Rnase of Classical Swine Fever Virus: Biochemical Characterization and Inhibition by Virus-Neutralizing Monoclonal Antibodies JO¨ RG M

JOURNAL OF VIROLOGY, Jan. 1996, p. 352–358 Vol. 70, No. 1 0022-538X/96/$04.00ϩ0 Copyright ᭧ 1996, American Society for Microbiology

RNase of Classical Swine Fever Virus: Biochemical Characterization and Inhibition by Virus-Neutralizing Monoclonal Antibodies JO¨ RG M. WINDISCH,1 RAINER SCHNEIDER,1 ROBERT STARK,2 EMILIE WEILAND,3 3 2 GREGOR MEYERS, AND HEINZ-JU¨ RGEN THIEL * Institute of Biochemistry, University of Innsbruck, A-6020 Innsbruck, Austria,1 and Federal Research Centre for Virus Diseases of Animals, D-72076 Tu¨bingen,3 and Institut fu¨r Virologie, Justus-Leibig-Universita¨t Giessen, D-35392 Giessen,2 Germany

Received 16 June 1995/Accepted 4 October 1995

The structural glycoprotein E0 of classical swine fever virus (CSFV) possesses an intrinsic RNase activity. Here we present the first comprehensive biochemical characterization of E0, using a recombinant glycoprotein expressed in insect cells. We were able to show that the presence of neither carbohydrate moieties nor disulfide bonds is a prerequisite for RNase activity. In addition, virus-neutralizing and nonneutralizing anti-E0 monoclonal antibodies were tested for their ability to influence RNase activity. In these experiments, the antibodies which effectively blocked the infection of STE cells also exerted a high degree of E0 RNase inhibition. This correlation suggests that the RNase activity of CSFV E0 plays a role in the viral life cycle.

Classical swine fever, also called hog cholera or European to neutralize CSFV infection of STE cells by monoclonal an- swine fever, is a viral disease leading to severe economic losses tibodies (MAbs) directed against E0 had been performed (32). worldwide (34). The causative agent is classical swine fever Some of these antibodies neutralized CSFV infectivity to var- virus (CSFV) (17, 18, 28, 30), a member of the Pestivirus genus, ious degrees, whereas others had no effect. Together with the family Flaviviridae (4, 7, 29). Other members of this genus are detection of RNase activity, these findings made E0 a promis- bovine viral diarrhea virus and border disease virus of sheep ing target for the development of antiviral drugs and vaccines. (4). The positive-stranded RNA genome comprises a single To this end, it is necessary to further study the E0 protein in long open reading frame (15, 19). Viral gene expression occurs biochemical as well as functional terms. The enzymatic prop- by translation of this open reading frame into a large polypro- erties of E0, the importance of glycosylation and dimerization, tein which is processed co- and posttranslationally by virus- as and the inhibition of the inherent RNase activity by divalent well as host cell-encoded proteases (23, 27, 35). The 5Ј-termi- cations and MAbs might give clues to alternative strategies to nal part of the pestiviral RNA encodes the structural proteins, combat CSFV and related viruses. For such experiments, large namely, the capsid protein C and three envelope glycoproteins quantities of E0 protein are required. Since only limited (E0, E1, and E2) (30). E0 and E2 induce virus-neutralizing amounts of E0 protein could be purified from pig cells infected antibodies (32, 33); both glycoproteins have been shown to with CSFV, the E0 glycoprotein was recombinantly expressed induce protective immunity in the natural host (9, 12, 22, 31). in insect cells within the boundaries defined by proteolytic The E0 glycoprotein forms a disulfide-bonded homodimer cleavages observed in vivo (23). with an apparent molecular mass of about 97 kDa. The corresponding monomers have slightly differing molecular masses MATERIALS AND METHODS (44 and 48 kDa, respectively), probably due to variation in glycosylation. Each monomer consists of 227 amino acids and Detection of RNase activity. The purified authentic (24) as well as recombinant corresponds to residues 268 to 494 of the CSFV polyprotein (30a) glycoproteins were tested for RNase activity as described by Schneider et al. (24). From 50 pg to 0.5 ␮g of E0 was incubated with 80 ␮g of poly(rU) in 40 (15, 23). This indicates that about 50% of the molecular mass mM Tris-acetate (pH 6.5)–0.5 mM EDTA–5 mM dithiothreitol (DTT) for 60 of the mature E0 glycoprotein is made up of carbohydrates. E0 min at 37ЊC. The reactions were stopped by the addition of 100 ␮l of 1.2 N is apparently not attached to the membrane by a transmem- HClO4–25 mM LaSO4. After 15 min of incubation on ice and 15 min of centrif- brane helix, and a considerable portion of the protein is actu- ugation (15,000 ϫ g,4ЊC), 185 ␮l of the supernatant was transferred to a new tube and diluted with 500 ␮l of distilled water, and the A260 was measured in ally secreted from infected cells (23). The mechanism by which 1-mm crystal cuvettes in a Hitachi U-2000 spectrophotometer. The reasons for E0 is bound to the virion surface is yet to be elucidated (32). including 5 mM DTT in the assays are (i) to standardize redox conditions and to Only recently, an additional function of E0 was discovered (8, protect the enzyme from forming ‘‘wrong’’ disulfide bonds and (ii) to avoid 24). Comparative amino acid sequence analysis with sensitive having a heterogeneous solution of dimeric and monomeric proteins. Temperature and pH dependence of the E0 RNase activity. An 80-␮g amount search algorithms revealed the existence of sequence features of 16/23S rRNA from Escherichia coli MRE600 (Boehringer Mannheim) was in the deduced primary structure of E0 characteristic of a incubated with 20 ng of E0 for 30 min at the different temperatures used (Fig. 1a) family of fungal and plant RNases (24). Subsequent biochem- in 100 ␮l of 40 mM Tris-acetate (pH 6.5)–0.5 mM EDTA–5 mM DTT. The ical analysis of E0 glycoprotein purified from cells infected reactions were stopped and processed as described above, and the A260 was measured. The experiments for pH dependence were carried out in an identical with CSFV demonstrated that E0 actually possesses RNase fashion. activity (24). All values were corrected for non-E0-specific RNA degradation by subtracting Before the enzymatic function of E0 was revealed, attempts the values obtained in parallel assays lacking E0. Non-E0-specific RNA degradation and variations between parallels were very low (Ͻ5% of the total value) in all experiments presented in this work. Influence of divalent cations and chelators. The assays were performed as for the temperature and pH curves. For the strongest inhibitors, the inhibition * Corresponding author. Mailing address: Institut fu¨r Virologie, Jus- curves were established, and the concentrations at which half-maximal inhibition tus-Liebig-Universita¨t Giessen, Frankfurter Strasse 107, D-35392 occurred were calculated. Giessen, Germany. Phone: 49-641-702-4990. Fax: 49-641-702 4990. Substrate specificity and reaction kinetics. (i) Polymeric substrates. The

352 VOL. 70, 1996 RNase OF CSFV 353

RNase assays were basically performed as described above. For specific experi- mental descriptions, see the figure legends. (ii) CSFV RNA. Harvesting of CSF virions and preparation of RNA were performed as described by Ru¨menapf et al. (21). About 0.5 ␮g of CSFV RNA was incubated in 10 ␮l of 40 mM Tris-acetate (pH 6.5)–0.5 mM EDTA–5 mM DTT with different amounts of authentic CSFV E0 for 30 min at 37ЊC. After phenol extraction and ethanol precipitation, the samples were subjected to RNA gel electrophoresis (21). Northern (RNA) hybridization was performed with a CSFV-specific cDNA probe as described before (16). (iii) Cyclic nucleotides. A total of 0.4 ␮g of the respective proteins was incubated at 37ЊC with 0.4 mM 2Ј,3Ј-cyclic CMP (cCMP) or 2Ј,3Ј-cyclic UMP (cUMP) in 100 ␮l of 40 mM Tris-acetate (pH 6.5)–0.5 mM EDTA–5 mM DTT. The hydrolysis of the cyclic nucleotides was traced photometrically as the increase in A286 (cCMP) or A276 (cUMP) over a period of3hasassessed by a time scan in the Hitachi U-2000 spectrophotometer. As a control, the same experiments were done with an equal molar amount of bovine RNase A (0.11 ␮g; purchased from Boehringer Mannheim). Influence of sulfhydryl agents and salt concentration on E0 activity. Authentic and recombinant E0 dimers (10 ng) were incubated with different concentrations (0 to 100 mM) of DL-DTT and 2-mercaptoethanol (both from Sigma) for 60 min at 37ЊC with gentle shaking in 80 ␮l of 40 mM Tris acetate–0.5 mM EDTA. After this, 80 ␮g of 16/23S rRNA dissolved in the same buffer was added, followed by incubation at 37ЊC for another 60 min. The reactions were stopped and processed as described above. In order to analyze the importance of dimer formation via noncovalent forces as well as the influence of different salt concentrations on the enzymatic activity in general, analogous experiments were performed with both enzymes (authentic and recombinant E0) at NaCl concentrations ranging from 0 to2Minthebufferdescribed above containing 20 mM DTT. Effect of deglycosylation on RNase activity of E0. Recombinant E0 (1 ␮g) was deglycosylated by incubation with 10 mU of endoglycosidase H (Boehringer Mannheim) in 30 ␮l of 50 mM sodium acetate (pH 5.5)–10 mM DTT–0.5 mM phenylmethylsulfonyl fluoride at 37ЊC for 15 h. As a control for all the experiments involving deglycosylated E0, another microgram of the protein was treated in an identical fashion, but instead of endoglycosidase H, the respective enzyme storage buffer was added. To obtain a reference point for the specific activity of the deglycosylated recombinant enzyme, a small amount of the authentic enzyme from virus-infected pig cells was also deglycosylated. The specific activities of the glycoprotein and deglycosylated forms of authentic as well as recombinant E0 were determined by incubating 80 ␮gofE. coli 16/23S rRNA with 0.5 ng of the respective E0 for 30 min at 37ЊCin100␮lof40 mM Tris-acetate (pH 6.5)–0.5 mM EDTA–5 mM DTT. The digests were stopped and processed as described above, and the A260 was determined. All values were corrected for non-E0-specific RNA degradation by subtracting the values obtained in parallel assays lacking the enzyme. The specific activities were calculated as A260 units per minute per milligram, corrected for non-E0-specific RNA degradation (controls) and set in relation to each other. Inhibition of E0 RNase activity by antibodies. The sera and hybridoma super- natants were diluted with phosphate-buffered saline, and the antibodies were FIG. 1. (a) Temperature dependence of E0 RNase activity. The activities of purified by protein A-Sepharose (Boehringer Mannheim) chromatography by authentic (F) and recombinant (E) E0 in degrading E. coli 16/23S rRNA were standard procedures. Buffer molecules and salt ions were removed by Sephadex determined at 0, 4, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65ЊC. All data points G10 gel filtration chromatography; the flowthrough was lyophilized, and the are means of duplicates. (b) pH dependence of E0 RNase activity. The mea- antibodies were dissolved in 50 ␮l of 40 mM Tris acetate (pH 6.5)–0.5 mM surements were performed as in panel a at pHs ranging from 4.0 to 9.0 in steps EDTA for each milliliter of hybridoma supernatant and in 0.5 ml of the same of 0.5 pH unit. For the lower pH range, sodium acetate buffer was used; for the buffer for each milliliter of polyclonal serum. This led to concentrations of about higher pH range, Tris-acetate buffer was used (both at 40 mM). For pH 6.0, 6.5, 0.5 mg of immunoglobulin G per ml for the MAb stock solutions and about 7 and 7.0, both buffers yielded identical results, allowing combination of the data mg/ml for the polyclonal antibody (PAb) stock solutions. For the inhibition sets. All data points are means of duplicates. assays, see the legend to Fig. 6.

RESULTS shown). A reaction time course experiment showed that RNA degradation slows only when the substrate becomes depleted Enzymatic activity of recombinant E0. A recombinant E0 (data not shown). protein encompassing CSFV amino acids 268 to 494 was ex- Temperature and pH dependence of the E0 RNase activity. pressed in insect cells from a baculovirus construct and purified The temperature dependence curves of the authentic and re- by immunoaffinity chromatography (30a). In this paper, E0 combinant E0 were nearly identical (Fig. 1a). E0 exhibits a expressed in insect cells is referred to as recombinant E0 and great degree of temperature stability, with RNase activity the form isolated from pig lymphoma cells infected with CSFV reaching a broad maximum between 50 and 60ЊC. At 65ЊC, the is referred to as authentic E0 for reasons of simplicity. RNase enzyme still showed approximately 75% of its maximum activ- assays showed that the enzyme was expressed in an active form ity; at 37ЊC, it was about 86%. by the insect cells. The detected activity was comparable to the The pH curves were also very similar for the authentic and one obtained for authentic E0 (24). For both enzymes (authen- recombinant enzymes and revealed a preference of E0 for tic and recombinant), 50 pg was sufficient to yield detectable slightly acidic milieus, as found for many RNases (Fig. 1b). RNA degradation in an RNase assay with poly(rU) as the Between pH 3.5 and 4.5, RNase activity leaped from ϳ25% to substrate, indicating high specific activities of the enzymes. greater than 90% of maximum activity but then increased only An enzyme activity versus enzyme concentration curve was slightly, reaching a maximum at pH 6.0 to 6.5. Similar to the carried out with E. coli 16/23S rRNA as a substrate; linear decrease below pH 4.5, an increase above pH 7.0 led to a correlation of both parameters was demonstrated (data not drastic reduction in enzymatic activity. 354 WINDISCH ET AL. J. VIROL.

FIG. 2. (a) Inhibition of E0 RNase activity by divalent cations and EDTA. The ability of divalent cations to inhibit the degradation of E. coli 16/23S rRNA by authentic (left, solid bars) and recombinant (right, shaded bars) E0 was tested. All data points are means of duplicates. (b) Inhibition of RNase activity of E0 by Mn2ϩ and Zn2ϩ. For the strongest inhibitors, Mn2ϩ (F) and Zn2ϩ (■), the inhibition curves were established with recombinant E0. Fifty percent inhibition was reached at about 137 ␮MMn2ϩand about 16.5 ␮MZn2ϩ. All data points are FIG. 3. (a) Substrate specificity of E0 RNase activity. The assays were per- means of duplicates. formed as described for Fig. 1 and 2 except that 5 ng of E0 was used. Because of the high specific activity towards uridine-rich substrates, the experiments had to be repeated for poly(rU), ds poly(rA)-poly(rU), ds poly(rG)-poly(rU), and poly(rGU) with 10-fold less (0.5 ng) E0. The A260 [A273 for poly(rC)] was Influence of divalent cations and chelators on E0 RNase determined. For the DNA substrates, positive controls with DNase I (Boehringer activity. Many different types of RNases can be substantially Mannheim) were performed. All values were corrected for non-E0-specific RNA inhibited by the addition or withdrawal of certain divalent degradation by subtracting the values obtained in parallel assays lacking E0. cations (6, 13). In an initial experiment, all relevant divalent Individual results are given as a percentage of the activity with yeast RNA (100%), taking into consideration the amount of enzyme present in each assay. cations or cation chelators were tested at high concentrations. All data points are means of duplicates. rRNA, 16/23S rRNA from E. coli The ones that showed some degree of inhibition were then MRE600; tRNA, tRNA from E. coli. (b) Northern blot analysis of CSFV RNA investigated in more detail. Most of the cations and EDTA did after incubation with E0. After incubation with different amounts of authentic not have a pronounced effect on enzymatic activity. High con- E0, RNA was separated electrophoretically. Samples were transferred to a mem- centrations of Ca2ϩ and Mg2ϩ led to a weak inhibition of RNA brane and hybridized with a CSFV Alfort-derived cDNA probe (16). degradation (Fig. 2a), which in the case of Mg2ϩ is not sur- prising, since this cation is known to be important for nucleic acid stability. The only cations that led to strong inhibitions of Substrate specificity and enzyme kinetics of the E0 RNase E0 activity even in the submillimolar range were Mn2ϩ and activity. The substrate specificity of an RNase sometimes gives Zn2ϩ, with Zn2ϩ being the most potent inhibitor. For these clues to its biological function, especially if the range of de- ions, the inhibition curves were established (Fig. 2b). Fifty graded substrates is narrow. The specific activities of authentic percent inhibition was observed at ϳ137 ␮MMn2ϩand ϳ16.5 and recombinant E0 in degrading 13 different potential sub- ␮MZn2ϩ, respectively. The values for recombinant E0 were strates were determined. Recombinant and authentic E0 ex- almost identical to those for the authentic enzyme. hibited the same substrate specificity, with a clear preference VOL. 70, 1996 RNase OF CSFV 355

form the 3Ј-phosphoryl product (25). Strong differences can be observed in the rate at which individual RNases catalyze this second step, making it a characteristic feature of an enzyme (3). Using a large quantity of recombinant E0, we could detect hardly any hydrolysis of 2Ј,3Ј-cUMP and 2Ј,3Ј-cCMP in our assay, while an equimolar amount of bovine RNase A led to complete hydrolysis in the same time span (data not shown). This indicates either that hydrolysis of 2Ј,3Ј-cNMPs by E0 occurs extremely slowly, that the 2Ј,3Ј-cNMPs represent the end product of E0 RNA degradation, or that E0 degrades its substrates via a different mechanism, possibly producing 3Ј- hydroxy nucleotides. Complementary experiments with authentic E0 ruled out the possibility that the inability of the recombinant enzyme to hydrolyze 2Ј,3Ј-cNMPs was due to improper folding or a missing modification (data not shown). E0 is active in the presence of high concentrations of sulf- FIG. 4. Reaction kinetics of the degradation of poly(rU) by E0. For the hydryl agents and over a wide range of salt concentrations. E0 determination of the reaction kinetics, 1 ng of recombinant E0 was incubated monomers form a disulfide-bonded homodimer with an appar- with poly(rU) at concentrations ranging from 1.64 ␮M to 3.25 mM (concentra- ent molecular mass of 97 kDa (24, 30, 32). Since dimer formation of cleavable dinucleotides) for 20 min at 37ЊC in the buffer described for the tion is a prerequisite for the activity of many enzymes, we detection of RNase activity (see Materials and Methods). After acid precipita- investigated whether this structural feature of E0 plays a role tion and centrifugation, the supernatant was diluted 1:2, and the A260 was measured. The curve for authentic E0 was identical to that of the recombinant in its enzymatic function as an RNase or if a monomer also enzyme. possesses this activity. Authentic E0 as well as recombinant E0 was preincubated with DL-DTT and 2-mercaptoethanol at concentrations of up to 100 mM to break existing disulfide bonds for uridine-rich sequences (Fig. 3a). Poly(rU) and poly(rGU) before the substrate was added. The enzymatic activities of were degraded with relative activities of about 1,700 and 260%, these proteins were tested in RNase assays which showed that respectively. Neither poly(rA), poly(rC), nor poly(rG) was sus- the formation of covalently bonded homodimers via intermo- ceptible to hydrolysis by E0. lecular disulfide bonds is not a prerequisite for E0 RNase To determine whether E0 is an RNase specific for single- activity (data not shown). Moreover, once correctly folded, E0 stranded (ss) RNA or if it also accepts double stranded (ds) does not depend on intramolecular disulfide bonds for the RNA (as in RNA secondary structures) as a substrate, E0 was maintenance of an active conformation. incubated with mixtures of poly(rA)-poly(rU), poly(rC)- This result, however, did not rule out the possibility that E0 poly(rG), and poly(rG)-poly(rU) that had previously been dimers remain intact via noncovalent forces even when disul- turned into double strands. Interestingly, the poly(rU) strand fide bonds have been broken. To induce dissociation of any in ds poly(rA)-poly(rU) and in ds poly(rG)-poly(rU) was pro- noncovalently bonded E0 dimers, E0 pretreated with DTT was tected from degradation by the formation of an RNA double incubated with concentrations of NaCl ranging from 0 M, at strand (Fig. 3a). In conformity with the stability of the base which hydrophobic protein-protein interactions are at a mini- pairs in these double strands, the hydrolysis of poly(rU) was mum, to 2 M, at which ionic interactions are at a minimum. reduced by 90% in ds poly(rA)-poly(rU) and by 76% in ds Neither very low nor very high concentrations of NaCl altered poly(rG)-poly(rU). As expected from the experiments with ss E0 RNase activity (data not shown). Therefore, the formation RNA, no degradation of ds poly(rC)-poly(rG) was found. E0 of dimers, covalently or noncovalently bonded, is not a prereq- does not possess any DNase activity, as assessed by incubation uisite for the enzymatic activity of E0. These results also show with ds and ss DNA prepared from E. coli. that the tertiary structure of E0 is very rigid and that the RNase In order to determine whether CSFV RNA is accessible to is enzymatically active over a wide range of salt concentrations. its own RNase, viral RNA was incubated with authentic E0 and Effect of deglycosylation on RNase activity of E0. Many analyzed by Northern hybridization (Fig. 3b). Increasing deg- glycoproteins depend on their carbohydrate moieties for opti- radation of CSFV RNA after incubation with increasing mal function. To elucidate whether this is also the case for E0, amounts of E0 can be clearly demonstrated. Incubation with 4 which contains about 50% carbohydrates, the necessity of gly- ng of E0 led to complete degradation of the viral RNA. cosylation for the enzymatic activity of E0 was investigated. To ensure that all the quantitative data presented in this For this purpose, recombinant E0 was deglycosylated with en- work were measured at substrate concentrations at which endoglycosidase H. To ensure that deglycosylation was complete zyme activity is in the linear range, the enzyme kinetics of the and that the specific RNase activities to be determined were degradation of the preferred E0 substrate poly(rU) were de- not due to remaining intact glycoprotein, the deglycosylated termined (Fig. 4). The Km of E0 for cleavable UpU dinucle- proteins as well as control proteins were subjected to reducing otides was 872.5 Ϯ 125.9 (standard deviation [SD]) ␮M, and SDS-PAGE. The molecular mass shifted from ϳ42 to 44 kDa Ϫ1 Ϫ1 the vmax was 1.123 Ϯ 0.364 (SD) mmol min mg . This (for the intact glycoprotein) toward ϳ26 kDa (the calculated means that for the preferred substrate poly(rU), our measure- molecular mass of the deglycosylated protein) (Fig. 5). In the ments for 80 ␮g of substrate were performed right around the lane containing deglycosylated protein, more than 95% of the

Km. For the other substrates, the working concentrations were enzymatic activity disappeared from 42/44 kDa and reap- even below the Km and therefore still within the linear range of peared as a novel peak of RNase activity at ϳ26 kDa (Fig. 5). enzyme activity. These results indicate that (i) deglycosylation was complete, RNases often degrade their RNA substrates by a two-step (ii) the carbohydrate moieties are not required for the proper mechanism (3). In the ﬁrst step, the RNA polymer is broken refolding of the denatured proteins, and (iii) the presence of down into 2Ј,3Ј-cyclic mononucleotides (2Ј,3Ј-cNMPs). Subse- carbohydrate moieties is dispensable for the RNase activity of quently, the intramolecular phosphoryl ester is hydrolyzed to E0, at least in qualitative terms. 356 WINDISCH ET AL. J. VIROL.

FIG. 6. Inhibition of E0 RNase activity by PAb and MAbs. Antibodies (5 ␮l; ϳ2.5 ␮g for MAbs and 35 ␮g for the PAb) were incubated with 0.5 ng of FIG. 5. Effect of deglycosylation on activity of E0. Recombinant E0 was recombinant E0 for 60 min at 37ЊC with gentle shaking in 80 ␮lof40mM deglycosylated by incubation with endoglycosidase H. Both deglycosylated re- Tris-acetate (pH 6.5)–0.5 mM EDTA. After this, 80 ␮g of poly(rU) dissolved in combinant E0 and a nondeglycosylated control (15 ␮l [0.5 ␮g]) were loaded onto 20 ␮l of the same buffer was added, followed by incubation at 37ЊC for 60 min. an SDS-polyacrylamide gel; on the second half of the gel, 5 ␮l each of the same The digests were stopped and processed as described above, and the A260 was samples was loaded. After electrophoresis under reducing conditions, the gel was measured. A positive and a negative control were included. All values were cut into halves, and the second half was silver stained. To allow refolding of the corrected for non-E0-specific RNA degradation, and the reduction in RNase denatured protein, the first half was incubated in 40 mM Tris-acetate (pH activity (as a percentage of the positive-control activity) was calculated. The 6.5)–0.5 mM EDTA–5 mM DTT with frequent changes of the buffer for 8 h. The antibodies were present at a high molar excess (about 5,000-fold) over E0, so all lanes containing deglycosylated E0 and the control were cut into horizontal slices available epitopes were occupied. In accordance with this, an increase in the about 1 mm in height. Each slice was homogenized, and an aliquot was subjected amount of antibodies by a factor of 3 did not result in enhanced inhibition of E0 to an RNase assay with poly(rU) as the substrate (60 min, 37ЊC; all other activity. The neutralization assays were performed as described by Weiland et al. procedures as above). As a control, a gel without E0 which had been processed (32). The ability of an individual antibody to block virus infection was scored on identically was used. The value for this control was subtracted from the observed a scale from 0 to 5. All data points are means of duplicates. pAb, polyclonal absorbance values. By assigning each slice to a distinct molecular mass, the anti-E0 serum. measured activities were set in relation to the proteins seen in the stained gel. gp, recombinant E0 glycoprotein; deglyc, deglycosylated recombinant E0 protein. (32). However, correlation between virus neutralization and inhibition of RNase activity was not seen with MAb 1d13, To quantitatively assess the importance of glycosylation for which neutralized virus but did not significantly inhibit E0 E0 RNase activity, the specific activities of authentic E0, re- activity. A control MAb (a18) directed against another coat combinant E0, and deglycosylated forms of both enzymes were protein of CSFV (E2) (Fig. 6) as well as several other control determined and set in relation to each other. Interestingly, MAbs (data not shown) did not inhibit E0 RNase activity. In treatment of authentic E0 (purified from CSFV-infected cells) addition, a monospecific serum against E0 completely blocked with endoglycosidase H led to almost complete removal of E0 RNase activity and efficiently neutralized virus infectivity. carbohydrate chains. The specific activities of nondeglycosylated authentic and recombinant E0 for yeast RNA were ba- DISCUSSION Ϫ1 Ϫ1 sically identical (485 and 520 A260 units min mg , respectively). The deglycosylated proteins exhibited a reduction of In this paper, we report a comprehensive biochemical char- only about 30 to 40% in enzymatic activity on an equimolar acterization of the E0 surface glycoprotein of CSFV. E0 pro- basis (data not shown). tein purified from pig lymphoma cells infected with CSFV as Inhibition of E0 RNase activity by antibodies. A crucial well as from insect cells infected with a recombinant baculovi- question about the CSFV E0 glycoprotein is whether it serves rus was characterized, and the biochemical properties of E0 any additional biological function besides being a structural from both sources were compared. The large quantities of component of the viral envelope. The importance of E0 as a functional recombinant protein obtained by expression in in- potential target to combat CSFV was clearly demonstrated by sect cells allowed detailed studies, including the relevance of virus neutralization assays with MAbs and by the induction of glycosylation of E0. It was shown that most virus-neutralizing protective immunity by E0 protein expressed via vaccinia virus anti-E0 MAbs also have a strong inhibitory effect on the RNase (12, 32). The only biochemical function that has been assigned activity of this protein. This suggests that the enzymatic activity to E0 so far is the RNase activity described by Schneider et al. of E0 plays a role in the viral life cycle. (24). The ability of several virus-neutralizing and nonneutral- In order to investigate whether E0 possesses RNase activity, izing anti-E0 MAbs to influence this intrinsic activity of E0 was Hulst et al. (8) previously expressed the region encoding amino investigated. acids 268 to 507 of the CSFV polyprotein as a recombinant Surprisingly, five of six MAbs which neutralized CSFV in the protein. The expressed region covers not only the entire se- infectious assays also significantly blocked the RNase activity quence of E0 but also the first 13 amino acids of the following of E0 (Fig. 6). The MAb that showed strong inhibition of E0 CSFV glycoprotein, E1. In this context, it is important to men- RNase activity, 24/16, also effectively neutralized virus infection that the sequence upstream of the cleavage site between tivity. One MAb (4b6) which has little effect on virus infectivity both proteins (Ala-494 and Leu-495) contains residues re- hardly influenced E0 enzymatic activity in spite of showing quired by the Ϫ1, Ϫ3 rule for signalase cleavage but lacks the good binding properties in immunoprecipitation experiments hydrophobic domain which is part of the signal peptide. It has VOL. 70, 1996 RNase OF CSFV 357 been speculated that this sequence resembles an incomplete et al. (8) and a thorough characterization of enzymatic param- (atypical) signalase cleavage site. Alternatively, cleavage may eters, a number of novel features of E0 were discovered. First, be achieved by a Golgi apparatus-associated protease. Both E0 was unable to degrade ds RNAs. The dogma that RNases suggestions provide explanations for the observed delayed of the pancreatic type are unable to degrade ds RNAs was cleavage between E0 and E1 (23). Preliminary data obtained recently questioned by results from several groups (10, 14), from our laboratory indicate that cleavage at the E0/E1 site is making this property of E0 even more unusual. It is also note- incomplete if short E1-derived sequences are present. The worthy that E0 does not seem to be capable of degrading its resulting C-terminally extended E0 used in the studies men- substrates all the way to the 3Ј-phosphoryl products but re- tioned above (8) may exhibit characteristics different from a leases the 2Ј,3Ј-cNMPs as its end products. However, there are recombinant E0 expressed in the authentic boundaries. In fact, a number of examples of RNases that do not degrade cyclic the biochemical characteristics of the aberrant E0 expressed by 2Ј,3Ј-cNMPs to the 3Ј-phosphoryl products, especially endo- Hulst et al. (8) differ significantly from those of the E0 used in nucleases that participate in RNA processing and turnover, our study (see below). which generate 3Ј-hydroxy termini. In these cases, the cyclic The most striking biochemical difference between our re- nucleotides represent the final stages of degradation (3). It is combinant protein and the protein expressed by Hulst et al. (8) also possible that E0 utilizes an as yet undiscovered mechanism is the pH optimum, which they determined to be 4.5. This led of degrading its substrates or that the rate of hydrolysis is them to speculate that E0 is sorted in the Golgi complex by simply too slow to be detected by conventional methods. binding to the mannose-6-phosphate receptor and moved to Most glycosylated enzymes investigated so far show drastic lysosomes. From deglycosylation experiments, they speculate reductions in enzymatic activity after deglycosylation (5). The that E0, in contrast to E2, is not localized to the endoplasmic molecular mechanisms by which the carbohydrate moieties reticulum or cis-Golgi. These hypotheses, however, are based influence enzymatic activity in these enzymes have not been on the finding that in the system they used, E0 is not secreted. investigated in detail. In E0, the carbohydrate moieties make Since E0 is in fact secreted from pig cells as well as insect cells up about half of the apparent molecular mass. Our results (23, 30a) and exhibits a pH optimum of 6.0 to 6.5, it appears demonstrate, however, that glycosylation is not required for unlikely that it is actually targeted to lysosomes. Also, we proper RNase function (Fig. 5), even though a reduction in cannot envision a possible function for E0 in a lysosome, es- specific activity of about one-third was observed. There are a pecially if one takes into consideration that the pH optimum of few other examples of enzymes whose enzymatic activity is 4.5 described by Hulst et al. (8) actually represents the lowest basically not affected by the absence or presence of carbohy- possible pH at which reasonable enzymatic activity is observed. drates (26). In the case of E0, the carbohydrate moieties are The rigidity of E0 is also reflected in the temperature depen- most likely not directly involved in the catalytic mechanism but dence curve of its enzymatic activity. Authentic as well as may play a role in (i) maintaining an active conformation, (ii) recombinant E0 exhibited a great degree of temperature sta- efficiently acquiring substrate, (iii) stability against proteolytic bility, with RNase activity reaching a broad maximum between degradation, or (iv) simply increasing the solubility of the pro- 50 and 60ЊC. The temperature stability of these proteins is tein. In the course of these experiments it became clear that E0 greater than that exhibited by the larger protein expressed by is also enzymatically active as a monomer. This is interesting, Hulst et al. (8). since there are prominent examples of RNases, such as bovine Another important biochemical feature of an RNase is its seminal RNase, that require the formation of an enzyme ho- dependence on or inhibition by divalent cations. For RNases modimer for the active site to be constituted (2). The experi- which depend upon Mg2ϩ as a cofactor, Ca2ϩ,Zn2ϩ,Mn2ϩ, ments involving sulfhydryl agents and various salt concentra- and Co2ϩ often have a competitively inhibitory effect, even tions provided further hints that the formation of dimers is not though Mn2ϩ and Co2ϩ can also compensate for Mg2ϩ in some a prerequisite for proper enzymatic function of the protein. cases (6, 13). For such RNases, EDTA and EGTA (ethylene With respect to the active site of the RNase, it was interest- glycol tetraacetic acid) serve as inhibitors. E0 does not appear ing to investigate whether any of the CSFV-neutralizing an- to be dependent on Mg2ϩ, since the addition of high concen- ti-E0 MAbs described previously (32) interacted with or influ- trations of EDTA had no major effect on enzyme activity. enced the conformation of this region and thus inhibited Interestingly, Zn2ϩ and Mn2ϩ showed inhibition of the enzyme RNase activity. These studies revealed an interesting although even at concentrations in the micromolar range. Mg2ϩ and not perfect correlation between virus neutralization and E0 Ca2ϩ showed some inhibition of the enzyme when applied at RNase inhibition. The fact that most virus-neutralizing MAbs high concentrations. E0 RNase activity is therefore inhibited inhibited the enzymatic activity of E0 makes it unlikely that this by divalent cations via a noncompetitive mechanism which correlation is coincidental. The biological function of the possibly disturbs protein conformation or access to the sub- RNase activity should be studied by site-directed mutagenesis strate, or the required cation cofactor remains bound to the of the CSFV E0 gene in the context of an infectious viral enzyme so tightly that an inhibition of catalytic activity cannot genome. These experiments will resolve the question of be observed even at high concentrations of EDTA. These whether the E0 RNase activity plays an indispensable role in findings indicate that E0 functions via an enzymatic mecha- the infection cycle of CSFV. nism distinct from the one described for RNases of the pancreatic type (1, 3). This notion is also supported by the selec- ACKNOWLEDGMENTS tivity of E0 RNase with respect to its substrates. While its We thank Bernhard Auer for help with preparing some of the enzymatic activity toward standard RNase assay substrates figures and inspiring discussions. such as yeast RNA and E. coli rRNA is fairly distinct, it is This study was supported by the Bundesministerium fu¨r Forschung specifically high toward uridine-rich substrates. This high sub- und Technologie and Intervet BV (project 0319028A). strate specificity of E0 in combination with the knowledge about the specific action of the fungal and plant RNases ho- ADDENDUM IN PROOF mologous to E0 (11, 20) implies a function for E0 other than just nonspecific degradation of host RNAs. 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