secretion in vivo might facilitate the release of neutrophilic lysosomal hydrolases, including NE, into the extracellular milieu. In this report, we have shown that mammalian reovirus can utilize this acid-independent serine protease for uncoating. Our data suggest that, in vivo, one consequence of reovirus-induced IL-8 expression would be the generation of infectious NE-SVPs. Like ISVPs, these particles would be predicted to have an expanded cellular host range because they can infect cells that restrict intracellular uncoating . Thus, inflammation might be predicted to exacerbate reovirus infection by promoting viral spread. Future studies using mice with deletions in the NE gene will be required to elucidate the role this protease plays during reovirus infection in the respiratory tract and other tissues. Finally, given the recent finding that endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection , our results raise the interesting possibility that NE or other neutrophil proteases may play a role in cell entry of other viruses.
Cells and viruses
Murine L929 cells were maintained as suspension cultures as described previously [Kedl, 1995 #94]. U937 cells were maintained in RPMI medium (GIBCO-BRL, Gaithersburg, MD) supplemented to contain 10% fetal calf serum (Gibco-BRL), 50-units/ml penicillin (GIBCO-BRL), 50 μg/ml streptomycin (GIBCO-BRL) and 2 mM glutamine (GIBCO-BRL). Where indicated, U937 cells were differentiated by treatment with 150 nM of PMA (Sigma) for 48 h prior to infection.
Third-passage lysate stocks of reovirus were prepared in L929 cell cultures. Purified virions were prepared by CsCl density gradient centrifugation of extracts from cells infected with third-passage lysate stocks [Furlong, 1988 #81]. ISVPs were prepared by treating purified virions with chymotrypsin as described elsewhere [Nibert, 1992 #95].
Measurement of cysteine protease activity
Cysteine protease activity was measured as described previously  with some minor modifications. Briefly, P388D U937 and L929 cells (2 × 106 each) were incubated in the presence or absence of 300 μM E-64, 5 nM LHVS, or 5 μM CA074 for the times indicated. After incubation, cells were trypsinized, collected by centrifugation at 179 × g for 10 min at 4°C and washed once in PBS. Cell pellets were resuspended in 100 μl of lysis buffer (100 mM sodium acetate [pH 5.5], 1 mM EDTA, and 0.5% Triton X-100), incubated on ice for 30 min and cell debris was pelleted by centrifugation at 89 × g for 10 min at 4°C. For each sample, 20 μl of clarified cell lysate was added to 80 μl of reaction buffer (100 mM sodium acetate [pH 5.5], 1 mM EDTA, 4 mM dithiothreitol) in a well of a black 96-well plate (Corning). To measure Cat B activity, 100 μM Z-Arg-Arg-7-amido-4-methylcoumarin (Z-Arg-Arg MCA) (Calbiochem) was included in the reaction buffer. To measure Cat L and Cat B activity, 100 μM Z-Phe-Arg-7-amido-4-methylcoumarin (Z-Phe-Arg-MCA) (Calbiochem) was added to the reaction buffer. Reactions were incubated for 30 min at room temperature with gentle tapping every 10 min. Fluorescence was measured using an FL600 microplate reader (Bio-Tek Instruments, Inc., Winooski, VT) with an excitation of 390 nm and emission at 460 nm.
Measurement of serine protease activity
NE activity was determined by incubating 3 × 106 U937 cells in the presence or absence of 200 μM NE inhibitor (N-(methoxysuccinyl)-Ala-Ala-Pro-Val-chloromethyl ketone) (Sigma) for the indicated times. After treatment, cells were collected by centrifugation at 179 × g for 10 min at 4°C, washed twice in PBS and lysed in TLB (10 mM Tris [pH 7.5], 2.5 mM MgCλ2, 100 NaCl, 0.5% Triton X-100, 5 μg/μl of leupeptin [Sigma], 1 mM PMSF) for 30 m on ice. The lysate was clarified by centrifugation at 89 × g for 10 min at 4°C. For each sample, 20 μl of cell lysate was added to 80 μl of virion dialysis buffer (VDB) (150 mM NaCl, 10 mM MgCl2, 10 mM Tris [pH 7.5]) containing 500 μM of NE substrate (MeOSuc-Ala-Ala-Pro-Val-pNA) (Calbiochem) and incubated for 30 min at room temperature with gentle tapping every 10 min. Absorbance was measured at 405 nm using an EL340 BioTek microplate reader (Bio-Tek Instruments).
Analysis of viral growth
Cells were infected (in triplicate) at the indicated MOI and adsorption was allowed to proceed for 1 h on ice at 4°C. After adsorption, cells were pelleted by low speed centrifugation and resuspended in fresh media. Virus and cells were then added to dram vials (2 × 105 cells/vial) containing 1 ml of chilled medium. Prior to infection, some cells were pre-treated for 3 h with 300 μM E64 and/or 25 nM Baf (Sigma), 20 mM NH4Cl (Sigma) and 200 μM NE inhibitor. Inhibitors were included in the medium throughout the time course for treated samples. Time zero samples were immediately frozen at -20°C and remaining samples were incubated at 37°C until the desired time point was reached. Samples were frozen and thawed three times and titrated by plaque assay on L929 cells as described elsewhere . Viral yields were calculated according to the following formula: log10(PFU/ml)t = x hr - log10 (PFU/ml)t = 0 +/- standard deviation (SD).
In vitro analysis of NE-mediated uncoating
NE digestions were performed as follows. Purified virions (7.5 × 1010) were incubated with 25 μg/ml of purified NE (Calbiochem) in 20 μL VDB at 37°C for the times indicated. Mock-treated samples were incubated in VDB for the longest time point. 1 mM PMSF and 200 μM of NE inhibitor were added to the samples to terminate the reactions. Protein sample buffer (0.125 M Tris [pH 8.0], 1% SDS, 0.01% bromphenol blue, 10% sucrose, and 5% β-mercaptoethanol) was added to each reaction mixture and samples were resolved on SDS-12% polyacrylamide gels. The protein gels were stained with Coomassie Brilliant Blue.
Immunoblot analysis of NE expression
To analyze NE expression, cell lysates were generated from U937 cells, either treated or untreated for 48 h with 150 nM PMA as described for the analysis of viral protein expression. Lysate from the equivalent of 1 × 106 cells was run on SDS-12% polyacrylamide gels and transferred to nitrocellulose. Membranes were blocked overnight in TBST containing 10% nonfat dry milk. NE expression was analyzed using a polyclonal antibody against NE (1:400 in TBST) (Santa Cruz Biotechnology Inc, Santa Cruz, CA). Membranes were washed with TBST and incubated with a horseradish peroxidase-conjugated anti-goat IgG (1:5000 in TBST). Bound antibody was detected by treating the nitrocellulose filters with enhanced chemiluminescence (ECL) detection reagents (Amersham) and exposing them to Full Speed Blue X-ray film (Henry Schein, Melville, NY).
Analysis of viral protein expression in infected cells
Cells were plated at 106/well in a 6-well plate 18–24 h prior to infection. Virus was allowed to adsorb to cells for 1.5 h at 4°C. At this temperature, virus binds to cells but is not internalized . After adsorption, the cultures were incubated at 37°C in fresh medium. Prior to some infections, cells were pre-treated for 3 h with 300 μM E64, 100 nM Baf, 25 μM monensin (Sigma), or 20 mM NH4Cl. In those instances inhibitors were also included in the post-adsorption culture medium. At the indicated times p.i., cells were collected by centrifugation at 179 × g, washed twice in chilled PBS and lysed in TLB. After centrifugation at 179 × g to remove cellular debris, samples were resuspended in sample buffer. Protein samples (representing 1 × 105 cells) were analyzed by electrophoresis on SDS-12% polyacrylamide gels and transferred to nitrocellulose membranes for 2 h at 100 V in 25 mM Tris-192 mM glycine-20% methanol. Nitrocellulose membranes (Bio-Rad Laboratories, Hercules, Calif.) were blocked overnight at 4°C in TBST (10 mM Tris [pH 8.0], 150 mM NaCl and 0.05% Tween) containing 5% nonfat dry milk, rinsed with TBST, and incubated with a rabbit anti-μNS polyclonal antiserum  (1:12500 in TBST) for 1 h. Membranes were subsequently washed with TBST and incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit immunogloblin G (IgG) (1:7500 in TBST) (Amersham, Arlington Heights, Ill.). Bound antibody was detected by treating the nitrocellulose filters with enhanced chemilumescence (ECL) detection reagents (Amersham) and exposing the filters to Full Speed Blue X-ray film (Eastman Kodak, Rochester, N.Y.).
Generation of NE subviral particles for infection
Purified virions (1.4 × 1011) were incubated with 25 μg/ml of purified neutrophil elastase (Calbiochem) in 40 μL of VDB at 37°C for 3 h. Reactions were terminated by adding 1 mM PMSF and 200 μM NE inhibitor to the reaction mixture. 5.0 × 1010 particles were run on SDS-12% polyacrylamide gels stained with Coomassie Brilliant Blue to confirm the removal of σ3. Viral infectivity was determined by plaque assay on L929 cell monlayers.
Analysis of virus titer after NE treatment of virions
Purified Lang virions (1.4 × 1011) were treated with 25 μg/ml of NE in 40 μL of VDB at 37°C for the times indicated. Reactions were terminated as described above. To verify σ3 removal, the proteins from 5.0 × 1010 particles were separated on SDS-12% polyacrylamide gels and visualized with Coomassie Brilliant Blue staining. Viral infectivity for each time point was determined by plaque assay on L929 cell monolayers.
We express thanks to Stephen Rice and Max Nibert for critically reviewing this manuscript. We also thank members of the Schiff, Rice and Bresnahan laboratories for their constructive input throughout these studies.
This work was supported by NIH grant AI45990 and University of Minnesota Graduate School Grant-In-Aid #20017 to L. A. S.
J. W. G. received support from NIH training grant 2T32 AI0742.
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