Veterinary Microbiology, 22 (1990) 137-152 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
137
Effects of Proteolytic Enzymes on the Infectivity, Haemagglutinating Activity and Protein Composition of Bluetongue Virus Type 20 J.A. COWLEY 1'2 and B.M. GORMAN 3
Queensland Institute of Medical Research, Bramston Terrace, Herston, 4006, Qld. (Australia) (Accepted 12 October 1989)
ABSTRACT Cowley, J.A. and Gorman, B.M., 1990. Effects of proteolytic enzymes on the infectivity, haemagglutinating activity and protein composition of bluetongue virus type 20. Vet. Microbiol., 29: 137-152. The effects on virus infectivity, haemagglutinating (HA) activity and polypeptide composition of bluetongue virus type 20 (BTV 20) were determined after digestion with the proteolytic enzymes, chymotrypsin, thermolysin and trypsin. Virus infectivity increased eight to 50-fold after exposure periods which reflected the activity of the proteases. Identical maximum increases in HA activity {i.e. 4096, 1024 and 128 HAU per 0.05 ml with sheep, bovine and human erythrocytes, respectively) occurred with each of the three proteases. Peak increases in virus infectivities and HA activities occurred after similar exposure periods. Outer capsid protein VP2 was the most sensitive virus protein to proteolytic digestion, being cleaved into a number of smaller polypeptides that remained attached to the virus particle. Digestion with chymotrypsin and thermolysin yielded four common cleavage products, designated P93, P76, P54 and P25 according to their estimated molecular weight, which suggested that they shared at least three cleavage sites. VP2 cleavage products resulting from digestion with trypsin differed somewhat from those of chymotrypsin and thermolysin, although the generation of polypeptides P93, P54 and P25.5 suggested the existence of common cleavage sites for the three proteases. Possible mechanisms whereby proteolytic cleavage of VP2 may enhance the infectivity and HA activity of BTV 20 are discussed.
INTRODUCTION
Early biochemical studies of the structure of bluetongue virus (BTV) demonstrated the existence of three distinct particle types (Martin and Zweerink, 1972; Verwoerd et al., 1972). Complete particles purified in sucrose gradients 'Author to whom correspondence should be addressed. 2Present address: Animal Research Institute, Queensland Department of Primary Industries, Yeerongpilly, 4105, Australia. 3Present address: 13 Daniel Street, Nambour, 4560, Australia.
0378-1135/90/$03.50
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possessed a double-layered protein capsid, with two viral proteins (VP2 and VP5 ) forming the outer capsid and two major (VP3 and VP7) and three minor proteins (VP1, VP4 and VP6) forming the nucleocapsid or core particle. Purification in CsC1 gradients, however, degraded BTV either into particles that lacked VP2, or into core particles that lacked both proteins (VP2 and VP5) associated with the outer capsid. In a detailed investigation of the BTV transcriptase, Van Dijk and Huismans (1980) showed that different concentrations of MgC12 acted similarly to CsC1 (at different pH values ) with respect to the selective removal of VP2 and VP5 from virus particles. Huismans et al. ( 1983, 1987) subsequently demonstrated that the action of MgC12 was also dependent on pH. At pH 5.0, dissociation of VP2 occurred with 0.2 M MgC12, while at pH 8.0, 1.0 M MgC12 was required to dissociate both VP2 and VP5; thus indicating that the BTV outer capsid was less stable at acidic pH than at basic pH. For some BTV isolates, dissociation of outer capsid proteins, with either CsCl or MgC12, was found to be enhanced by digestion with chymotrypsin (Van Dijk and Huismans, 1980, 1982; Mertens et al., 1986, 1987). However, digestion with chymotrypsin in the absence of cations resulted in the formation of so-called "intermediate" or "infectious sub-viral particles" (ISVPs) (Van Dijk and Huismans, 1980; Mertens et al., 1986, 1987). Mertens et al. (1986, 1987) characterised some effects of chymotrypsin on BTV 1 and BTV 4. They found that the resulting ISVPs retained their infectivity, lost haemagglutinating (HA) activity and shifted from a highly aggregated state to a mono-dispersed suspension. In addition, ISVPs possessed a VP2 which, when analysed in SDS-polyacrylamide gels, was found to be cleaved into at least three major polypeptides, designated VP2a, VP2b and VP2c. This cleavage of VP2 was suggested as the probable mechanism whereby ISVPs lost their ability to aggregate to themselves and to agglutinate erythrocytes. Changes in the molecular and biological properties of BTV resulting from enzymatic digestion with other proteases have, however, not been investigated. In this paper we have characterised the effects of chymotrypsin, and two other proteases (trypsin and thermolysin) on the infectivity, HA activity and protein composition of BTV 20. In contrast to Mertens et al. (1987), our results indicate that both biological activities can be initially enhanced with each of the three proteases. In addition, some preliminary data on the kinetic appearance of cleavage products of protease-sensitive proteins of BTV 20 are presented for each enzyme. MATERIALSAND METHODS Cells and virus
The PS-EK line of pig kidney cells (Gorman et al., 1975) was grown in Hanks' medium-199 containing 14 mM H E P E S pH 7.2, 100 U ml-1 penicillin,
EFFECTS OF PROTEOLYTIC ENZYMES ON BLUETONGUE VIRUS TYPE 20
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100 pg ml-1 streptomycin, 2 pg m l - ' amphotericin B and 10% fetal bovine serum (FBS). BTV 20 (isolate CSIRO 19) was originally obtained from Dr. T.D. St. George, CSIRO Division of Tropical Animal Science, Brisbane. A small plaque-forming type was isolated from this BTV 20 pool and plaque cloned twice more in PS-EK cells.
Virus infectivity titration BTV 20 was titrated for plaques in PS-EK cell monolayers in 9.8 cm 2 plastic dishes. Tenfold dilutions of virus were prepared in Hanks' medium-199 containing 5% FBS, 100 ttl inoculated per dish and the virus adsorbed 1 h at room temperature as previously described (Gorman et al., 1975). Excess inoculum was removed, cells overlaid with 3 ml per dish of Minimal Essential Medium (MEM AutoPowTM; Flow Labs.) containing 14 mM HEPES pH 7.2; 1% NaHCO3, 20 mM L-glutamine and 0.9% agarose (Seakem ME; FMC BioProducts) and incubated for 7 days at 32 ° C in a humidified airtight chamber.
Virus purification Ten roller flasks of confluent PS-EK cells ( ~ l0 s cells per flask) were inoculated with BTV 20 at a m.o.i, of ~ 0.1 PFU ml-1. Virus was adsorbed for 1 h, Hanks' medium-199 containing 5% FBS added and the cells incubated at 32 ° C until the monolayers had degenerated. Virus was purified from cell debris by extraction with the fluorocarbon Arklone 113 (Daikin Kogyo, Japan) as previously described (Hubschle, 1980), except that virus was treated with 1% sodium lauryl sarcosine (NLS), 10 mM dithiothreitol (DTT) for 1 h on ice (Mertens et al., 1987) before velocity centrifugation (25 000 r.p.m., 1 h, 4°C, Beckman SW41 rotor) through 10-50% (w/v) sucrose gradients prepared with 2 mM Tris-HC1 pH 8.8. Virus bands were collected, immediately snap frozen in a solid CO2-ethanol bath and stored at - 7 0 ° C. Protein concentrations of purified BTV 20 were determined using the ratio 1 OD26o:400/lg protein as previously reported with purified BTV 1 (Mertens et al., 1986, 1987).
Protease digestion Trypsin, alpha-chymotrypsin (Boehringer Mannheim) and thermolysin (3 × crystalline, Calbiochem) were prepared at 2 mg ml-1 in protease buffer (0.2 M Tris-HC1 pH 8.0, 0.02 M CaC12), frozen at - 7 0 ° C and thawed once only at 37 °C immediately before use. Purified BTV 20, used at final protein concentrations of either ~ 295 pg m l - 1 or ~ 585 ttg m l - ' , was thawed at 37 ° C. It was then added to an equal volume of protease buffer containing trypsin, alpha-chymotrypsin or thermolysin to give final enzyme concentrations of 10, 10 or 100 pg m l - 1 respectively, or to other concentrations described in the text. Incubation at 37 ° C was continued and aliquots removed after exposure periods of 0.2, 0.5, 1, 2, 3, 4, 5, 10 and 20 h. ( 1 ) For analysis of the protein compositions of protease-digested BTV par-
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J.A. COWLEY AND B.M. GORMAN
ticles, 50 ]~1 aliquots were removed, snap frozen in a solid CO2-ethanol bath and stored at - 7 0 ° C. Groups of six samples were thawed rapidly at 37 ° C, diluted with 150 zl ice-cold 2 mM Tris-HC1 pH 8.8 and virus particles pelletted through a 4.8 ml cushion of 10% (w/v) sucrose by centrifugation at 40 000 r.p.m., 30 min, 4 °C in a Beckman SW55 rotor. Virus pellets were resuspended in 50 zl electrophoresis sample buffer (Laemmli, 1970), boiled for 3 rain and stored at - 70 ° C until analysed. (2) For analysis of HA activities, 25 pl aliquots were added to 375 #l ice-cold borate saline pH 9.0, 0.2% bovine serum albumin (Cowley and Gorman, 1987 ), snap frozen and stored as above. (3) For analysis of virus infectivities, 5/tl aliquots were added to 245 ~l icecold Hanks' medium-199 containing 5% FBS, snap frozen and stored at - 70 ° C until titrated for plaques in PS-EK cells as described above.
Polyacrylamide gel electrophoresis BTV proteins were analysed in 7.5, 10 or 12.5% SDS-polyacrylamide gels using the Tris-glycine buffer system of Laemmli (1970). Protein molecular weight standards (in kilodaltons (kDa)) (Pharmacia) consisted of phosphorylase b (94), bovine serum albumin (67), catalase (60), ovalbumin (45/46), lactate dehydrogenase (36), carbonic anhydrase ( 30 ), trypsin inhibitor ( 20.1 ) and alpha-lactalbumin (14.4). Proteins were detected by silver staining as described by Marshall (1984). Molecular weight estimations were made by the method of Weber and Osburn (1969).
HA titrations Sheep, bovine and human erythrocytes were collected in Alsever's solution, washed in dextrose-gelatin-veronal solution and prepared as a 0.5% suspension in adjusting diluent pH 7.0 as described by Clarke and Casals (1959). HA titrations were performed in U-bottom microtitre plates using a diluent of borate saline pH 9.0, 0.2% bovine serum albumin as described previously (Cowley and Gorman, 1987). RESULTS
Enhanced in[ectivities alter digestion with chymotrypsin, thermolysin or trypsin Purified BTV 20 ( ~ 295 ~g m1-1) was digested with chymotrypsin, thermolysin or trypsin (i.e. 10, 100 or 10/tg m1-1, respectively) at 37°C over an exposure period of 0.2 to 20 h. The resulting effects on virus infectivities were determined by plaque titration in PS-EK cells (Fig. 1). Initial increases in virus infectivities were observed with each of the three proteases. The increases occurred rapidly, being detectable after the short exposure period nec-
EFFECTS OF PROTEOLYTIC ENZYMES ON BLUETONGUEVIRUSTYPE 20
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essary for addition of the protease, storage and processing of the samples (i.e. 0.2 h). Plaque titres recorded at later times demonstrated that there were differences in the rates of action of the three proteases. The rate of enhancement of virus infectivity was most rapid with trypsin, followed by chymotrypsin and thermolysin, in that order. Compared to the infectivity of untreated BTV 20 at the initial sampling point (i.e. 0.2 h), maximum increases occurred after 0.2 h with trypsin (0.9 loglo P F U ml-1), 1.0 h with chymotrypsin (1.7 loglo P F U m1-1) and 2.0 h with thermolysin (1.7 loglo P F U ml-1). However, after the initial increases, virus infectivities decreased more rapidly with protease-digested virus than with untreated virus. Enhanced HA activities The HA activity of BTV 20 digested with chymotrypsin, thermolysin or trypsin was determined using aliquots of the same reaction mixtures used to determine virus infectivities. HA titrations were performed with sheep, bovine
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J.A. COWLEY AND B.M. GORMAN
and human erythrocytes (Fig. 2). U n t r e a t e d B T V 20 possessed H A activity that could only be detected above the minimum virus dilution tested (i.e. 16 H A U per 0.05 ml) with sheep erythrocytes at the initial sampling point (i.e. 0.2 h). Digestion with each of the three proteases resulted in initial increases in HA titres with the three erythrocyte species tested. Except for agglutination of human erythrocytes with chymotrypsin-digested virus, all other increases in H A titres were detectable within the time required to process the initial sample (i.e. 0.2 h). Digestion of B T V 20 with each of the three proteases resulted in identical maximum HA titres with sheep (4096 H A U per 0.05 ml), bovine (1024 H A U per 0.05 ml) and h u m a n erythrocytes (128 H A U per 0.05 ml). Moreover, the rate at which H A titres increased and decreased closely resembled that observed with virus infectivities with the respective protease (cf. Fig. 1 and Fig. 2 ). Incubation periods giving maximum H A titres (Fig. 2 ) and virus infectivity titres (Fig. 1) coincided for B T V 20 digested with chymotrypsin (1.0 h) and 4O96 F
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EFFECTS OF PROTEOLYTIC ENZYMES ON BLUETONGUE VIRUS TYPE 20
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thermolysin (2.0 h). With trypsin, however, maximum infectivity titres occuffed at 0.2 h, while maximum HA titres occurred at 0.5 h.
Protease cleavage of B T V 20 proteins The protein composition of BTV 20 particles ( ~ 585 ~g ml-1), collected after digestion with 10 ttg m1-1 trypsin, chymotrypsin or thermolysin for 1 h at 37°C, was analysed in 7.5 and 12.5% SDS-polyacrylamide gels (Fig. 3). Untreated BTV 20 possessed the seven structural proteins characteristic of BTV (Martin and Zweerink, 1972; Verwoerd et al., 1972). However, VP2 migrated above VP3 in the 12.5% gel (Fig. 3b) but below VP3 in the 7.5% gel. This phenomenon has been previously observed in gels of different polyacrylamide concentrations (Cowley and Gorman, 1989). Silver-stained gels consistently revealed a number of minor proteins in purified BTV preparations; the most prominent of these migrated at 90 kDa, 52 kDa and 41 kDa. The 41 kDa protein, that migrated immediately above VP7, appears to be equivalent to protein 'x' described with other BTV isolates (Mertens et al., 1984). The 52 kDa protein may represent a cleavage product of VP5 (Cowley and Gorman, 1989). Digestion of BTV 20 with trypsin resulted in the cleavage of VP2 into a number of smaller polypeptides; all other virus proteins appeared to remain intact (Fig. 3, lane 2 ). The most prominent cleavage products (designated P ) were assigned according to their estimated molecular weights (in kDa ), as P93, P91, P66, P62, P54, P39, P38 and P25.5. Chymotrypsin also cleaved VP2 specifically to give major cleavage products designated P93, P76, P55, P54, P27 and P25 (Fig. 3, lane 3). Treatment of BTV 20 with 10 ~g ml -~ thermolysin did not cleave VP2 significantly (Fig. 3, lane 4). However, close analysis revealed three cleavage products, P93, P76 and P54, that were also observed with chymotrypsin. Subsequent digestion of BTV 20 with a higher thermolysin concentration (i.e. 100 ]lg ml -~) confirmed the formation of these major VP2 cleavage products (see Fig. 5). Analysis in a 12.5% gel of the cleavage products resulting from digestion of BTV 20 with each of the three proteases revealed that there were no detectable products below 25 kDa (Fig. 3b). Kinetics o[protease digestion of B T V 20 proteins An attempt was made to correlate directly the appearance of VP2 cleavage products with the observed increases in virus infectivity (Fig. 1 ) and HA activity (Fig. 2 ) resulting from digestion of BTV 20 with trypsin, chymotrypsin or thermolysin. Aliquots of virus were taken from the same reaction mixture, virus particles collected and their protein composition determined in 10% SDSpolyacrylamide gels. The protein compositions of BTV 20 ( ~ 295/tg m1-1 ) digested with 10 ]~g m l - 1 chymotrypsin for between 0.2 and 20 h are shown in Fig. 4a. Cleavage of VP2 was observed at 0.2 h, indicating that it occurred in
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145
EFFECTS OF PROTEOLYTIC ENZYMES ON BLUETONGUE VIRUS TYPE 20
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Fig. 4. Kinetics of the appearance of cleavage products of BTV 20 collected after in vitro digestion with 10/zg ml - 1 chymotrypsin. B T V 20 was used at two protein concentrations (a) ~ 295/~g m l - 1 and (b) ~ 585/zg m l - 1. Virus was digested for (a) untreated, 0.2 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 10 h and 20 h (lanes 1 to 10, respectively; lane 11, protein molecular weight standards), and (b) untreated, 1 h, 2 h, 3 h and 4 h (lanes 1 to 5, respectively; lane 6, protein molecular weight standards). After incubation at 37 °C for these time periods, virus particles were collected by centrifugation through a 10% sucrose cushion (the 0.5 h sample, (a) lane 3, was lost due to a technical problem) and analysed in a 10% polyacrylamide ge]. Proteins were visualised by silver staining.
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J,A. COWLEY AND B.M. GORMAN
the time required to process the sample. Of the chymotrypsin cleavage described earlier (see Fig. 3a), P93, P54 and P27 were detected after a short exposure period (Fig. 4a, 0.2 h). After digestion for 1 h two additional polypeptides, P76 and P25, became more obvious. Increased incubation duration resulted in decreased amounts of P76 and P27, some increased in the amount of P25; the amounts of P93 and P54 remained relatively unchanged. A similar experiment was conducted using 10/~g ml-1 chymotrypsin and double the concentration of BTV 20 (i.e. ~ 585/~g m1-1 ) (Fig. 4b). Here, polypeptides P76 and P27 represented the major early VP2 cleavage products, and P55, P54 and P25 the minor products detectable after 1 h incubation (Fig. 4b, lane 2). Polypeptides P76, P55 and P27 disappeared with increased exposure and by 4 h (Fig. 4b, lane 5) had been replaced by P54 and P25 as the major cleavage products. The highest molecular weight cleavage product, P93, detected in Fig. 3 and Fig. 4a, could not be accurately identified in this gel. Digestion of BTV 20 ( ~295 #g ml -~) with thermolysin (100/~g m1-1) resulted in the cleavage of VP2 into three major polypeptides, P76, P54 and P25 (Fig. 5). The migrational properties of these polypeptides correspond closely to the three major cleavage products resulting from cleavage with chymotrypsin (see Fig. 4a and b). As was also the case with chymotrypsin, P93 (obscured in this gel by the smearing of VP2), P54 and P25 and were generated in the 1
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EFFECTS OF PROTEOLYTIC ENZYMES ON BLUETONGUE VIRUS TYPE 20
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time elapsed between addition of the protease and purification of virus particles (0.2 h; Fig. 5, lane 2 ). Another common cleavage product, P76, was prominent by 0.5 h and subsequently disappeared by 5 h. Other minor cleavage products (P53, P35 and P31) were detected in varying amounts from 0.5 h onwards. The most obvious cleavage product that was processed further was P76, while the amounts of P53, P31 and P25 appeared to increase slightly with increased exposure. Digestion with 10/lg m1-1 trypsin resulted in by far the most rapid and extensive cleavage of BTV 20 proteins (Fig. 6). Although VP2 and VP3 comigrated in this gel, the visual decrease in the intensity of this band suggested that VP2 was processed very rapidly and to an extent where it may have been digested completely in the time interval required to process the initial sample (0.2 h; Fig. 6, lane 2 ). In addition, VP1 was almost completely absent after 0.2 h, while further exposure resulted in some loss of VP5. The fact that proteins other than VP2 were susceptible to trypsin cleavage at these concentrations of virus and enzyme undoubtedly contributed to the numerous cleavage products. For this reason, only those cleavage products (i.e. P93, P91, P66, P62, P54, P39, P38 and P25.5) detected previously using a higher virus concentration (see Fig. 3, lane 2 ) are indicated in Fig. 6. Higher concentrations of BTV 20, reduced protease concentrations, or shorter exposure periods may be required to define the kinetics of trypsin cleavage of VP2.
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DISCUSSION The results presented in this paper demonstrate that in vitro digestion of BTV 20 with the proteases chymotrypsin, thermolysin or trypsin can initially enhance virus infectivity and HA activity. The primary action of these proteases involves cleavage of the outer capsid protein VP2 into a number of similar, but characteristic polypeptides. In addition, kinetic studies revealed that some VP2 cleavage products are only transiently associated with the ISVPs resulting from digestion of BTV 20 with chymotrypsin or thermolysin. Digestion of BTV 20 with chymotrypsin resulted in the progressive loss of VP2 and six smaller polypeptides appearing at different stages of cleavage: designated P93, P76, P55, P54, P27 and P25. Previous studies have also shown that VP2 of other BTV isolates and orbiviruses is susceptible to chymotrypsin cleavage, although some inconsistencies have occurred with regard to the number of cleavage products detected (Van Dijk and Huismans, 1980, 1982; Mertens et al., 1986, 1987). Van Dijk and Huismans (1980) made reference to two VP2 cleavage products, migrating above VP4 and just below VP5, that remained attached to ISVPs of BTV 10. However, the more recent studies of Mertens et al. (1986, 1987) reported four primary cleavage products of VP2 for ISVPs of BTV 1 and BTV 4. In order of decreasing molecular weight, the most obvious were designated VP2a, VP2b and VP2c; one polypeptide migrating below VP2c, now designated VP2d (P.P.C. Mertens, pers. comm., 1989), could be detected by silver staining but poorly by incorporation of [3~S]-methionine. Polypeptides VP2a, VP2b and VP2c migrated below VP3, VP5 and VP7, respectively, with BTV 4 and similarly with BTV 1 except that polypeptide VP2b migrated slightly above VP5. An additional minor polypeptide, migrating just above VP4 with BTV 4, and closer to VP3 with BTV 1, has also been detected in differing amounts depending on whether ISVPs were purified on CsC1 or sucrose gradients (P.P.C. Mertens, pers. comm., 1989). Thus, it appears that the number and relative size of VP2 cleavage products resulting from chymotrypsin digestion of BTV 4, and to a lesser extent BTV 1, are similar to those reported here for BTV 20. Kinetic studies revealed that VP2 of BTV 20 underwent progressive cleavage upon extended digestion with chymotrypsin. Using virus and enzyme concentrations that allowed investigation of the early stages of VP2 cleavage, polypeptides P93, P76 and P27 represented the major primary cleavage products. Further digestion revealed that P76 and P27 were only transiently associated with ISVPs and were replaced by two secondary cleavage products, P54 and P25. A major objective of this study was to determine whether proteases of different specificities to chymotrypsin would also cleave VP2, and to what extent this would affect virus infectivity and HA activity. Two other proteases tested, thermolysin and trypsin, both cleaved VP2 of BTV 20. Thermolysin in partic-
EFFECTS OF PROTEOLYTIC ENZYMES ON BLUETONGUE VIRUS TYPE 20
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ular generated VP2 cleavage products that were similar to those observed with chymotrypsin, except that two polypeptides, P35 and P31, were detected instead of P27. As was also the case with chymotrypsin, the P76 cleavage product generated by thermolysin was only transiently associated with ISVPs, and disappeared after extended exposure. Trypsin generated a number of major VP2 cleavage products that were quite distinct from those detected with chymotrypsin or thermolysin. Nevertheless two polypeptides, P93 and P54, were generated by all three proteases. At the same time however a major low molecular weight trypsin cleavage product, P25.5, was only marginally different to the P25 cleavage product generated by chymotrypsin and thermolysin. Unfortunately, any processing that occured with these VP2-specific cleavage products could not be determined with the BTV 20 and trypsin concentrations used in kinetic analyses. Under the conditions used, VP2 cleavage was essentially complete in the time that elapsed between addition of protease and processing of the sample. Moreover, the presence of numerous other polypeptides that resulted from cleavage of other virus proteins obscured those resulting from the specific cleavage of VP2. However, our results show that in vitro digestion of BTV 20 with chymotrypsin, thermolysin or trypsin generates three cleavage products of similar size, suggesting that at least two common regions on VP2 are sensitive to cleavage with these proteases. Mertens et al. (1986) have also reported that, for both BTV 1 and BTV 4, trypsin produced similar VP2 cleavage products to chymotrypsin, except the polypeptide equivalent to VP2c was slightly smaller. Therefore, the close similarities between the trypsin and chymotrypsin cleavage products reported for BTV 1 and BTV 4 (Mertens et al., 1986, 1987) and those found with BTV 20, suggest that there are conserved cleavage sites in the VP2 outer capsid proteins of serologically and geographically distinct BTV isolates. The biological significance of proteolytic cleavage of VP2 of BTV has only recently been addressed (Mertens et al., 1986, 1987). In these studies, digestion with chymotrypsin resulted in the mono-dispersion of virus particles, maintenance of similar virus infectivities to disaggregated untreated particles, maintenance of serotype-specificity in virus-neutralisation tests and the loss of HA activity. In addition, Mertens et al. (1986, 1987) reported that purification of BTV using sodium lauryl sarcosine (NLS) and dithiothreitol (DTT), followed by storage in the absence of NLS, caused virus particles to become highly aggregated. The BTV 20 preparation used here was purified in a similar manner, stored without NLS, and possessed a specific infectivity (i.e. P F U / OD26o unit) of 1.5 × 109. This specific infectivity was lower than that reported for BTV 10 (i.e. 1.1× 101°) purified in sucrose gradients without treatment with NLS or DTT (Verwoerd et al., 1972 ), but approximated that of disaggregated BTV 1 and BTV 4 (i.e. 1.8× 109 TCIDso/OD26o unit) maintained in 1% NLS after treatment with DTT (Mertens et al., 1986). This suggested that
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BTV 20 particles were aggregated, though to a lesser extent than that found with BTV 1 or BTV 4. Mertens et al. (1986, 1987) reported a 10-fold increase in infectivity when BTV 1 or BTV 4 was treated with NLS in combination with either D T T or chymotrypsin. With BTV 20, trypsin digestion resulted in an eightfold increase in infectivity while 50-fold maximum increases were observed with both chymotrypsin and thermolysin. Due to the rapid action of trypsin, however, the enhancement in infectivity observed had most probably passed its maximum. It appears likely that the increases in virus infectivities induced by chymotrypsin, thermolysin or trypsin resulted from disaggregation of BTV 20 ISVPs associated with cleavage of VP2, rather than from the activation of non-infectious particles. However, the extent of VP2 cleavage observed when significant increases in virus infectivities had occurred suggested that limited cleavage may not adversely affect the ability of BTV 20 particles to infect PS-EK cells. In contrast to the findings of Mertens et al. (1987), HA titres with three erythrocyte species were enhanced, similarly to infectivity titres, in the early stages of digestion of BTV 20 with the three proteases analysed. Kinetic studies of the effects of proteases on the HA activity and protein composition of BTV 20 suggested possible explanations for this discrepancy. HA titres increased initially, then decreased rapidly upon extended exposure to proteases, therefore, either the extent of VP2 cleavage, or the degree of particle disaggregation at the time of analysis could affect HA activity. The use of preparations of mono-dispersed particles will be required to ascertain a direct relationship between the observed increases in HA activity and infectivity of BTV 20 and proteolytic cleavage of VP2. Cleavage of outer capsid protein VP2 of BTV is comparable in many respects to similar phenomena observed with other Reoviridae members. For instance, with reoviruses, progressive digestion of three outer capsid proteins is observed upon extended exposure to chymotrypsin (Joklik, 1972; Borsa et al., 1973). This digestion of the reovirus outer capsid can have variable effects on virus infectivity and HA activity. It is also dependent on factors, such as the purity of virus preparations, enzyme concentrations and the presence of cations, that affect the extent of digestion. Limited digestion either maintains or enhances infectivity titres and HA activity, while extended digestion greatly reduces these activities (see review, Joklik, 1983). With rotaviruses, a major non-glycosylated outer capsid protein (VP3) is highly susceptible to protease digestion (Clark et al., 1981; Estes et al., 1981; Sato et al., 1987). With some rotavirus isolates, trypsin digestion can enhance virus infectivity, but cause the loss of HA activity (Barnett et al., 1979; Graham and Estes, 1981; Holmes, 1983). Chymotrypsin similarly reduces HA activity but does not enhance infectivity titres significantly (Graham and Estes, 1981; Kitaoka et al., 1984, 1986, 1987). Holmes (1983) has suggested that the cleavage of VP3 by trypsin may be important to rotavirus infection of intestinal epithelial cells, where contact with
EFFECTS OF PROTEOLYTIC ENZYMES ON BLUETONGUE VIRUS TYPE 20
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this enzyme is inevitable. Therefore it may be of some interest to examine the mechanism of infection of BTV in insect hosts, where primary contact would be with salivary gland and gut secretions, environments rich in proteases. ACKNOWLEDGEMENTS
Bluetongue virus research at the Queensland Institute of Medical Research was supported by a grant from the Australian Meat Research Committee. We thank Mr. R.W. Campbell for provision of cell cultures and Dr. P.P.C. Mertens for making available unpublished data and for helpful suggestions on the manuscript.
REFERENCES Barnett, B.B., Spendlove, R.S. and Clark, M.L., 1979. Effect of enzymes on rotavirus infectivity. J. Clin. Microbiol., 10: 111-113. Borsa, J., Copps, T.P., Sargent, M.D., Long, D.G. and Chapman, J.D., 1973. New intermediate subviral particles in the in vitro uncoating of reovirus virions by chymotrypsin. J. Virol., 11: 552-564. Clarke, D.M. and Casals, J., 1959. Techniques for haemagglutination and haemagglutinationinhibition with arthropod-borne viruses. Am. J. Trop. Med. Hyg., 7: 561-573. Clark, S.M., Roth, J.R., Clark, M.L., Barnett, B.B. and Spendlove, R.S., 1981. Trypsin enhancement of rotavirus infectivity: mechanism of enhancement. J. Virol., 39: 816-822. Cowley, J.A. and Gorman, B.M., 1987. Genetic reassortants for identification of the genome segment coding for the bluetongue virus hemagglutinin. J. Virol., 61: 2304-2306. Cowley, J.A. and Gorman, B.M., 1989. Cross-neutralization of genetic reassortants of bluetongue virus serotypes 20 and 21. Vet. Microbiol., 19: 37-51. Estes, M.K., Graham, D.Y. and Mason, B.B., 1981. Proteolytic enhancement of rotavirus infectivity: molecular mechanisms. J. Virol., 39: 879-888. Gorman, B.M., Leer, J.T., Filippich, C., Goss, P.D. and Doherty, R.L., 1975. Plaquing and neutralising of arboviruses in the PS-EK line of cells. Aust. J. Med. Technol., 6: 65-71. Graham, D.Y. and Estes, M.K., 1980. Proteolytic enhancement of rotavirus infectivity: biological mechanisms. Virology, 101: 432-439. Holmes, I.H., 1983. Rotaviruses. In: W.K. Joklik (Editor), The Reoviridae. Plenum, New York, pp. 359-423. Hubschle, O.J.B., 1980. Bluetongue virus haemagglutination and its inhibition by specific antiserum. Arch. Virol., 64: 133-140. Huismans, H. Van Der Walt, N.T., Cloete, M. and Erasmus, B.J., 1983. The biological and immunological characterization of bluetongue virus outer capsid polypeptides. In: R.W. Compans and D.H.L. Bishop (Editors), Double-Stranded RNA Viruses. Elsevier, Amsterdam, pp. 165172. Huismans, H., Van Der Walt, N.T., Cloete, M. and Erasmus, B.J., 1987. Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. Virology, 157: 172-179. Joklik, W.K., 1972. Studies on the effect of chymotrypsin on reovirons. Virology, 49: 700-715. Joklik, W.K., 1983. The reovirus particle. In: W.K. Joklik (Editor), The Reoviridae, Plenum, New York, pp. 9-78.
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Kitaoka, S., Suzuki, H., Numazaki, Y., Sato, T., Konno, T., Ebina, T. and Ishida, N., 1984. Hemagglutination by human rotavirus strains. J. Med. Virol., 13: 215-222. Kitaoka, S., Suzuki, H., Numazaki, Y., Konno, T. and Ishida, N., 1986. The effect of trypsin on the growth and infectivity of human rotavirus. Tohoku J. Exp. Med., 149:437 447. Kitaoka, S., Suzuki, H., Sato, T., Numazaki, Y., Konno, T. and Ishida, N., 1987. Failure in chymotrypsin enhancement of human rotavirus infectivity. Tohoku J. Exp. Med., 151: 127-128. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature {London), 227: 680-685. Marshall, T., 1984. Detection of protein in polyacrylamide gels using an improved silver stain. Anal. Biochem., 136: 340-346. Martin, S.A., and Zweerink, H.J., 1972. Isolation and characterisation of two types of bluetongue virus particles. Virology, 50: 495-506. Mertens, P.P.C., Brown, F. and Sangar, D.V., 1984. Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode. Virology, 135: 207-217. Mertens, P.P.C., Burroughs, J.N. and Anderson, J., 1986. Purification procedures and structural analyses of bluetongue virus particles and sub-viral particles of serotyes 1 and 4. In: P. Roy, C.E. Schore and B.I. Osburn {Editors ), Orbiviruses and Birnaviruses: Proceedings of the Double-Stranded RNA Virus Symposium, Oxford, pp. 53-73. Mertens, P.P.C., Burroughs, J.N. and Anderson, J., 1987. Purification and properties of virus particles, infectious sub-viral particles and cores of bluetonge virus serotypes 1 and 4. Virology, 157: 375-386. Sato, T., Kitaoka, S., Suzuki, H., Konno, T. and Ishida, N., 1987. Effect of trypsin and chymotrypsin on polypeptides of human rotavirus KUN strain. Med. Microbiol. Immunol., 176:65 73. Van Dijk, A.A. and Huismans, H., 1980. The in vitro activation and further characterisation of the bluetongue virus-associated transcriptase. Virology, 104: 347-356. Van Dijk, A.A. and Huismans, H., 1982. The effect of temperature on the in vitro transcriptase reaction of bluetongue virus, epizootic haemorrhagic disease virus and African horsesickness virus. Onderstepoort J. Vet. Res., 49: 227-232. Verwoerd, D.S. and Huismans, H., 1972. Studies on the in vitro and the in vivo transcription of the bluetongue virus genome. Onderstepoort J. Vet. Res., 39: 185-192. Verwoerd, D.W., Els, H.J., De Villiers, E.M. and Huismans, H., 1972. Structure of the bluetongue virus capsid. J. Virol., 10: 783-794. Weber, K. and Osburn, M., 1969. The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J. Biol. Chem., 24: 4406-4412.
137
Effects of Proteolytic Enzymes on the Infectivity, Haemagglutinating Activity and Protein Composition of Bluetongue Virus Type 20 J.A. COWLEY 1'2 and B.M. GORMAN 3
Queensland Institute of Medical Research, Bramston Terrace, Herston, 4006, Qld. (Australia) (Accepted 12 October 1989)
ABSTRACT Cowley, J.A. and Gorman, B.M., 1990. Effects of proteolytic enzymes on the infectivity, haemagglutinating activity and protein composition of bluetongue virus type 20. Vet. Microbiol., 29: 137-152. The effects on virus infectivity, haemagglutinating (HA) activity and polypeptide composition of bluetongue virus type 20 (BTV 20) were determined after digestion with the proteolytic enzymes, chymotrypsin, thermolysin and trypsin. Virus infectivity increased eight to 50-fold after exposure periods which reflected the activity of the proteases. Identical maximum increases in HA activity {i.e. 4096, 1024 and 128 HAU per 0.05 ml with sheep, bovine and human erythrocytes, respectively) occurred with each of the three proteases. Peak increases in virus infectivities and HA activities occurred after similar exposure periods. Outer capsid protein VP2 was the most sensitive virus protein to proteolytic digestion, being cleaved into a number of smaller polypeptides that remained attached to the virus particle. Digestion with chymotrypsin and thermolysin yielded four common cleavage products, designated P93, P76, P54 and P25 according to their estimated molecular weight, which suggested that they shared at least three cleavage sites. VP2 cleavage products resulting from digestion with trypsin differed somewhat from those of chymotrypsin and thermolysin, although the generation of polypeptides P93, P54 and P25.5 suggested the existence of common cleavage sites for the three proteases. Possible mechanisms whereby proteolytic cleavage of VP2 may enhance the infectivity and HA activity of BTV 20 are discussed.
INTRODUCTION
Early biochemical studies of the structure of bluetongue virus (BTV) demonstrated the existence of three distinct particle types (Martin and Zweerink, 1972; Verwoerd et al., 1972). Complete particles purified in sucrose gradients 'Author to whom correspondence should be addressed. 2Present address: Animal Research Institute, Queensland Department of Primary Industries, Yeerongpilly, 4105, Australia. 3Present address: 13 Daniel Street, Nambour, 4560, Australia.
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possessed a double-layered protein capsid, with two viral proteins (VP2 and VP5 ) forming the outer capsid and two major (VP3 and VP7) and three minor proteins (VP1, VP4 and VP6) forming the nucleocapsid or core particle. Purification in CsC1 gradients, however, degraded BTV either into particles that lacked VP2, or into core particles that lacked both proteins (VP2 and VP5) associated with the outer capsid. In a detailed investigation of the BTV transcriptase, Van Dijk and Huismans (1980) showed that different concentrations of MgC12 acted similarly to CsC1 (at different pH values ) with respect to the selective removal of VP2 and VP5 from virus particles. Huismans et al. ( 1983, 1987) subsequently demonstrated that the action of MgC12 was also dependent on pH. At pH 5.0, dissociation of VP2 occurred with 0.2 M MgC12, while at pH 8.0, 1.0 M MgC12 was required to dissociate both VP2 and VP5; thus indicating that the BTV outer capsid was less stable at acidic pH than at basic pH. For some BTV isolates, dissociation of outer capsid proteins, with either CsCl or MgC12, was found to be enhanced by digestion with chymotrypsin (Van Dijk and Huismans, 1980, 1982; Mertens et al., 1986, 1987). However, digestion with chymotrypsin in the absence of cations resulted in the formation of so-called "intermediate" or "infectious sub-viral particles" (ISVPs) (Van Dijk and Huismans, 1980; Mertens et al., 1986, 1987). Mertens et al. (1986, 1987) characterised some effects of chymotrypsin on BTV 1 and BTV 4. They found that the resulting ISVPs retained their infectivity, lost haemagglutinating (HA) activity and shifted from a highly aggregated state to a mono-dispersed suspension. In addition, ISVPs possessed a VP2 which, when analysed in SDS-polyacrylamide gels, was found to be cleaved into at least three major polypeptides, designated VP2a, VP2b and VP2c. This cleavage of VP2 was suggested as the probable mechanism whereby ISVPs lost their ability to aggregate to themselves and to agglutinate erythrocytes. Changes in the molecular and biological properties of BTV resulting from enzymatic digestion with other proteases have, however, not been investigated. In this paper we have characterised the effects of chymotrypsin, and two other proteases (trypsin and thermolysin) on the infectivity, HA activity and protein composition of BTV 20. In contrast to Mertens et al. (1987), our results indicate that both biological activities can be initially enhanced with each of the three proteases. In addition, some preliminary data on the kinetic appearance of cleavage products of protease-sensitive proteins of BTV 20 are presented for each enzyme. MATERIALSAND METHODS Cells and virus
The PS-EK line of pig kidney cells (Gorman et al., 1975) was grown in Hanks' medium-199 containing 14 mM H E P E S pH 7.2, 100 U ml-1 penicillin,
EFFECTS OF PROTEOLYTIC ENZYMES ON BLUETONGUE VIRUS TYPE 20
139
100 pg ml-1 streptomycin, 2 pg m l - ' amphotericin B and 10% fetal bovine serum (FBS). BTV 20 (isolate CSIRO 19) was originally obtained from Dr. T.D. St. George, CSIRO Division of Tropical Animal Science, Brisbane. A small plaque-forming type was isolated from this BTV 20 pool and plaque cloned twice more in PS-EK cells.
Virus infectivity titration BTV 20 was titrated for plaques in PS-EK cell monolayers in 9.8 cm 2 plastic dishes. Tenfold dilutions of virus were prepared in Hanks' medium-199 containing 5% FBS, 100 ttl inoculated per dish and the virus adsorbed 1 h at room temperature as previously described (Gorman et al., 1975). Excess inoculum was removed, cells overlaid with 3 ml per dish of Minimal Essential Medium (MEM AutoPowTM; Flow Labs.) containing 14 mM HEPES pH 7.2; 1% NaHCO3, 20 mM L-glutamine and 0.9% agarose (Seakem ME; FMC BioProducts) and incubated for 7 days at 32 ° C in a humidified airtight chamber.
Virus purification Ten roller flasks of confluent PS-EK cells ( ~ l0 s cells per flask) were inoculated with BTV 20 at a m.o.i, of ~ 0.1 PFU ml-1. Virus was adsorbed for 1 h, Hanks' medium-199 containing 5% FBS added and the cells incubated at 32 ° C until the monolayers had degenerated. Virus was purified from cell debris by extraction with the fluorocarbon Arklone 113 (Daikin Kogyo, Japan) as previously described (Hubschle, 1980), except that virus was treated with 1% sodium lauryl sarcosine (NLS), 10 mM dithiothreitol (DTT) for 1 h on ice (Mertens et al., 1987) before velocity centrifugation (25 000 r.p.m., 1 h, 4°C, Beckman SW41 rotor) through 10-50% (w/v) sucrose gradients prepared with 2 mM Tris-HC1 pH 8.8. Virus bands were collected, immediately snap frozen in a solid CO2-ethanol bath and stored at - 7 0 ° C. Protein concentrations of purified BTV 20 were determined using the ratio 1 OD26o:400/lg protein as previously reported with purified BTV 1 (Mertens et al., 1986, 1987).
Protease digestion Trypsin, alpha-chymotrypsin (Boehringer Mannheim) and thermolysin (3 × crystalline, Calbiochem) were prepared at 2 mg ml-1 in protease buffer (0.2 M Tris-HC1 pH 8.0, 0.02 M CaC12), frozen at - 7 0 ° C and thawed once only at 37 °C immediately before use. Purified BTV 20, used at final protein concentrations of either ~ 295 pg m l - 1 or ~ 585 ttg m l - ' , was thawed at 37 ° C. It was then added to an equal volume of protease buffer containing trypsin, alpha-chymotrypsin or thermolysin to give final enzyme concentrations of 10, 10 or 100 pg m l - 1 respectively, or to other concentrations described in the text. Incubation at 37 ° C was continued and aliquots removed after exposure periods of 0.2, 0.5, 1, 2, 3, 4, 5, 10 and 20 h. ( 1 ) For analysis of the protein compositions of protease-digested BTV par-
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J.A. COWLEY AND B.M. GORMAN
ticles, 50 ]~1 aliquots were removed, snap frozen in a solid CO2-ethanol bath and stored at - 7 0 ° C. Groups of six samples were thawed rapidly at 37 ° C, diluted with 150 zl ice-cold 2 mM Tris-HC1 pH 8.8 and virus particles pelletted through a 4.8 ml cushion of 10% (w/v) sucrose by centrifugation at 40 000 r.p.m., 30 min, 4 °C in a Beckman SW55 rotor. Virus pellets were resuspended in 50 zl electrophoresis sample buffer (Laemmli, 1970), boiled for 3 rain and stored at - 70 ° C until analysed. (2) For analysis of HA activities, 25 pl aliquots were added to 375 #l ice-cold borate saline pH 9.0, 0.2% bovine serum albumin (Cowley and Gorman, 1987 ), snap frozen and stored as above. (3) For analysis of virus infectivities, 5/tl aliquots were added to 245 ~l icecold Hanks' medium-199 containing 5% FBS, snap frozen and stored at - 70 ° C until titrated for plaques in PS-EK cells as described above.
Polyacrylamide gel electrophoresis BTV proteins were analysed in 7.5, 10 or 12.5% SDS-polyacrylamide gels using the Tris-glycine buffer system of Laemmli (1970). Protein molecular weight standards (in kilodaltons (kDa)) (Pharmacia) consisted of phosphorylase b (94), bovine serum albumin (67), catalase (60), ovalbumin (45/46), lactate dehydrogenase (36), carbonic anhydrase ( 30 ), trypsin inhibitor ( 20.1 ) and alpha-lactalbumin (14.4). Proteins were detected by silver staining as described by Marshall (1984). Molecular weight estimations were made by the method of Weber and Osburn (1969).
HA titrations Sheep, bovine and human erythrocytes were collected in Alsever's solution, washed in dextrose-gelatin-veronal solution and prepared as a 0.5% suspension in adjusting diluent pH 7.0 as described by Clarke and Casals (1959). HA titrations were performed in U-bottom microtitre plates using a diluent of borate saline pH 9.0, 0.2% bovine serum albumin as described previously (Cowley and Gorman, 1987). RESULTS
Enhanced in[ectivities alter digestion with chymotrypsin, thermolysin or trypsin Purified BTV 20 ( ~ 295 ~g m1-1) was digested with chymotrypsin, thermolysin or trypsin (i.e. 10, 100 or 10/tg m1-1, respectively) at 37°C over an exposure period of 0.2 to 20 h. The resulting effects on virus infectivities were determined by plaque titration in PS-EK cells (Fig. 1). Initial increases in virus infectivities were observed with each of the three proteases. The increases occurred rapidly, being detectable after the short exposure period nec-
EFFECTS OF PROTEOLYTIC ENZYMES ON BLUETONGUEVIRUSTYPE 20
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essary for addition of the protease, storage and processing of the samples (i.e. 0.2 h). Plaque titres recorded at later times demonstrated that there were differences in the rates of action of the three proteases. The rate of enhancement of virus infectivity was most rapid with trypsin, followed by chymotrypsin and thermolysin, in that order. Compared to the infectivity of untreated BTV 20 at the initial sampling point (i.e. 0.2 h), maximum increases occurred after 0.2 h with trypsin (0.9 loglo P F U ml-1), 1.0 h with chymotrypsin (1.7 loglo P F U m1-1) and 2.0 h with thermolysin (1.7 loglo P F U ml-1). However, after the initial increases, virus infectivities decreased more rapidly with protease-digested virus than with untreated virus. Enhanced HA activities The HA activity of BTV 20 digested with chymotrypsin, thermolysin or trypsin was determined using aliquots of the same reaction mixtures used to determine virus infectivities. HA titrations were performed with sheep, bovine
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J.A. COWLEY AND B.M. GORMAN
and human erythrocytes (Fig. 2). U n t r e a t e d B T V 20 possessed H A activity that could only be detected above the minimum virus dilution tested (i.e. 16 H A U per 0.05 ml) with sheep erythrocytes at the initial sampling point (i.e. 0.2 h). Digestion with each of the three proteases resulted in initial increases in HA titres with the three erythrocyte species tested. Except for agglutination of human erythrocytes with chymotrypsin-digested virus, all other increases in H A titres were detectable within the time required to process the initial sample (i.e. 0.2 h). Digestion of B T V 20 with each of the three proteases resulted in identical maximum HA titres with sheep (4096 H A U per 0.05 ml), bovine (1024 H A U per 0.05 ml) and h u m a n erythrocytes (128 H A U per 0.05 ml). Moreover, the rate at which H A titres increased and decreased closely resembled that observed with virus infectivities with the respective protease (cf. Fig. 1 and Fig. 2 ). Incubation periods giving maximum H A titres (Fig. 2 ) and virus infectivity titres (Fig. 1) coincided for B T V 20 digested with chymotrypsin (1.0 h) and 4O96 F
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EFFECTS OF PROTEOLYTIC ENZYMES ON BLUETONGUE VIRUS TYPE 20
143
thermolysin (2.0 h). With trypsin, however, maximum infectivity titres occuffed at 0.2 h, while maximum HA titres occurred at 0.5 h.
Protease cleavage of B T V 20 proteins The protein composition of BTV 20 particles ( ~ 585 ~g ml-1), collected after digestion with 10 ttg m1-1 trypsin, chymotrypsin or thermolysin for 1 h at 37°C, was analysed in 7.5 and 12.5% SDS-polyacrylamide gels (Fig. 3). Untreated BTV 20 possessed the seven structural proteins characteristic of BTV (Martin and Zweerink, 1972; Verwoerd et al., 1972). However, VP2 migrated above VP3 in the 12.5% gel (Fig. 3b) but below VP3 in the 7.5% gel. This phenomenon has been previously observed in gels of different polyacrylamide concentrations (Cowley and Gorman, 1989). Silver-stained gels consistently revealed a number of minor proteins in purified BTV preparations; the most prominent of these migrated at 90 kDa, 52 kDa and 41 kDa. The 41 kDa protein, that migrated immediately above VP7, appears to be equivalent to protein 'x' described with other BTV isolates (Mertens et al., 1984). The 52 kDa protein may represent a cleavage product of VP5 (Cowley and Gorman, 1989). Digestion of BTV 20 with trypsin resulted in the cleavage of VP2 into a number of smaller polypeptides; all other virus proteins appeared to remain intact (Fig. 3, lane 2 ). The most prominent cleavage products (designated P ) were assigned according to their estimated molecular weights (in kDa ), as P93, P91, P66, P62, P54, P39, P38 and P25.5. Chymotrypsin also cleaved VP2 specifically to give major cleavage products designated P93, P76, P55, P54, P27 and P25 (Fig. 3, lane 3). Treatment of BTV 20 with 10 ~g ml -~ thermolysin did not cleave VP2 significantly (Fig. 3, lane 4). However, close analysis revealed three cleavage products, P93, P76 and P54, that were also observed with chymotrypsin. Subsequent digestion of BTV 20 with a higher thermolysin concentration (i.e. 100 ]lg ml -~) confirmed the formation of these major VP2 cleavage products (see Fig. 5). Analysis in a 12.5% gel of the cleavage products resulting from digestion of BTV 20 with each of the three proteases revealed that there were no detectable products below 25 kDa (Fig. 3b). Kinetics o[protease digestion of B T V 20 proteins An attempt was made to correlate directly the appearance of VP2 cleavage products with the observed increases in virus infectivity (Fig. 1 ) and HA activity (Fig. 2 ) resulting from digestion of BTV 20 with trypsin, chymotrypsin or thermolysin. Aliquots of virus were taken from the same reaction mixture, virus particles collected and their protein composition determined in 10% SDSpolyacrylamide gels. The protein compositions of BTV 20 ( ~ 295/tg m1-1 ) digested with 10 ]~g m l - 1 chymotrypsin for between 0.2 and 20 h are shown in Fig. 4a. Cleavage of VP2 was observed at 0.2 h, indicating that it occurred in
144
J.A. COWLEY AND B.M. GORMAN
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145
EFFECTS OF PROTEOLYTIC ENZYMES ON BLUETONGUE VIRUS TYPE 20
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J,A. COWLEY AND B.M. GORMAN
the time required to process the sample. Of the chymotrypsin cleavage described earlier (see Fig. 3a), P93, P54 and P27 were detected after a short exposure period (Fig. 4a, 0.2 h). After digestion for 1 h two additional polypeptides, P76 and P25, became more obvious. Increased incubation duration resulted in decreased amounts of P76 and P27, some increased in the amount of P25; the amounts of P93 and P54 remained relatively unchanged. A similar experiment was conducted using 10/~g ml-1 chymotrypsin and double the concentration of BTV 20 (i.e. ~ 585/~g m1-1 ) (Fig. 4b). Here, polypeptides P76 and P27 represented the major early VP2 cleavage products, and P55, P54 and P25 the minor products detectable after 1 h incubation (Fig. 4b, lane 2). Polypeptides P76, P55 and P27 disappeared with increased exposure and by 4 h (Fig. 4b, lane 5) had been replaced by P54 and P25 as the major cleavage products. The highest molecular weight cleavage product, P93, detected in Fig. 3 and Fig. 4a, could not be accurately identified in this gel. Digestion of BTV 20 ( ~295 #g ml -~) with thermolysin (100/~g m1-1) resulted in the cleavage of VP2 into three major polypeptides, P76, P54 and P25 (Fig. 5). The migrational properties of these polypeptides correspond closely to the three major cleavage products resulting from cleavage with chymotrypsin (see Fig. 4a and b). As was also the case with chymotrypsin, P93 (obscured in this gel by the smearing of VP2), P54 and P25 and were generated in the 1
2
3
4
5
6
7
8
9
10
11
94
I d
67 60
|
46
E
36 30
20 !i!!i!!!!~:,:
14
Fig. 5. Kinetics of the appearance of cleavage products of BTV 20 collected after in vitro digestion with 100/~g ml-1 thermolysin. BTV 20 protein concentration, incubation periods, electrophoresis conditions and lane designations are identical to those in Fig. 4a.
EFFECTS OF PROTEOLYTIC ENZYMES ON BLUETONGUE VIRUS TYPE 20
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time elapsed between addition of the protease and purification of virus particles (0.2 h; Fig. 5, lane 2 ). Another common cleavage product, P76, was prominent by 0.5 h and subsequently disappeared by 5 h. Other minor cleavage products (P53, P35 and P31) were detected in varying amounts from 0.5 h onwards. The most obvious cleavage product that was processed further was P76, while the amounts of P53, P31 and P25 appeared to increase slightly with increased exposure. Digestion with 10/lg m1-1 trypsin resulted in by far the most rapid and extensive cleavage of BTV 20 proteins (Fig. 6). Although VP2 and VP3 comigrated in this gel, the visual decrease in the intensity of this band suggested that VP2 was processed very rapidly and to an extent where it may have been digested completely in the time interval required to process the initial sample (0.2 h; Fig. 6, lane 2 ). In addition, VP1 was almost completely absent after 0.2 h, while further exposure resulted in some loss of VP5. The fact that proteins other than VP2 were susceptible to trypsin cleavage at these concentrations of virus and enzyme undoubtedly contributed to the numerous cleavage products. For this reason, only those cleavage products (i.e. P93, P91, P66, P62, P54, P39, P38 and P25.5) detected previously using a higher virus concentration (see Fig. 3, lane 2 ) are indicated in Fig. 6. Higher concentrations of BTV 20, reduced protease concentrations, or shorter exposure periods may be required to define the kinetics of trypsin cleavage of VP2.
1
2
3
4
5
6
7
8
9
10
11
1 2,3
94
4 5
67 60
E
46
7
36
30
'
....................
~1.......
'~
,:
20
Fig. 6. K i n e t i c s o f t h e a p p e a r a n c e o f cleavage p r o d u c t s o f B T V 20 collected after in vitro digestion w i t h 10 pg m l - 1 trypsin. B T V 20 p r o t e i n c o n c e n t r a t i o n , i n c u b a t i o n periods, electrophoresis cond i t i o n s a n d lane d e s i g n a t i o n s are identical to t h o s e in Fig. 4a.
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DISCUSSION The results presented in this paper demonstrate that in vitro digestion of BTV 20 with the proteases chymotrypsin, thermolysin or trypsin can initially enhance virus infectivity and HA activity. The primary action of these proteases involves cleavage of the outer capsid protein VP2 into a number of similar, but characteristic polypeptides. In addition, kinetic studies revealed that some VP2 cleavage products are only transiently associated with the ISVPs resulting from digestion of BTV 20 with chymotrypsin or thermolysin. Digestion of BTV 20 with chymotrypsin resulted in the progressive loss of VP2 and six smaller polypeptides appearing at different stages of cleavage: designated P93, P76, P55, P54, P27 and P25. Previous studies have also shown that VP2 of other BTV isolates and orbiviruses is susceptible to chymotrypsin cleavage, although some inconsistencies have occurred with regard to the number of cleavage products detected (Van Dijk and Huismans, 1980, 1982; Mertens et al., 1986, 1987). Van Dijk and Huismans (1980) made reference to two VP2 cleavage products, migrating above VP4 and just below VP5, that remained attached to ISVPs of BTV 10. However, the more recent studies of Mertens et al. (1986, 1987) reported four primary cleavage products of VP2 for ISVPs of BTV 1 and BTV 4. In order of decreasing molecular weight, the most obvious were designated VP2a, VP2b and VP2c; one polypeptide migrating below VP2c, now designated VP2d (P.P.C. Mertens, pers. comm., 1989), could be detected by silver staining but poorly by incorporation of [3~S]-methionine. Polypeptides VP2a, VP2b and VP2c migrated below VP3, VP5 and VP7, respectively, with BTV 4 and similarly with BTV 1 except that polypeptide VP2b migrated slightly above VP5. An additional minor polypeptide, migrating just above VP4 with BTV 4, and closer to VP3 with BTV 1, has also been detected in differing amounts depending on whether ISVPs were purified on CsC1 or sucrose gradients (P.P.C. Mertens, pers. comm., 1989). Thus, it appears that the number and relative size of VP2 cleavage products resulting from chymotrypsin digestion of BTV 4, and to a lesser extent BTV 1, are similar to those reported here for BTV 20. Kinetic studies revealed that VP2 of BTV 20 underwent progressive cleavage upon extended digestion with chymotrypsin. Using virus and enzyme concentrations that allowed investigation of the early stages of VP2 cleavage, polypeptides P93, P76 and P27 represented the major primary cleavage products. Further digestion revealed that P76 and P27 were only transiently associated with ISVPs and were replaced by two secondary cleavage products, P54 and P25. A major objective of this study was to determine whether proteases of different specificities to chymotrypsin would also cleave VP2, and to what extent this would affect virus infectivity and HA activity. Two other proteases tested, thermolysin and trypsin, both cleaved VP2 of BTV 20. Thermolysin in partic-
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ular generated VP2 cleavage products that were similar to those observed with chymotrypsin, except that two polypeptides, P35 and P31, were detected instead of P27. As was also the case with chymotrypsin, the P76 cleavage product generated by thermolysin was only transiently associated with ISVPs, and disappeared after extended exposure. Trypsin generated a number of major VP2 cleavage products that were quite distinct from those detected with chymotrypsin or thermolysin. Nevertheless two polypeptides, P93 and P54, were generated by all three proteases. At the same time however a major low molecular weight trypsin cleavage product, P25.5, was only marginally different to the P25 cleavage product generated by chymotrypsin and thermolysin. Unfortunately, any processing that occured with these VP2-specific cleavage products could not be determined with the BTV 20 and trypsin concentrations used in kinetic analyses. Under the conditions used, VP2 cleavage was essentially complete in the time that elapsed between addition of protease and processing of the sample. Moreover, the presence of numerous other polypeptides that resulted from cleavage of other virus proteins obscured those resulting from the specific cleavage of VP2. However, our results show that in vitro digestion of BTV 20 with chymotrypsin, thermolysin or trypsin generates three cleavage products of similar size, suggesting that at least two common regions on VP2 are sensitive to cleavage with these proteases. Mertens et al. (1986) have also reported that, for both BTV 1 and BTV 4, trypsin produced similar VP2 cleavage products to chymotrypsin, except the polypeptide equivalent to VP2c was slightly smaller. Therefore, the close similarities between the trypsin and chymotrypsin cleavage products reported for BTV 1 and BTV 4 (Mertens et al., 1986, 1987) and those found with BTV 20, suggest that there are conserved cleavage sites in the VP2 outer capsid proteins of serologically and geographically distinct BTV isolates. The biological significance of proteolytic cleavage of VP2 of BTV has only recently been addressed (Mertens et al., 1986, 1987). In these studies, digestion with chymotrypsin resulted in the mono-dispersion of virus particles, maintenance of similar virus infectivities to disaggregated untreated particles, maintenance of serotype-specificity in virus-neutralisation tests and the loss of HA activity. In addition, Mertens et al. (1986, 1987) reported that purification of BTV using sodium lauryl sarcosine (NLS) and dithiothreitol (DTT), followed by storage in the absence of NLS, caused virus particles to become highly aggregated. The BTV 20 preparation used here was purified in a similar manner, stored without NLS, and possessed a specific infectivity (i.e. P F U / OD26o unit) of 1.5 × 109. This specific infectivity was lower than that reported for BTV 10 (i.e. 1.1× 101°) purified in sucrose gradients without treatment with NLS or DTT (Verwoerd et al., 1972 ), but approximated that of disaggregated BTV 1 and BTV 4 (i.e. 1.8× 109 TCIDso/OD26o unit) maintained in 1% NLS after treatment with DTT (Mertens et al., 1986). This suggested that
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BTV 20 particles were aggregated, though to a lesser extent than that found with BTV 1 or BTV 4. Mertens et al. (1986, 1987) reported a 10-fold increase in infectivity when BTV 1 or BTV 4 was treated with NLS in combination with either D T T or chymotrypsin. With BTV 20, trypsin digestion resulted in an eightfold increase in infectivity while 50-fold maximum increases were observed with both chymotrypsin and thermolysin. Due to the rapid action of trypsin, however, the enhancement in infectivity observed had most probably passed its maximum. It appears likely that the increases in virus infectivities induced by chymotrypsin, thermolysin or trypsin resulted from disaggregation of BTV 20 ISVPs associated with cleavage of VP2, rather than from the activation of non-infectious particles. However, the extent of VP2 cleavage observed when significant increases in virus infectivities had occurred suggested that limited cleavage may not adversely affect the ability of BTV 20 particles to infect PS-EK cells. In contrast to the findings of Mertens et al. (1987), HA titres with three erythrocyte species were enhanced, similarly to infectivity titres, in the early stages of digestion of BTV 20 with the three proteases analysed. Kinetic studies of the effects of proteases on the HA activity and protein composition of BTV 20 suggested possible explanations for this discrepancy. HA titres increased initially, then decreased rapidly upon extended exposure to proteases, therefore, either the extent of VP2 cleavage, or the degree of particle disaggregation at the time of analysis could affect HA activity. The use of preparations of mono-dispersed particles will be required to ascertain a direct relationship between the observed increases in HA activity and infectivity of BTV 20 and proteolytic cleavage of VP2. Cleavage of outer capsid protein VP2 of BTV is comparable in many respects to similar phenomena observed with other Reoviridae members. For instance, with reoviruses, progressive digestion of three outer capsid proteins is observed upon extended exposure to chymotrypsin (Joklik, 1972; Borsa et al., 1973). This digestion of the reovirus outer capsid can have variable effects on virus infectivity and HA activity. It is also dependent on factors, such as the purity of virus preparations, enzyme concentrations and the presence of cations, that affect the extent of digestion. Limited digestion either maintains or enhances infectivity titres and HA activity, while extended digestion greatly reduces these activities (see review, Joklik, 1983). With rotaviruses, a major non-glycosylated outer capsid protein (VP3) is highly susceptible to protease digestion (Clark et al., 1981; Estes et al., 1981; Sato et al., 1987). With some rotavirus isolates, trypsin digestion can enhance virus infectivity, but cause the loss of HA activity (Barnett et al., 1979; Graham and Estes, 1981; Holmes, 1983). Chymotrypsin similarly reduces HA activity but does not enhance infectivity titres significantly (Graham and Estes, 1981; Kitaoka et al., 1984, 1986, 1987). Holmes (1983) has suggested that the cleavage of VP3 by trypsin may be important to rotavirus infection of intestinal epithelial cells, where contact with
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this enzyme is inevitable. Therefore it may be of some interest to examine the mechanism of infection of BTV in insect hosts, where primary contact would be with salivary gland and gut secretions, environments rich in proteases. ACKNOWLEDGEMENTS
Bluetongue virus research at the Queensland Institute of Medical Research was supported by a grant from the Australian Meat Research Committee. We thank Mr. R.W. Campbell for provision of cell cultures and Dr. P.P.C. Mertens for making available unpublished data and for helpful suggestions on the manuscript.
REFERENCES Barnett, B.B., Spendlove, R.S. and Clark, M.L., 1979. Effect of enzymes on rotavirus infectivity. J. Clin. Microbiol., 10: 111-113. Borsa, J., Copps, T.P., Sargent, M.D., Long, D.G. and Chapman, J.D., 1973. New intermediate subviral particles in the in vitro uncoating of reovirus virions by chymotrypsin. J. Virol., 11: 552-564. Clarke, D.M. and Casals, J., 1959. Techniques for haemagglutination and haemagglutinationinhibition with arthropod-borne viruses. Am. J. Trop. Med. Hyg., 7: 561-573. Clark, S.M., Roth, J.R., Clark, M.L., Barnett, B.B. and Spendlove, R.S., 1981. Trypsin enhancement of rotavirus infectivity: mechanism of enhancement. J. Virol., 39: 816-822. Cowley, J.A. and Gorman, B.M., 1987. Genetic reassortants for identification of the genome segment coding for the bluetongue virus hemagglutinin. J. Virol., 61: 2304-2306. Cowley, J.A. and Gorman, B.M., 1989. Cross-neutralization of genetic reassortants of bluetongue virus serotypes 20 and 21. Vet. Microbiol., 19: 37-51. Estes, M.K., Graham, D.Y. and Mason, B.B., 1981. Proteolytic enhancement of rotavirus infectivity: molecular mechanisms. J. Virol., 39: 879-888. Gorman, B.M., Leer, J.T., Filippich, C., Goss, P.D. and Doherty, R.L., 1975. Plaquing and neutralising of arboviruses in the PS-EK line of cells. Aust. J. Med. Technol., 6: 65-71. Graham, D.Y. and Estes, M.K., 1980. Proteolytic enhancement of rotavirus infectivity: biological mechanisms. Virology, 101: 432-439. Holmes, I.H., 1983. Rotaviruses. In: W.K. Joklik (Editor), The Reoviridae. Plenum, New York, pp. 359-423. Hubschle, O.J.B., 1980. Bluetongue virus haemagglutination and its inhibition by specific antiserum. Arch. Virol., 64: 133-140. Huismans, H. Van Der Walt, N.T., Cloete, M. and Erasmus, B.J., 1983. The biological and immunological characterization of bluetongue virus outer capsid polypeptides. In: R.W. Compans and D.H.L. Bishop (Editors), Double-Stranded RNA Viruses. Elsevier, Amsterdam, pp. 165172. Huismans, H., Van Der Walt, N.T., Cloete, M. and Erasmus, B.J., 1987. Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. Virology, 157: 172-179. Joklik, W.K., 1972. Studies on the effect of chymotrypsin on reovirons. Virology, 49: 700-715. Joklik, W.K., 1983. The reovirus particle. In: W.K. Joklik (Editor), The Reoviridae, Plenum, New York, pp. 9-78.
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Kitaoka, S., Suzuki, H., Numazaki, Y., Sato, T., Konno, T., Ebina, T. and Ishida, N., 1984. Hemagglutination by human rotavirus strains. J. Med. Virol., 13: 215-222. Kitaoka, S., Suzuki, H., Numazaki, Y., Konno, T. and Ishida, N., 1986. The effect of trypsin on the growth and infectivity of human rotavirus. Tohoku J. Exp. Med., 149:437 447. Kitaoka, S., Suzuki, H., Sato, T., Numazaki, Y., Konno, T. and Ishida, N., 1987. Failure in chymotrypsin enhancement of human rotavirus infectivity. Tohoku J. Exp. Med., 151: 127-128. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature {London), 227: 680-685. Marshall, T., 1984. Detection of protein in polyacrylamide gels using an improved silver stain. Anal. Biochem., 136: 340-346. Martin, S.A., and Zweerink, H.J., 1972. Isolation and characterisation of two types of bluetongue virus particles. Virology, 50: 495-506. Mertens, P.P.C., Brown, F. and Sangar, D.V., 1984. Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode. Virology, 135: 207-217. Mertens, P.P.C., Burroughs, J.N. and Anderson, J., 1986. Purification procedures and structural analyses of bluetongue virus particles and sub-viral particles of serotyes 1 and 4. In: P. Roy, C.E. Schore and B.I. Osburn {Editors ), Orbiviruses and Birnaviruses: Proceedings of the Double-Stranded RNA Virus Symposium, Oxford, pp. 53-73. Mertens, P.P.C., Burroughs, J.N. and Anderson, J., 1987. Purification and properties of virus particles, infectious sub-viral particles and cores of bluetonge virus serotypes 1 and 4. Virology, 157: 375-386. Sato, T., Kitaoka, S., Suzuki, H., Konno, T. and Ishida, N., 1987. Effect of trypsin and chymotrypsin on polypeptides of human rotavirus KUN strain. Med. Microbiol. Immunol., 176:65 73. Van Dijk, A.A. and Huismans, H., 1980. The in vitro activation and further characterisation of the bluetongue virus-associated transcriptase. Virology, 104: 347-356. Van Dijk, A.A. and Huismans, H., 1982. The effect of temperature on the in vitro transcriptase reaction of bluetongue virus, epizootic haemorrhagic disease virus and African horsesickness virus. Onderstepoort J. Vet. Res., 49: 227-232. Verwoerd, D.S. and Huismans, H., 1972. Studies on the in vitro and the in vivo transcription of the bluetongue virus genome. Onderstepoort J. Vet. Res., 39: 185-192. Verwoerd, D.W., Els, H.J., De Villiers, E.M. and Huismans, H., 1972. Structure of the bluetongue virus capsid. J. Virol., 10: 783-794. Weber, K. and Osburn, M., 1969. The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J. Biol. Chem., 24: 4406-4412.
