1 Virologie Moléculaire et Structurale, CNRS-INRA, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
2 Biochimie et Structure des Protéines, INRA, 78352 Jouy-en-Josas Cedex, France
Correspondence
Didier Poncet
poncet{at}vms.cnrs-gif.fr
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ABSTRACT |
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INTRODUCTION |
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Rotaviruses particles have a complex architecture of three concentric capsid layers (Prasad et al., 2001). The innermost capsid layer, composed of VP2, is surrounded by the intermediate capsid layer composed of VP6. The outermost layer is composed of the glycoprotein VP7 and the spike protein VP4. VP7 is a calcium-binding glycoprotein that interacts with integrins during the final virus adsorption step (Lopez & Arias, 2004
).
Trypsin treatment of viral particles modifies the structure of VP4 and is correlated with an enhancement of infectivity by a mechanism that is not yet clearly established (Crawford et al., 2001). Conversely, protease inhibitors can prevent the development of rotavirus-induced diarrhoea and reduce virus replication in cell culture (Katyal et al., 2001
; Vonderfecht et al., 1988
). The two trypsin cleavage products of VP4 (VP8* and VP5*) remain associated with the virion (Dormitzer et al., 2004
). VP5* contains a putative fusion domain and VP8* mediates initial cell attachment (Lopez & Arias, 2004
).
In this study, we show that trypsin molecules are tightly associated with the virion. Trypsin activity is inhibited when associated with virions, but is activated upon outer capsid layer solubilization. Inhibition of virus-associated trypsin by addition of protease inhibitors to cell culture medium reduces primary transcription of the genome, and thus virus replication.
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METHODS |
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Purification of rotavirus triple- (TLPs) and double- (DLPs) layer particles.
Rotavirus-infected MA104 cells were incubated for 4 days at 37 °C and frozen when a complete cytopathic effect (CPE) was reached. The thawed tissue-culture suspension containing the virus and cell debris was subjected to ultracentrifugation for 60 min in a 45TI rotor at 90 000 g at 4 °C. The supernatant was discarded and the pellet treated with Vertrel XF as described by Mendez et al. (2000). The aqueous phase was then mixed with CsCl, adjusted to obtain a refractive index of 1·369 and centrifuged for 16 h at 80 000 g at 4 °C. The upper (TLPs) and lower (DLPs) bands were collected separately and each centrifuged in CsCl again as described above. Particles were kept suspended in CsCl at 4 °C and salt was removed by a spin column (Sephadex G25; Pharmacia).
Detection of trypsin and trypsin activity.
Trypsin activity was analysed by zymogram assay or SDS-PAGE. A combination of 1 µg BSA, EGTA (500 µM final concentration) and 2 µg aprotinin [4·4 trypsin inhibitor units (TIU) mg1] was added to 4 µg desalted TR-TLP or DLP in 20 mM Tris/HCl, pH 7·4, 50 mM NaCl and 100 µM CaCl2, and incubated for 15 min at 37 °C. EGTA was added before incubation but after addition of BSA or aprotinin. Samples, in Laemmli sample buffer under non-reducing (2 % SDS, 10 % glycerol, 0·025 % bromophenol blue) or reducing (plus 700 mM 2-mercaptoethanol) conditions, were incubated at room temperature for 5 min and analysed by PAGE. PAGE was performed using the Nupage system (Invitrogen), in Tris-MES (pH 7) running buffer as recommended by the manufacturer. Following electrophoresis, gels were stained with 1 % Coomassie blue R-250. Gels were destained completely by incubation in 10 % acetic acid in 40 % ethanol and then silver-stained (Silver Stained Plus kit; Bio-Rad). Zymogram analysis was performed in Novex 416 % gels (Invitrogen) with blue-stained -casein incorporated as a substrate. Zymograms were stained with ethidium bromide (10 µg ml1) to visualize dsRNA.
Reverse-phase HPLC.
Two-hundred micrograms purified TLPs, or DLPs as a control, was diluted fivefold in 10 mM Tris/HCl buffer, pH 7·4, containing 1 mM CaCl2 and sedimented by centrifugation (100 000 g, 10 min) at 4 °C to remove CsCl. The pellet was acidified in 2·5 % formic acid (Fluka) and incubated overnight at room temperature. Subsequently, the reaction mixture was centrifuged (13 000 g, 10 min). The supernatant was analysed by reverse-phase HPLC (RPLC) with an Applied Biosystems device on a C4 LC-Packings capillary column. A linear acetonitrile gradient (4·585·5 %) was made by mixing 0·1 % (v/v) formic acid and 4 mM ammonium acetate (Fluka) in water with 90 % acetonitrile, 0·1 % (v/v) formic acid and 4 mM ammonium acetate in water, and applied at a flow rate of 4 µl min1.
Sequencing and mass spectrometry.
Peptides present in fractions collected from RPLC analysis were characterized by N-terminal sequencing using Edman chemistry, with a Perkin-Elmer Procise 494 HT protein sequencer. Proteins were identified by searching amino acid sequence databases using the MS-Pattern search engine (http://prospector.ucsf.edu/).
Rotavirus infection and inhibition assays.
For viral infection, 5x105 MA104 cells were plated in 6-well plates, incubated for 72 h and then washed three times with EMEM and incubated for 4 h with EMEM. Cells were infected with purified rotavirus TR-TLPs (RF bovine strain) at an m.o.i. of 0·5 p.f.u. per cell. Virus adsorption was performed for 1 h at 15 or 37 °C, supplemented or not with 624 TIU aprotinin l1 (Sigma-Aldrich). The inoculum was then removed and cells were washed three times with EMEM and incubated for 5 or 18 h with EMEM at 37 °C with 5 % CO2 in the presence of various concentrations of aprotinin (1·6252496 TIU l1).
For preparation of cell lysates, cells were harvested by treatment with trypsin at 5 or 18 h post-infection (p.i.) and washed twice with EMEM by centrifugation at 200 g for 5 min at room temperature. Cell pellets were subjected to three freezethaw cycles, resuspended in 1 ml EMEM and clarified by centrifugation at 16 000 g for 5 min. Infectious virus titres were determined by indirect IF assay using MA104 cell monolayers grown in 96-well microtitre plates (Ciarlet et al., 1994).
For detection of rotavirus antigens, cell lysates were prepared at 5 h p.i. using the protocol described above. ELISA was used as described previously (Schwartz-Cornil et al., 2002). To determine the toxicity of protease inhibitors, infected and mock-infected cell monolayers were incubated with 0·1 % Trypan blue prepared in PBS for 30 min at 37 °C and live cells were counted.
Preparation of total mRNA and real-time PCR assays.
MA104 cells (1·5x105 cells per well) were plated in 24-well plates for 72 h. Cells were infected with RF rotavirus at an m.o.i. of 0·5 p.f.u. per cell for 1 h at 15 or 37 °C. Cells were then washed three times with EMEM and incubated in EMEM containing different amounts of aprotinin (62·4624 TIU l1) for 3 h at 37 °C. The total RNA in the pellets was extracted using TRIzol reagent (Invitrogen). One-tenth volume of the total cellular RNA was heat-denatured at 65 °C for 5 min and used as template for reverse transcription (RT). RT reactions were performed using 50 U Superscript II reverse transcriptase (Invitrogen) at 42 °C for 50 min in the presence of 20 mM Tris/HCl (pH 8·4), 50 mM KCl, 5 mM magnesium acetate, 10 mM DTT, 40 U RNaseOut (Invitrogen) and 12·5 ng random hexanucleotide primers µl1 (Invitrogen). A 1 : 125 dilution of the RT reaction was used for quantitative RT-PCR (Q-RT-PCR) analysis.
Primers were generated for Q-RT-PCR amplification of rotavirus RF segment 6 [sense, 5'-GCTTTAAAACGAAGTCTTCAAC-3' (positions 224); antisense, 5'-GGTAAATTACCAATTCCTCCAG-3' (positions 166188)] and the human cellular housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [sense, 5'-GGGCGCCTGGTCACCAGGGCTGC-3' (positions 118141); antisense, 5'-GAGCCCCAGCCTTCTCCATGGTGG-3' (positions 383407)]. Each primer pair was used at a concentration of 150 nM in a 25 µl reaction mixture. Real-time PCR was performed using the Stratagene MX3000p apparatus and the Brilliant SYBR Green Q-PCR Master Mix (Stratagene). The method and the quantification procedure have been extensively described elsewhere (Overbergh et al., 1999). The conditions used for Q-PCR consisted of denaturation for 15 s at 95 °C, annealing for 18 s at 55 °C and amplification for 15 s at 72 °C for 40 cycles, with an initial step of denaturation at 95 °C for 10 min. Following each cycle, accumulation of PCR products was detected by monitoring the increase of fluorescence of the dsDNA-binding SYBER Green reporter dye. Each PCR amplification was performed in duplicate, in optical 96-well reaction plates with optical caps (Stratagene). Analysis and quantification were performed using the Stratagene MX3000p 2.00 software. A range from 1 : 25 to 1 : 250 000 dilutions of total cDNA was used to define standard curves for each primer pair and to quantify primer efficiency, which reflects the capacity to amplify small amounts of target. Efficiency of primers for rotavirus gene 6 and the GAPDH gene was measured to be 102·3 and 100·8 %, respectively. The amount of gene 6-specific mRNA was related to that of GAPDH gene-specific mRNA in each sample. Standard curves were used to estimate the relative amounts of DNA. Following the PCRs, a melting curve was established in the range of 60 to 95 °C, to identify amplified products by their melting temperature (Tm). No amplification was observed in the absence of template.
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RESULTS |
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These data show clearly that the trypsin molecules are neither free nor active in mature TR-TLPs, as would be expected if the purified virion preparations were contaminated with trypsin from the cell culture medium, but are closely associated with the virus outer layer. Trypsin can be activated only when the TR-TLP outer layer is solubilized, resulting in the degradation of outer capsid proteins VP7 and VP5* and of exogenously added substrates, such as BSA.
Trypsin is not accessible to protease inhibitors in TLPs
To clarify the location of the trypsin within TR-TLPs, we attempted to inhibit its proteolytic activity in vitro by addition of aprotinin, a powerful serine protease inhibitor. After electrophoresis under non-reducing conditions, proteins with protease activity can be visualized on a zymogram as white bands. Using this methodology, trypsin molecules can be observed as a white band migrating with the appropriate molecular mass (Fig. 3a, lane 1) and a minor band, which is most likely due to the non-reducing conditions of the electrophoresis. Proteins without protease activity can be visualized after Coomassie blue staining of the zymogram (Fig. 3a
, lane M). Inhibition of trypsin activity by addition of aprotinin results in the disappearance of the white bands correlated with the protease activity of trypsin and appearance of a low molecular mass band corresponding to aprotinin (Fig. 3a
, lanes 2 and 3). Addition of EGTA inhibited neither trypsin (Fig. 3a
, lane 4) nor aprotinin (Fig. 3a
, lane 5). Thus, treatment at room temperature with 2 % SDS in the presence of EGTA and electrophoresis under non-reducing conditions did not alter the activity of trypsin or aprotinin.
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When TR-TLPs treated for 5 min at room temperature in 2 % SDS were subjected to zymogram analysis, trypsin activity could be seen as a white smear in addition to the sharp white band described above (Fig. 3a, lane 7). This smear may be interpreted as a progressive release of the trypsin associated with the viral outer proteins, due to incubation with 2 % SDS, or migration into the gel. Addition of aprotinin to TR-TLPs prior to incubation in 2 % SDS did not significantly reduce the trypsin activity (Fig. 3a
, lanes 7 and 8). The presence of a smear suggests that the TR-TLPs were not totally disrupted after incubation at room temperature, and that the capsid proteins either did not enter the gel or migrated as complexes of various molecular masses. This interpretation was confirmed by ethidium bromide staining to visualize the genomic dsRNA present in the gel. The majority of nucleic acids, probably associated with viral proteins, was present at the top of the gel, whereas after treatment at 80 °C all the individual dsRNA segments could be visualized (Fig. 3b
, lanes 6 and 710).
When TR-TLPs were treated at room temperature with EGTA in 2 % SDS prior to zymogram analysis, trypsin activity could also be seen as a sharp white band, but none of the viral proteins were visible (Fig. 3a, lane 9). When aprotinin was added prior to EGTA treatment, the bands corresponding to VP7 and VP5* were again visible (Fig. 3a
, lane 10). Thus, aprotinin can inhibit trypsin only when present during solubilization of the outer capsid proteins by EGTA.
The smearing of the protease activity confirmed the tight binding of trypsin to TR-TLPs, since their association resists 2 % SDS at room temperature but not at 80 °C, and indicated that the trypsin is not accessible to aprotinin before solubilization of the outer protein layer.
Effect of aprotinin on rotavirus multiplication
To determine the functional role of trypsin molecules associated with infectious virus, we examined whether protease inhibitors have an effect on virus replication. After adsorption of purified TR-TLPs onto cells, aprotinin was added to the cell culture medium. The effect of the addition of this protease inhibitor was evaluated by determining the amount of rotaviral antigen present in infected cells 5 h p.i. by ELISA. The doseresponse curve of aprotinin on antigen production is shown in Fig. 4. Aprotinin significantly reduced the production of viral antigen and addition of 624 TIU l1 resulted in an eightfold reduction of the viral titre recovered at 18 h p.i. (data not shown). Even at the highest dose used, aprotinin did not have a toxic effect on MA104 cells, as estimated by trypan blue exclusion assay (data not shown). This last observation is in accordance with previous studies showing the very low cytotoxicity of this serine protease inhibitor (Shah et al., 2004
). These findings show that addition of serine protease inhibitors to cell culture medium reduces rotavirus antigen production in a dose-dependent manner.
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Early viral transcription is reduced in the presence of aprotinin
To address which early step of the viral life cycle is affected by aprotinin, transcription of viral mRNAs was examined. MA104 cells were infected with purified TR-TLPs and incubated in the presence of different concentrations of aprotinin. Total mRNA was then extracted, denatured at 65 °C and then the random-primed RT reaction was performed. To estimate viral transcription, the amount of rotavirus gene 6 mRNA present in the experimental samples was determined by Q-RT-PCR and normalized using the amount of cellular GAPDH mRNA present in each sample.
To evaluate the amount of viral dsRNA which could be detected under the conditions of denaturation used, we performed a control experiment by extracting total RNA following viral adsorption at 15 °C (0 h p.i.). This control experiment showed that the viral dsRNA remaining associated with the cells were not amplified by Q-RT-PCR when RNA was heated to 65 °C prior to RT (data not shown). Thus, the signal detected under our experimental conditions corresponds to the gene 6 mRNA present in the cell cytoplasm and not the gene 6 dsRNA. It is a measure of the viral mRNAs synthesized in the cell and, hence, an evaluation of the quantity of virus which has passed the cellular membrane.
After infection of cells followed by incubation with various amounts of aprotinin, total RNA was extracted at 3 h p.i. and viral mRNAs were quantified. As the concentration of aprotinin added to the medium increased, viral transcription decreased (Fig. 6). Because trypsin was not added during infection, this result may indicate that inhibition of virion-associated trypsin interferes with viral penetration and thus viral transcription.
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DISCUSSION |
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How and when does trypsin gain access to the virus?
The porcine origin of the trypsin associated with TR-TLPs was established unambiguously by protein sequencing. We have shown that trypsin is not active when the viral outer capsid is intact. It is possible that, following virus assembly, extracellular trypsin binds to VP4 and/or VP7, which could block the enzymic activity of the protease in a manner similar to trypsin inhibitors like serpins (Ye & Goldsmith, 2001). Another possibility is that trypsin enters cells due to altered cell permeability (Obert et al., 2000
) or during virus entry, as was shown for the toxin
-sarcin (Liprandi et al., 1997
), and is then incorporated into the virus particle during virus maturation. A similar hypothesis has already been presented to explain the differences in VP4 conformation between TR-TLPs and NTR-TLPs seen by electron cryomicroscopy (Crawford et al., 2001
).
Virus-associated trypsin: nature versus nurture?
In this study we used the bovine rotavirus RF strain adapted to cell culture, but trypsin was also found in other cell cultured-adapted rotavirus strains. Rotavirus infectivity in cell culture is enhanced by trypsin treatment, and cleavage of VP4 by trypsin occurs in vivo in the lumen of the intestine prior to infection of enterocytes in animal models (Ludert et al., 1996). It is possible that viruses capable of incorporating trypsin molecules have a selective advantage during adaptation to cell culture.
Our results raise the question of the possible function of virus-associated trypsin in the viral life cycle of rotavirus. During rotavirus infection, viruses could acquire trypsin while passing through the gastrointestinal tract, an environment very rich in proteases. Again, rotaviruses capable of acquiring trypsin would have a selective advantage by being able to infect intestinal cells efficiently. Further studies need to be performed to verify whether trypsin can be detected in TLPs prepared from rotavirus-infected animals, in other enteric viruses or in other viruses requiring trypsin for optimal growth (e.g. influenza viruses; Klenk et al., 1975).
A role for the virus-associated trypsin in rotavirus entry
The results presented here indicate that addition of aprotinin to the cell culture medium inhibits viral multiplication, as seen by the decrease in RNA and viral protein synthesis in the presence of aprotinin. The protease inhibitor did not interfere with virus attachment, since the presence of aprotinin during adsorption alone was not sufficient to obtain maximal inhibition. These observations are in accordance with previous results showing that trypsin treatment of rotavirus does not attenuate viral attachment to cell membranes (Kaljot et al., 1988). The inhibition by aprotinin was more efficient when the protease inhibitor was present during recovery of endosomal activity during the transition from 15 to 37 °C. This suggests that, to be efficient, aprotinin has to enter the cell simultaneously with TR-TLPs. Q-RT-PCR results indicated that early viral transcription was reduced in the presence of aprotinin following adsorption, suggesting that aprotinin, and hence trypsin, acts after virus binding, but prior to initiation of viral transcription. These results are in agreement with previous observations showing that (i) infection with trypsin-treated virus leads to greater levels of RNA synthesis early in infection, (ii) trypsin converts non-infectious virions into infectious virions by allowing them to be decapsidated into the cell (Clark et al., 1981
), (iii) NTR-TLPs enter the cells more slowly than TR-TLPs and produce less virus (Crawford et al., 2001
; Kaljot et al., 1988
) and (iv) NTR-TLPs never produce virus titres as high as those produced by TR-TLPs, even when NTR-TLPs are treated exogenously with a high concentration of trypsin (Crawford et al., 2001
).
Although we cannot exclude an effect of trypsin or aprotinin on the cell itself, our results showing activation of trypsin upon solubilization of the viral outer capsid agree with previous experiments that have suggested a role for outer capsid proteins in membrane solubilization. TR-TLPs are able to solubilize pig jejunum brush border membrane vesicles charged with carboxyfluorescein (CF) after addition of EGTA or heating at 60 °C (Ruiz et al., 1994). Membrane solubilization was not observed with NTR-TLPs, unless they were treated first with EGTA and then with trypsin. Similar results were observed with intact cells by measuring incorporation of ethidium bromide (Ruiz et al., 1997
). These results were explained by the effect of VP5*, which is able to solubilize membranes (Denisova et al., 1999
), but membrane permeabilization can also be obtained in the absence of VP4. Trypsin treatment of VP7, solubilized from virus-like particles composed of VP2, VP6 and VP7 (VLP 2/6/7), is required to induce permeabilization of membrane vesicles detected by measurement of the release of CF (Charpilienne et al., 1997
). Conversely, it has been shown that addition of a monoclonal antibody specific for VP7 neutralizes rotavirus infection by impairing virus outer capsid solubilization (Ludert et al., 2002
). Altogether, these results indicate that VP7 solubilization is required for rotavirus infection and that solubilized and trypsinized VP7 is capable of permeabilizing cellular membranes in the absence of VP4.
We propose that, during rotavirus infection, trypsin molecules are bound to the outer capsid layer in an inactive form, and are activated with the solubilization of VP7 and VP4 due to a drop in calcium concentration in the endosomal vesicle (Chemello et al., 2002) (Fig. 7
). Activated trypsin may then cleave VP7 and VP4 into fragments capable of disrupting cellular membranes. However, virions could possibly enter the cell by an endosome-independent mechanism (Lopez & Arias, 2004
). We propose that, following rotavirus attachment, and a local drop in calcium concentration near the plasma membrane which partially solubilizes the viral outer capsid, VP7 or VP4 cleaved by trypsin could then permeabilize the plasma membrane. This step could be inhibited in the presence of aprotinin, which may enter cells along with the virus and gain access to trypsin during outer capsid solubilization. Entry of macromolecules present in the medium during rotavirus infection has already been described (Liprandi et al., 1997
). This would lead to DLPs gaining access to the cytoplasm to begin actively transcribing viral mRNA to complete the next step in the viral life cycle.
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ACKNOWLEDGEMENTS |
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Received 18 March 2005;
accepted 19 July 2005.
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