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Virology 273, 351–363 (2000)doi:10.1006/viro.2000.0425, available online at http://www.idealibrary.com on

NS3 Serine Protease of Bovine Viral Diarrhea Virus: Characterization of Active Site Residues,NS4A Cofactor Domain, and Protease–Cofactor Interactions

Norbert Tautz,1 Astrid Kaiser, and Heinz-Jurgen Thiel

Institut fur Virologie (FB Veterinarmedizin), Justus-Liebig-Universitat Gieben, D-35392 Gieben, Germany

Received March 7, 2000; returned to the author for revision April 20, 2000; accepted May 16, 2000

The gene expression of bovine viral diarrhea virus (BVDV), a pestivirus, occurs via translation of a hypothetical polyproteinthat is processed cotranslationally and posttranslationally by viral and cellular enzymes. A protease located in the N-terminalregion of nonstructural (NS) protein NS3 catalyzes the cleavages, leading to the release of NS4A, NS4B, NS5A, and NS5B.Our study provides experimental evidence that histidine at position 1658 and aspartic acid at position 1686 constitutetogether with the previously identified serine at position 1752 (S1752) the catalytic triad of the pestiviral NS3 serine protease.Interestingly, a mutant protease encompassing an exchange of the active site S1752 to threonine still showed residualactivity. This finding links the NS3 protease of pestiviruses to the capsid protease of Sindbis virus. Furthermore, we observedthat the minimal protease domain of NS3 encompasses about 209 amino acids. The NS3 protease was found to be sensitiveto N-terminal truncation because a deletion of 6 amino acids significantly reduced the cleavage efficiency at the NS4A/4Bsite. Larger N-terminal deletions also impaired the activity of the enzyme with respect to the other cleavage sites but to adifferent degree at each site. The NS3 protease of BVDV has previously been shown to depend on NS4A as cofactor. Wedemonstrate here that the central region of NS4A represents the cofactor domain. Furthermore, coprecipitation studiesstrongly suggest an interaction between NS4A and the N-terminal region of NS3. Besides the remarkable similarities
observed between the pestiviral NS3 protease and the corresponding enzyme of hepatitis C virus (HCV), our results suggesta common ancestry between these enzymes and the capsid protease of Sindbis virus. © 2000 Academic Press
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INTRODUCTION

The genera Flavivirus, Hepacivirus, and Pestivirus con-stitute the family Flaviviridae (Rice, 1996). Members ofthe genus Pestivirus include important pathogens of live-stock, such as classical swine fever virus (CSFV), bovineviral diarrhea virus (BVDV-1 and BVDV-2), and borderdisease virus (BDV). The single-stranded RNA genomesof these enveloped viruses are of positive polarity andhave in general a length of 12.3 kb (Becher et al., 1998;Collett et al., 1988b; Deng and Brock, 1992; Meyers et al.,1989; Renard et al., 1987). The genome contains one longopen reading frame (ORF) flanked by untranslated re-gions (UTRs). The order of the viral proteins in the hypo-thetical polyprotein is as follows: Npro, C, Erns, E1, E2, p7,

S2-3 (NS2, NS3), NS4A, NS4B, NS5A, NS5B (Collett etl., 1988a, 1991; Elbers et al., 1996; Meyers et al., 1992;umenapf et al., 1993; Stark et al., 1993; Tautz et al., 1997;u et al., 1997). Npro represents a nonstructural (NS)utoprotease, which releases itself from the polyproteinnd thereby generates the N-terminus of the capsidrotein (C) (Stark et al., 1993; Wiskerchen et al., 1991).he envelope glycoproteins Erns, E1, and E2, as well as

1 To whom reprint requests should be addressed at Institut furirologie (FB 10). Justus-Liebig-Universitat Gieben, Frankfurter Strasse
07, D-35392 Gieben, Germany. Fax: 49-(641)-99-3-83-59. E-mail:[email protected].
351

the NS protein p7, are presumably released from thepolyprotein via cellular proteases (Rumenapf et al., 1993;Elbers et al., 1996; N. Tautz, unpublished data).

The degree of processing at the NS2/3 site showsremarkable variation among different pestivirus speciesand strains (reviewed in Meyers and Thiel, 1996). NS2-3and NS3 represent multifunctional proteins with pro-tease, RNA binding, NTPase, and helicase activity(Grassmann et al., 1999; Tamura et al., 1993; Warrener

nd Collett, 1995; Wiskerchen and Collett, 1991). Therotease located in the N-terminal domain of NS3 leads

o the release of NS4A, NS4B, NS5A, and NS5BWiskerchen, 1991; see later). NS4A has recently beenstablished as cofactor of the NS3 protease (Xu et al.,997). The functions of NS4B and NS5A are not known.S5B represents the viral RNA-dependent RNA polymer-se (Kao et al., 1999; Lai et al., 1999; Steffens et al., 1999;

Zhong et al., 1998).Hepaciviruses represent the closest relatives of pes-

tiviruses, particularly with regard to the genome organi-zation of the NS protein region and apparently also thefunction or functions of individual NS proteins (Rice,1996). In addition, for HCV NS3 represents a serineprotease that catalyzes all cleavages required for thegeneration of NS4A, NS4B, NS5A, and NS5B (Barten-schlager et al., 1993; Grakoui et al., 1993; Hijikata et al.,
1993; Tomei et al., 1993; reviewed in Bartenschlager,
0042-6822/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

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352 TAUTZ, KAISER, AND THIEL

1997). Although processing at the 3/4A site is catalyzedexclusively in cis, all other cleavages can be mediated intrans. The observed consensus sequence for the cleav-age sites is: E/D-X-X-X-X-C/T 2 S/A (where X is anyamino acid and 2 symbolizes the cleavage site). Thecatalytic triad of the protease is composed of histidine,aspartic acid, and serine. Furthermore, a structurallyimportant Zn-binding motif was identified in the proteasedomain (de Francesco et al., 1996; Kim et al., 1996; Loveet al., 1996; Stempniak et al., 1997). The minimal fragmentof NS3 still capable of cleaving at all sites was shown toconsist of a 169-amino-acid peptide located in the N-terminal region of the protein (Bartenschlager et al., 1994;Failla et al., 1995; Tanji et al., 1994). The NS3 protease is

ssisted by NS4A as a cofactor that stimulates the cleav-ges at the four sites to significantly different extents

Bartenschlager et al., 1994; Failla et al., 1994; Lin et al.,994; Steinkuhler et al., 1996; Tanji et al., 1995a). In NS4A,

central 12-amino-acid peptide was identified that pre-erves cofactor activity (Bartenschlager et al., 1995b; Lint al., 1995; Steinkuhler et al., 1996; Tanji et al., 1995b).oprecipitation analyses indicated a stable interactionetween NS4A and the extreme N-terminus of NS3 (Bar-

enschlager et al., 1995a; Failla et al., 1995; Lin et al.,1994; Satoh et al., 1995). This interaction was confirmedby the resolution of the three-dimensional structure ofthe crystallized HCV NS3 protease domain complexedwith a synthetic NS4A peptide (Kim et al., 1996).

In contrast to the NS3 protease of HCV, knowledgeabout the corresponding enzyme of pestiviruses is verylimited. Two computer models, established for the pes-tiviral enzyme (Bazan and Fletterick, 1989; Gorbalenya etal., 1989), have been experimentally challenged so faronly by the exchange of the putative active site serine;mutation to alanine abolished the release of NS4A,NS4B, NS5A, and NS5B from the BVDV polyprotein(Wiskerchen and Collett, 1991). Identification of the othermembers of the catalytic triad has not been approachedexperimentally. Mapping of the protease domain wasattempted (Wiskerchen and Collett, 1991) but did not leadto valid data because the activity of the NS2-3 fragmentswas determined at a site that is not processed by NS3(Petric et al., 1992; Xu et al., 1997). At the NS3/4A site, arestriction to cis cleavage was postulated because pro-cessing was not observed in trans; all other cleavagescan be mediated by NS3 in trans (Wiskerchen and Col-lett, 1991). At the NS3-dependent cleavage sites of BVDV,a leucine residue is strictly conserved in the P1 position(nomenclature according to Berger and Schlechter,1970), and serine or alanine predominantly resides at theP19 position (Tautz et al., 1997; Xu et al., 1997). Thus the

equences at the cleavage sites used by the NS3 pro-eases of BVDV and HCV differ significantly. Moreover,he overall degree of similarity between these enzymes
s very low on amino acid level. In contrast to HCV, non-binding motif can be delineated from the primary
equence of BVDV NS3. Both proteases are stimulated inheir activity by NS4A. NS3 of BVDV strictly depends on

S4A in its activity at the NS4B/5A and NS5A/5B sitesXu et al., 1997). The role of NS4A in the other NS3-ependent cleavages, as well as the cofactor domain ofS4A, has not been studied so far. Our aim in theresent study was a comprehensive characterization of

he pestiviral NS3 protease, its NS4A cofactor, and pro-ease–cofactor interactions.

RESULTS

ctive center of the NS3 serine protease

The models proposed for the NS3 protease predict anctive role for serine at amino acid position 1752 (S1752)nd for histidine at position 1658 (H1658) (Bazan andletterick, 1989; Gorbalenya et al., 1989). Interestingly,

the models differed with respect to the third member ofthe catalytic triad by predicting aspartic acid at eitherposition 1686 (D1686) (Gorbalenya et al., 1989) or 1695(D1695) (Bazan and Fletterick, 1989). From the proposedresidues only S1752 was experimentally verified by anexchange to alanine (Mendez et al., 1998; Tautz et al.,1996; Wiskerchen and Collett, 1991).

We started our analysis of the NS3 protease of BVDVstrain CP7 with the characterization of its active center ina transient expression study by applying the T7 vacciniavirus system (Fuerst et al., 1986; Sutter et al., 1995). Thefirst set of mutants aimed at the identification of residuesthat constitute the catalytic triad as well as at theirfunctional characterization. Mutations of S1752 to cys-teine (S1752C) or threonine (S1752T) were introduced todetermine whether either of these amino acids couldfunctionally replace S1752, which serves as nucleophile.The exchange of H1658 and aspartic acids at positions1686/1695 by small nonpolar amino acids (glycine oralanine, respectively) served to identify the other mem-bers of the catalytic triad. The mutations were initiallytested in the context of a polyprotein fragment, extendingfrom the C-terminal region of E2 to the N-terminal part ofNS4B encoded by plasmid pE2*-4B* (Fig. 1 and Tautz etal., 1996). The release of authentic NS2, NS3, NS2-3, andNS4A from the polyprotein encoded by E2*-4B* (* indi-cates truncated proteins) has been demonstrated beforein the T7 vaccinia system (Tautz et al., 1996). Moreover,the use of this short polyprotein fragment facilitated thedetection of noncleaved protein species. In the S1752Amutant, included as a control, the partial cleavage ofNS2-3, which is independent of the NS3 protease, stilloccurred (Tautz et al., 1996), whereas the NS3 protease-dependent cleavages at the NS3/4A, NS2-3/4A andNS4A-4B* sites were abrogated. As a consequence,NS3-4B* and NS2-4B* with apparent molecular massesof 92 and 150 kDa, respectively, appeared. These pro-
teins could be precipitated by antisera specific for NS3or NS4 (Tautz et al., 1996, and Fig. 2, lanes 3 and 4). In the

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353BVDV NS3 SERINE PROTEASE

analyses shown in Fig. 2, the amount of NS3-4B* wasused as a parameter to monitor the impairment of theserine protease.

The S1752C exchange in construct pE2*-4B* led to anefficient release of NS3 and to the generation of a smallamount of NS3-4B* (Fig. 2, lane 5). By comparison, ex-change S1752T caused an accumulation of NS3-4B*,which illustrates the severely reduced activity of this

FIG. 1. Schematic diagram of the BVDV CP7 polyprotein (upper parruncated proteins are marked by *. The shadowed bar INSERTION symas been shown to induce NS2-3 cleavage independent of the NS3 s

FIG. 2. Single amino acid substitutions in the serine protease domainof NS3 and cleavage at the NS3/4A site. Expression of the E2*-4B*region of the BVDV polyprotein from transfected plasmids wasachieved by the use of a recombinant vaccinia virus expressing the T7RNA polymerase (MVA-T7pol; Sutter et al., 1995). The metabolicallylabeled proteins were immunoprecipitated with antisera against NS3 orNS4, respectively, and further analyzed by SDS–PAGE (Doucet andTrifaro, 1988) and fluorography. The transfected expression plasmidsas well as the antisera used are indicated above the correspondinglanes. Mutations in the encoded CP7 polypeptides are indicated in thenames of the corresponding plasmids. Precipitates derived from cellsinfected with MVA-T7pol without plasmid transfection are separated inlane 13 (Mock). The positions of NS2-3, NS3, and NS3-4B* are indicated
by arrowheads on the left. The positions of the mass standards([14C]MW markers) are shown on the right; k 5 kDa.
mutant. Unexpectedly, a substantial amount of NS3 wasstill generated (Fig. 2, lane 7). The H1658A exchange incontrast completely abrogated the generation of matureNS proteins (Fig. 2, lanes 9 and 10); this result was in linewith the postulated catalytic role of H1658 in the NS3protease.

Surprisingly, neither mutation D1686G (Fig. 2, lanes 11and 12) nor exchange D1695A (data not shown) inducedobvious changes in processing. Only for mutant D1686Gdid a minor band corresponding to NS3-4B* appear. Thisresult, which was obtained mainly by monitoring thecleavage activity at the NS3/4A site, thus did not allowestablishment of the third member of the catalytic triad.To identify the respective residue, we introduced ex-changes D1686G and D1695A into a polyprotein thatextended from E2* to NS5B encoded by plasmid pE2*-5B(Fig. 1). This setup allows to monitor the effect of muta-tions on the trans cleavages at sites NS4A/4B, NS4B/5A,and NS5A/5B. The transfection of plasmids pE2*-5B andpE2*-5B/D1695A led to the generation of all viral NSproteins (Figs. 3A and 3B, lanes 2 and 6, and data notshown), whereas the D1686G exchange completely in-hibited the trans cleavages of the NS3 protease (Figs. 3Aand 3B, lane 5); as expected, the cleavage at the NS3/4Asite was almost unaffected (data not shown).

Furthermore, we examined the effect of mutationsS1752C and S1752T on the trans cleavage activity of theprotease. After the transient expression of pE2*-5B/S1752C or pE2*-5B/S1752T, minor amounts of NS2-3 andNS3 (data not shown), but neither NS4A, NS4B, NS5A,nor NS5B, could be detected (Figs. 3A and 3B, lanes 3and 4, respectively). According to this assay system, therespective NS3 protease mutants exhibited only cleav-age activity at the NS3/4A site.

Taken together, our results are in agreement with themodel of Gorbalenya et al. (1989) that predicted for the

he parts encoded by T7 expression plasmids pE2*-4B* and pE2*-5B;the 9-amino-acid insertion present in NS2 of BVDV CP7. This sequence

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Characterization of the NS3 protease domain

For several BVDV strains, it has been experimentallydemonstrated (Kummerer et al., 1998; Meyers et al.,1998) or suggested (Mendez et al., 1998; Meyers andThiel, 1996; Tautz et al., 1993) that the first amino acid ofNS3 is glycine 1590. Accordingly, authentic NS3 encom-passes amino acids 1590–2272 of the BVDV polyprotein(Tautz et al., 1997; Xu et al., 1997). Our next aim was tourther define the protease domain of NS3. We startedhe corresponding analysis by the construction of ex-

FIG. 3. NS3 protease mutants and processing of E2*-5B. (A) Detec-tion of NS4A and NS4B. Immunoprecipitates obtained by the use ofNS4 specific antiserum were separated by Tricine–SDS–PAGE (Schag-ger and Jagow, 1987). Samples derived from cells infected with MVA-T7pol without plasmid transfection were separated in lane 7 (Mock).Arrowheads indicate the position of NS4A and NS4B. The 38-kDaprotein (lower band of the doublet) in lanes 2 and 6 most likelyrepresents noncleaved NS4A-4B (see also Fig. 5C). The amount of NS2detected on transfection of the different plasmids was used to stan-dardize transfection efficiencies (data not shown). The S1752A ex-change was included as a control to show inhibition of NS3-dependentcleavages. (B) Detection of NS5A-5B, NS5A, and NS5B. The transfectedexpression plasmids are depicted in the figure above the correspond-ing lanes. For immunoprecipitation, NS5-specific antisera were used.Arrowheads at the left indicate positions of NS5A-5B, NS5A, and NS5B.Precipitates derived from cells infected with MVA-T7pol without plas-mid transfection were separated in lane 7 (Mock).

ression plasmids encoding C-terminally truncated NS3olecules, which encompass 251, 211, or 171 amino

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cids from the NS3 region; the C-terminal amino acid ofhese NS3 derivatives encoded by pGST-NS3-1840,GST-NS3-1800, or pGST-NS3-1760 (Fig. 4A) is located in

he polyprotein at the positions indicated by the plasmidames. The NS3 fragments were expressed as glutathi-ne-S-transferase (GST) fusion proteins to ease theiretection (Figs. 4A and 4B). The substrate for the pro-

ease was expressed from pNS4A-5B-myc (Fig. 4A) en-oding an NS4A-5B polyprotein fragment with a C-termi-al myc-tag; the latter facilitates protein detection. On theoexpression of NS4A-5B-myc with GST-NS3-1800 orST-NS3-1840, a polypeptide with an apparent molecu-

ar mass expected for NS5B-myc (about 75 kDa) could beetected with the myc-tag-specific antibody (Fig. 4C,

anes 3 and 4). In contrast, cotransfection of pGST-NS3-760 and pNS4A-5B-myc did not result in the cleavage ofS4A-5B-myc (Fig. 4C, lane 2). The expression of theST-NS3 fusion proteins in the respective cell lysatesas also verified by Western blotting (data not shown).he use of identically truncated NS3 molecules devoid of

he GST fusion led to comparable results, which demon-trates that the fusion partner had no influence on thebtained results (data not shown). Accordingly, the C-

erminal border of the NS3 serine protease domain re-ides between amino acid positions 1760 and 1800 at

east with respect to cleavage at the NS5A/5B site.To determine the N-terminal border of the protease

omain, we chose a strategy that allowed the expressionf N-terminally truncated NS3 molecules without anyector-derived amino acid at their N-termini. For thisurpose, one ubiquitin monomer was inserted upstreamf the BVDV polyprotein. On expression, cellular ubiq-itin-specific proteases (Jonnalagadda et al., 1989;ayer and Wilkinson, 1989; Rose, 1988) will cleave after

he C-terminal amino acid of ubiquitin (glycine 76) andhereby generate the N-terminus of NS3. It has previ-usly been demonstrated that this cleavage also occursfficiently in the context of the BVDV polyprotein (Tautz etl., 1993). The BVDV polyprotein encoded by plasmidUb1590-5B starts with the first amino acid of NS3 (G1590)nd proceeds to the end of NS5B (Fig. 5A). In a series oferivatives of pUb1590-5B, the N-terminus of NS3 was

runcated by 2, 6, 20, 35, and 45 amino acids (Fig. 5A); theumbers in the names of the constructs reflect the rela-

ive position of the first amino acid of the encoded BVDVolypeptide with respect to the viral polyprotein. After the

ransfection of pUb1590-5B and pUb1592-5B, viral pro-eins NS3, NS4A, NS4B, NS5A, and NS5B could be de-ected (Figs. 5B and 5C). The deletion of the 6 N-terminalmino acids resulted in the appearance of an additionalrotein of about 38 kDa, which could be precipitated byntibodies against NS4 (Fig. 5C, lane 3). Due to itspparent molecular mass and the strong decline in themount of NS4A and NS4B, this protein most likely rep-

esents noncleaved NS4A-4B. On the deletion of 20 N-erminal amino acids, only two mature viral proteins were

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a c-myc–specific mAb (Invitrogen). The positions of unprocessedNS4A-5B-myc, NS5B-myc, and the cellular myc protein (c-myc) are


released from the polyprotein, namely NS3 (Fig. 5B, lane7) and a minor amount of NS5B (Fig. 5B, lane 8). Theadditional proteins with apparent molecular masses ofabout 95 and 160 kDa, respectively, could be precipitatedwith an NS5-specific antiserum (Fig. 5B, lane 8) and bythe serum directed against NS4 (data not shown) but notwith antibodies specific for NS3 (Fig. 5B, lane 7). Inaccordance with their apparent molecular masses andreactivity with defined antibodies, these proteins mostlikely represent NS4A-5A and NS4A-5B. The transfectionof pUb1625-5B (deletion of 35 N-terminal amino acids)resulted in the generation of the truncated NS3 as theonly mature viral protein; this NS3 derivative migratesdue to its truncation significantly faster than authenticNS3 (Fig. 5B, lane 9). In addition; NS4A-5A and NS4A-5Bwere precipitated by the antisera specific for NS5 (Fig.5B, lane 10). The appearance of these proteins indicatesthat cleavage still takes place at the NS3/4A andNS5A/5B sites, albeit at a very low level. The failure todetect NS5B might be due to the low cleavage efficiencyand the described inherent instability of NS5B (Collett etal., 1991). The deletion of the first 45 amino acids of NS3abolished the activity of the protease because neitherone of the mature BVDV proteins nor NS4A-5A andNS4A-5B could be identified (Figs. 5B, lanes 11 and 12,and 5C, lane 6).

Cofactor domain of NS4A

NS4A, which encompasses amino acids 2273–2336 ofthe BVDV polyprotein (Tautz et al., 1997; Xu et al., 1997),

cts as an essential cofactor of the NS3 protease, ateast for the cleavages at sites NS4B/5A and the

S5A/5B sites (Xu et al., 1997). To determine whether theomplete NS4A protein is necessary to activate the pro-

ease, we established an appropriate cleavage assay.NS5A-5B-myc (Fig. 6A) was used for the expression of

suitable substrate. On the transfection of plasmidNS2*-3* (Fig. 6A), an NS3-specific antibody detectedroteins NS2*-3* and NS3* (data not shown). The NS4A-erived peptides were expressed as fusions to GST tollow their detection by Western blotting (Fig. 6C). Asxpected, the cotransfection of pNS2*-3* and pNS5A-5B-yc in the absence of the cofactor NS4A did not lead to

etectable cleavage at the NS5A/5B site (Fig. 6B, lane 1).fter the cotransfection of plasmids pNS2*-3* andNS5A-5B-myc with pGST-NS4A fusion proteins encom-assing either complete NS4A together with a smallart of the NS4B coding region (pGST-NS4A-2349,

indicated at the left. In lane 5 (Mock), lysates of cells infected only withMVA-T7pol virus were separated. The lanes combined in the figure

FIG. 4. C-terminal border of the NS3 protease domain. (A) Scheme ofthe used cDNA constructs. The C-terminally truncated NS3 derivativeswere expressed from plasmids pGST-NS3-1840, pGST-NS3-1800, orpGST-NS3-1760 as fusions to GST. Plasmid pNS4A-5B-myc encodesthe NS protein region from NS4A-5B with an myc-tag fused to theC-terminus. The numbers in brackets indicate the amino acids of theBVDV polyprotein encoded by the respective construct. (B) Western blotanalysis of the GST-NS3* fusion proteins. Plasmids pGST-NS3-1840,pGST-NS3-1800, or pGST-NS3-1760 were transfected into cells previ-ously infected by MVA-T7pol virus. The respective cell lysates wereseparated by Tricine–SDS–PAGE (Schagger and Jagow, 1987) and an-alyzed by Western blotting with a mouse mAb directed against GST(Santa Cruz Biotechnology). The lanes combined in the figure werederived from the same gel. Lysates of cells only infected with MVA-T7pol virus were separated in lane 4 (Mock). The positions of the massstandards (prestained MW markers) are shown on the right; k 5 kDa.(C) Proteolytic activity of the GST-NS3* fusion proteins. On coexpres-sion of GST-NS3-1840, GST-NS3-1800, or GST-NS3-1760 and the NS4A-5B-myc substrate, the respective lysates were analyzed by Tricine–SDS–PAGE (Schagger and Jagow, 1987) and Western blotting applying

were derived from the same gel. On the right, the positions of the massstandards (prestained MW markers) are shown; k 5 kDa.

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pGSTNS4A-2339) (Fig. 6B, lanes 2 and 3) or an NS4Afragment with a 7-amino-acid deletion at its C-termi-nus (pGST-NS4A-2329) (Fig. 6B, lane 4), NS5B-myc

FIG. 5. N-terminal border of the NS3 protease domain. (A) Schemrganization of the polyprotein encoded by the expression plasmids is df the figure, the amino acid sequence at the cleavage site between u

irst amino acid of the encoded NS3 derivative is indicated by the plasellular ubiquitin specific proteases are known to cleave downstream

ollows the C-terminal amino acid of ubiquitin. We did therefore not g-terminus. The right part of the figure summarizes the obtained resultsxpression of NS3–5B and N-terminally truncated derivatives. After tranaccinia virus system, the cell lysates were subjected to a radioimmuno

ndicated. In lanes 12 and 13 (Mock), precipitates generated from lysaS5B, NS4A-5A, and NS4A-5B are indicated by arrowheads. The positio

(C) Processing at NS3/4A and NS4A/4B sites. After transient expressantibodies. Precipitates were separated by Tricine–SDS–PAGE (Schainfected with MVA-T7pol virus. The positions of NS4A, NS4B, and NS4markers) are shown on the right; k 5 kDa. The results concerning proplasmids described above; the corresponding constructs encoded NS

was released. Larger C-terminal truncations (17 aminoacids or more) of NS4A abrogated its cofactor activity.

NS4A truncated N-terminally by 9 amino acids stillassisted in NS5A-5B-myc cleavage (Fig. 6B, lane 8). Asindicated by the very weak appearance of NS5B-myc

wing of the cDNA constructs. In the upper part of the scheme, thed. In all clones, ubiquitin resides upstream of NS3. In the lower left partn and NS3 is given in the one-letter code. The relative position of theme. The C-terminal tripeptide of ubiquitin, namely (RGG), is indicated.sequence. This cleavage is strongly impaired only if a proline residue

an NS3 derivative in which just 1 amino acid was deleted from theicient cleavage; 2, no cleavage; (1), inefficient cleavage. (B) Transientxpression in the presence of [35S]methionine/cysteine by use of the T7itation analysis. The transfected plasmids and the utilized antisera areVA-T7pol-infected cells were separated. The positions of NS3, NS5A,e mass standards ([14C]MW markers) are shown on the right; k 5 kDa.

ll lysates were further processed by precipitation using NS4-specificd Jagow, 1987). Lane 7 (Mock) represents an analysis of cells only

e indicated on the left. The positions of the mass standards ([14C]MWg at the NS3/4A site were confirmed by the use of derivatives of the

data not shown).

atic draepictebiquitimid naof thisenerate: 1, effsient eprecip

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NS4A with a deletion of 18 amino acids from theN-terminus still retained residual cofactor activity (Fig.

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6B, lane 9). The minimal NS4A fragment that preservesthe cofactor function in cleavage at the NS5A/5B sitethus is probably composed of not more than aminoacids 2291–2329 even though a direct test with acorresponding peptide was not performed. Compara-ble results were also obtained with NS4A fragments

FIG. 6. Cofactor domain of NS4A. (A) Scheme of expressed proteinspNS5A-5B-myc served for generation of a suitable substrate for cofactGST fused to NS4A served for detection of individual proteins. Numberespective plasmids. (B) NS4A-dependent cleavage assay. After coexprlysates were subjected to Western blot analysis. Proteins were separamyc-tag-specific mAb. In lane 11 (Mock), lysates of cells infected only wthe cellular myc protein (c-myc) are indicated. On the right, the positio

etection of the GST-NS4A fusion proteins. The cell lysates describeestern blot analysis applying an mAb directed against GST. In lane

ositions of the mass standards (prestained MW markers) are shown

without GST-tag (data not shown).

Interaction between NS4A and the N-terminal domainof NS3

In the HCV system, a stable interaction between theNS4A cofactor domain and the N-terminal region of NS3has been established (Bartenschlager et al., 1995a; Failla

*-3* was used to produce NS2*-3* and NS3* in the absence of NS4A.ndent proteolysis. The myc-tag at the C-terminus of NS5B as well as

arentheses indicate the part of the BVDV polyprotein encoded by theof NS2*-3*, NS5A-5B-myc, and the different NS4A derivatives, the cellTricine–SDS–PAGE and further analyzed by Western blotting using an

-T7pol were analyzed. The positions of NS5A-5B-myc, NS5B-myc, ande mass standards (prestained MW markers) are shown; k 5 kDa. (C)e (see legend for Fig. 6B) were analyzed by Tricine–SDS–PAGE andck), lysates of cells infected only with MVA-T7pol were analyzed. Theright; k 5 kDa.

. pNS2or depers in pessionted byith MVA

ns of thd abov11 (Mo

et al., 1995; Hijikata et al., 1993a,b; Kim et al., 1996; Lin etal., 1995; Satoh et al., 1995). It was intended to test

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whether a similar NS3/NS4A interaction occurs in theBVDV system. Accordingly, plasmids pUb1590-4A and itsderivatives encoding N-terminally truncated NS3-4A mol-ecules were generated by the deletion of the NS4B-5Bcoding region from the series of plasmids based onpUb1590-5B. Proteins expressed in the presence of[35S]methionine/cysteine by pUb1590-4A and its deriva-ives were immunoprecipitated under nondenaturingonditions with a monoclonal antibody (mAb) directedgainst NS3. SDS–PAGE analysis and fluorography of

he precipitated proteins revealed that a 7-kDa proteinoprecipitated with full-length NS3 (Fig. 7, lane 1). Aestern blot analysis carried out in parallel identified the

-kDa protein as NS4A (data not shown). Although NS4Aould also be coprecipitated with the NS3 derivative,hich was N-terminally truncated by 2 amino acids (Fig.

, lane 2), all NS3 species with larger N-terminal trunca-ions did not allow pulling down NS4A under the chosenonditions (Fig. 7). These data are in line with an essen-

ial role of the N-terminal region of NS3 in the complexormation between NS3 and NS4A.

DISCUSSION

The aim of the present study was the characterizationf the pestiviral NS3 protease. As a first step, the resi-ues constituting the catalytic triad were determined.he obtained results are in agreement with the model oforbalenya et al. (1989), in which the catalytic triad is

omposed of H1658, D1686, and S1752. There was nondication for an essential role of D1695 in the proteaseomain as proposed in an alternative model (Bazan andletterick, 1990).

The NS3 serine protease catalyzes two types of

FIG. 7. Coprecipitation of NS3 and NS4A. Proteins encoded bypUbNS3-4A and its derivatives were transiently expressed in the pres-ence of [35S]methionine/cysteine by using MVA-T7pol virus. Cell lysateswere prepared under nondenaturing conditions and subjected to im-munoprecipitation by an NS3-specific mAb. The obtained aggregateswere denatured, separated by Tricine–SDS–PAGE, and processed forfluorography. The positions of NS3 and NS4A are indicated.

leavages. Although processing at the NS3/NS4A sites believed to occur exclusively in cis, all other cleav-

ti

ges can be mediated in trans (Wiskerchen and Col-ett, 1991). Studies on the kinetics of proteolysis of the

S region of the BVDV polyprotein in virus-infectedells indicated that the cleavage at the NS3/4A site

akes place with significantly higher efficiency andaster kinetics than the other NS3-mediated cleavagesCollett et al., 1991). Our study revealed that the NS3

utants carrying exchanges S1752C, S1752T, or1686G, respectively, still catalyzed the NS3-4A cleav-ge, whereas they showed no activity at the otherleavage sites in our assay system. With respect to theigher activity of the protease at the NS3/4A site, wessume that an overall reduction in the enzymaticctivity due to the introduced mutations can result in

he observed selective loss of trans cleavage activity.The finding that serine and cysteine are interchange-

ble as nucleophiles, at least to some extent, has al-eady been described for other viral serine proteasesHahn and Strauss, 1990; see later) as well as viralerine-like cysteine proteases such as the 3C proteasef picornaviruses and the 27-kDa NIa protease of theotyvirus tobacco etch virus (Dougherty et al., 1989; Law-on and Semler, 1991). It was surprising, however, thatS3 with the S1752T substitution exhibited a significant

esidual protease activity at the NS3/4A site, which indi-ates that the hydroxyl group of threonine serves in theucleophilic attack during proteolysis. There is only onether example of a serine protease where the exchangef the active site serine by threonine gave rise to annzyme with substantial protease activity (10% of wild

ype), namely, the capsid protease of Sindbis virus (Hahnnd Strauss, 1990). This enzyme is an autoprotease thatatalyzes its release from the viral polyprotein, therebyenerating its own C-terminus. It was supposed that thectivity of the capsid protease with a threonine in thective site is due to the intramolecular nature of the

eaction and the very fast reaction kinetics of the wild-ype protease (Hahn and Strauss, 1990). Furthermore, forhe Sindbis virus capsid protease as well as the BVDVnzyme, the exchange of the active site aspartic acid didot significantly reduce their cis cleavage activities. Inddition to the similarities observed in this study with

egard to the biochemistry of these enzymes, it turnedut recently that the three-dimensional structure of theS3 protease of HCV, a close relative of pestiviruses,

hares its greatest structural similarity with the Sindbisirus capsid protease (Kim et al., 1996). These findingsicely corroborate the relationship between the de-cribed proteases, which remarkably was postulatedeveral years ago on the basis of computer-assistedequence comparisons of flaviviruses, pestiviruses, andlphaviruses (Gorbalenya et al., 1989).

According to our study, a 209-amino-acid fragment ofhe BVDV NS3 serine protease is still capable of exerting
he NS5A-NS5B cleavage. A protease domain of this sizes in agreement with the data obtained for the NS3

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protease domains of HCV (169 amino acids) (Barten-schlager et al., 1994; Failla et al., 1995; Tanji et al., 1994)and members of the genus Flavivirus (e.g., yellow fevervirus, 181 amino acids; Chambers et al., 1990). Becausefusions of GST to the N-terminus of NS3 had no delete-rious effect on the activity of the enzyme, the folding ofthe NS3 protease domain can apparently occur indepen-dent of the preceding peptide sequence.

The sensitivity of the protease domain to N-terminaltruncations and the stepwise loss of activity are remark-able: a deletion of 6 N-terminal amino acids resulted in astrongly reduced activity at the NS4A/4B site (Figs. 5Band 5C). Larger N-terminal truncations of NS3 furtherreduced the activity of the enzyme at the individual cleav-age sites to different extents. The greatest tolerance toN-terminal truncation of the protease was observed forthe cis cleavage and processing at the NS5A/5B site.Interestingly, similar results have been obtained in theHCV system for N-terminally truncated NS3 proteasederivatives. Short deletions selectively interfered withNS4A-NS4B and NS4B-NS5A cleavage, whereas moreextensive truncations of NS3 also inhibited cleavage atthe NS5A/5B site and ultimately the cis cleavage at theNS3/4A site (reviewed in Bartenschlager, 1997).

Our coprecipitation study revealed an interaction be-tween BVDV proteins NS3 and NS4A, which was abro-gated by a deletion of the first 6 amino acids of NS3 (Fig.7). This finding strongly suggests a role of the N-terminaldomain of NS3 in NS4A binding. Interestingly, such atruncated protease was proteolytically active at all sites.For HCV, binding of NS4A to the N-terminal region of NS3has also been demonstrated by coprecipitation and wasconfirmed at the structural level (Kim et al., 1996). Thisinteraction could be abrogated by N-terminal truncationsof NS3 (Bartenschlager et al., 1995a; Failla et al., 1995;Lin et al., 1994; Satoh et al., 1995). N-terminally truncatedas well as mutagenized NS3 derivatives could be iden-tified that show proteolytic activity despite impairedNS4A binding. These data and the results obtained inthis study suggest that a weak transient protease–cofac-tor interaction seems to be sufficient for activation of theNS3 proteases of HCV (for review, see de Francesco andSteinkuhler, 2000) and BVDV.

Taken together, our study revealed striking parallelsbetween the NS3 proteases of BVDV and HCV, especiallywith respect to active site residues, size of proteasedomain, NS4A cofactor dependency, and NS3–NS4A in-teraction. These findings again underline the fascinat-ingly close relationship of pestiviruses and hepacivi-ruses. In addition, the similarities observed between thecapsid protease of Sindbis virus, a member of the genusAlphavirus, and the NS3 protease of a member of theFlaviviridae suggest a common ancestor for the serine
proteases of these positive-strand RNA viruses.
cp

MATERIALS AND METHODS

Cells and viruses

BHK-21 cells were kindly provided by J. Cox (FederalResearch Center for Virus Diseases of Animals, Tubingen,Germany). Cells were grown in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% fetal calf serum(FCS). The recombinant vaccinia virus expressing the T7RNA polymerase [modified virus Ankara (MVA)-T7pol] wasgenerously provided by G. Sutter (Institut fur MolekulareVirologie, GSF-Forschungszentrum fur Umwelt und Ge-sundheit GmbH, Oberschleibheim, Germany).

ransient expression with the T7 vaccinia virusystem

BHK-21 cells (5 3 105, 3.5-cm dish) were infected withVA-T7pol at a multiplicity of infection of 5 in medium

acking FCS. After 1 h of incubation at 37°C, cells wereashed twice with medium lacking FCS followed byNA transfection with 2 mg plasmid DNA by the use ofuperfect (Qiagen, Hilden, Germany) according to therotocol of the manufacturer. For Western blot analysis,

he transfected cell cultures were harvested about 6 hosttransfection (p.t.).

Radioactive labeling of cells. At 2 h p.t., cells wereashed two times with PBS and incubated with 1 ml of

abel medium lacking cysteine and methionine for 0.5 ht 37°C. After the addition of 500 mCi of [35S]methionine/

cysteine ([35S]-Pro Mix; Amersham, Freiburg, Germany),rotein expression was allowed to proceed for an addi-

ional 4 h at 37°C. After washing with PBS, cells weretored at 270°C until they were further processed.

adioimmunoprecipitation and SDS–PAGE

Immunoprecipitation under denaturing conditions. Cellxtracts prepared in the presence of 2% SDS were di-

uted and incubated with 5 ml of undiluted antiserum. Forhe formation of precipitates, protein A–agarose (Calbio-hem, La Jolla, CA) was used. SDS–PAGE analysis ofroteins was performed by the use of Tricine gels

Schagger and Jagow, 1987) or according to Doucet andrifaro (1988). Gels were processed for fluorography us-

ng En3Hance (New England Nuclear, Boston, MA).Immunoprecipitation under nondenaturing conditions.

ells were lysed with buffer IBP150 [20 mM Tris–HCl, pH.0, 150 mM KCl, 1% Nonidet P-40, 1 mM EDTA, pH 8.0, 1M EDTA, 0.1 mM Pefablock SC (Roche Diagnostics,annheim, Germany)] kept on ice for 5 min and slowly

otated for 30 min at 4°C. Insoluble parts were removedy two consecutive centrifugations at 300 and 10,000 g.S3-specific mAb 8.12.7 (Corapi et al., 1990) was bound

ia Fc-specific anti-mouse IgG (Sigma Chemical Co., St.ouis, MO) to protein A–agarose (Calbiochem) and
ross-linked by diaminopimelic acid according to therotocol of Harlow and Lane (1988). The cross-linked

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pB


antibody was incubated with the cleared cell lysates for1 h at 4°C. Complexes were collected by centrifugationat 4000 g and washed once with IBP150 and twice withBP500 (20 mM Tris–HCl, pH 8.0, 500 mM KCl, 1% Non-det P-40, 1 mM EDTA, pH 8.0, 1 mM EDTA, 0.1 mMefablock SC). After a final washing step with IBP150, therecipitate was diluted in gel loading buffer containing% SDS and 5% mercaptoethanol and boiled for 5 min.

olymerase chain reaction (PCR)

For PCR, a GENIUS thermocycler (Techne/thermo-UX, Wertheim, Germany) was used. Standard condi-

ions used for DNA-based amplifications have been de-cribed previously (Tautz et al., 1999).

ucleotide sequencing

Sequencing was carried out with the Thermo Seque-ase Cycle Sequencing Kit (Amersham Buchler, Braun-chweig, Germany). The primers used for sequencingere labeled with infrared dye IRD41 (MWG-Biotech,bersberg, Germany). Analyses of sequencing gels werearried out with an automatic sequencer LI-COR 4000L

MWG-Biotech, Ebersberg, Germany).

estern blotting

After SDS–PAGE proteins were transferred onto nitro-ellulose membranes (Optitran BA-S83 Reinforced NC;chleicher & Schuell, Duren, Germany). Blocking wasarried out with 2% dry milk in PBS for 1 h. The proteinsere visualized by chemiluminescence (SuperSignal;ierce Chemical, Rockford, IL).

onstruction of the expression plasmids

All BVDV cDNAs used for the construction of the ex-ression plasmids throughout this study are derived fromVDV strain CP7 (Meyers et al., 1996; Tautz et al., 1996).

For PCR amplifications used in the construction of theexpression plasmids, the already described BVDV CP7cDNA clones were used as templates (Meyers et al.,1996; Tautz et al., 1996, 1997). Numbering of nucleotidesor amino acids occurs throughout this report with re-spect to the BVDV SD-1 sequence because the completesequence of BVDV CP7 has not been published yet andSD-1 represents the only complete BVDV sequence de-void of a strain-specific insertion (Deng and Brock, 1992).

pE2*-4B*. This plasmid has already been describedunder the name pC7.1 (Tautz et al., 1996); in the samepublication, the generation of construct pC7PM1 wasdescribed. The latter construct is termed pE2*-4B*/S1752A in this report to obtain a uniform nomenclature.The other mutations in the active center of the NS3protease were generated by standard PCR techniques
using oligonucleotide primers that encompass the indi-vidual nucleotide exchanges.
pE2*-5B. Elongation of the BVDV CP7 cDNA in pC7.1 atits 39 end by a fragment derived from construct pC7.3898(Tautz et al., 1997) resulted in plasmid pE2*-5B. Thus theBVDV ORF encoded by this plasmid starts with codon967 and ends with the authentic stop codon encom-passed in the BVDV genome.

pNS4A-5B-myc. This plasmid is based on pCITE-2A(Novagene, La Jolla, CA), which downstream of a T7 RNApolymerase promoter encompasses an internal ribo-somal entry site (IRES) of the encephalomyocarditis virus(EMCV). The 59 end of the cDNA insert in this plasmidwas generated by PCR and starts downstream of thevector-derived AUG codon with an alanine codon fol-lowed by the BVDV ORF beginning with the codon forserine 2274, the second amino acid of NS4A; the cDNAproceeds downstream to the NS5B gene. Into a PvuII site(nucleotides 12260–12265) in the 39 region of the 5Bgene, an EcoRV–PvuII fragment derived from pSecTag(Invitrogen, Groningen, Netherlands) was cloned; the re-spective fragment encompassed part of the polylinkerand the coding sequence for an myc- and a His-tagfollowed by a stop codon. In the polyprotein encoded bypNS4A-5B-myc, accordingly the 6 C-terminal amino ac-ids of NS5B are substituted by 34 pSecTag-derivedamino acids, including the myc-epitope.

pGST-NS3-1760/1800/1840. The GST gene frompGEX-2T (Pharmacia) was amplified by PCR and inte-grated into the NcoI site of pCITE-2A by standard cloningtechniques. Downstream of the GST gene, the resultingplasmid, pCITE-GST, contains a singular NcoI site en-compassing an in-frame AUG codon. After restrictionwith NcoI–XbaI, this vector was ligated to an NcoI–XbaIfragment of pARC7 (Tautz et al., 1997). The resultingplasmid, pGST-NS3-2351, encoded for the CP7 polypro-tein from amino acids 1587–2351. To generate the finalconstructs, PCR-derived fragments of the NS3 gene ofCP7 that encode stop codons downstream of serine1760, lysine 1800, or leucine 1840, respectively, wereused to substitute an HpaI–XbaI (nucleotides 5434–5439/polylinker) fragment in pGST-NS3-2351.

pUb1590-5B. From plasmid FsubiNS3 (Tautz et al.,1999), which encompasses the mouse ubiquitin gene(ubi), an NcoI (first codon of ubiquitin)–AgeI (BVDV nu-cleotides 5312–5316) fragment was used to replace acorresponding NcoI–AgeI fragment of pC7.3898 (Tautz etal., 1997), which resulted in the final construct pUb1590-5B. In this plasmid, the NS3 gene, starting with the codonfor glycine 1590, is preceded by the 39 end of the ubiq-uitin gene, including an SacII site. To generate the de-rivatives that encode N-terminally truncated NS3 mole-cules downstream of the ubiquitin, the SacII (39 end ofubiquitin gene)–AgeI (BVDV nucleotides 5312–5316) frag-ment was replaced by corresponding PCR-derived frag-ments; the sense primers used for the generation of the
respective PCR products encompassed the 39 end of ubi,including an SacII site and the 59 region of the truncated

d

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NS3 gene. According to this scheme, plasmids pUb1592-5B, pUb1596-5B, pUb1596-5B, pUb1610-5B, pUb1625-5B,and pUb1635-5B were established. The plasmid namesindicate the first amino acid of the BVDV polyprotein thatis located downstream of ubiquitin.

For cloning of pUb1590-4A, an NcoI–MscI (ubi startcodon/BVDV nucleotides 7136–7141) fragment ofpUb1590-5B was inserted into pCITE-2A restricted withNcoI–HincII. Into this plasmid, the CP7 cDNA down tothe 39 end of the NS4A gene (codon 2336) was insertedtogether with a stop codon by PCR and standard cloningtechniques. Constructs pUb1592-4A, pUb1596-4A,pUb1610-4A, pUb1625-4A, and pUb1635-4A were gener-ated as described for the derivatives of pUb1590-5B bythe use of SacII and AgeI fragments.

pNS5A-5B-myc. For PCR amplification of the NS5A-Bregion, pE2*-5B-myc served as DNA template. The re-sulting PCR product encompassed a primer derived NcoIsite (including the start AUG) that is followed by a glycinecodon and the BVDV ORF beginning with codon 2586.The final construct, pNS5A-5B-mcy, was created by thereplacement of an NcoI (start AUG)–PstI (8682–8686)fragment of pNS4A-5B-myc by an identically restrictedfragment of the PCR fragment described earlier.

pGST-NS4-2350. The basic plasmid for this constructand its derivatives is pCITE-GST (see ear.ier). Into theORF of this vector, PCR-derived fragments that containparts of the NS4A-NS4B region were integrated by stan-dard cloning techniques. In clones pGST-NS4A-2349/-2339/-2329/-2319/-2309/-2299, the 59 end of the BVDVcDNA is identical to that of pNS4A-5B-myc (see earlier).The last amino acid of the BVDV polyprotein encoded bythe respective construct is illustrated by the plasmidname. For pGST-2281-2349, pGST-2291-2349, and pGST-2300-2349, the names of the constructs indicate theencoded fragments of the BVDV polyprotein downstreamof the GST gene.

Antisera and mAbs

Rabbit antiserum A3 generated against a bacterialfusion protein containing part of NS3 of CSFV strainAlfort Tubingen was used for immunoprecipitation ofNS3 under denaturing conditions (Thiel et al., 1991); fordetection of NS3 in Western blot analysis, hybridomasupernatant of mouse mAb 8.12.7 (Corapi et al., 1990)was applied. Rabbit antiserum P1 was used for immu-noprecipitations of NS4A and NS4B (Meyers et al., 1992)and for the detection of NS4A by Western blotting. TheNS5-specific serum used in immunoprecipitation analy-sis was a 1:1 mixture of rabbit antisera 62D and B3Bdirected against NS5A or NS5B, respectively (Collett etal., 1988a); the latter two sera were generously providedby M. S. Collett (ViroPharma Incorporated, Exton, PA).
The mAb directed against GST is distributed by SantaCruz Biotechnology (Santa Cruz, CA). Detection of pro-
teins carrying the myc-tag was achieved by a tag-spe-cific mAb purchased from Invitrogen.

ACKNOWLEDGMENTS

The authors thank Alexander E. Gorbalenya for critical reading of themanuscript and Gregor Meyers and Karin Kegreiss for their support inthe initial phase of this project. The authors are grateful to M. S. Collettfor supplying the NS5-specific antiserum. This study was supported bythe SFB535 “Invasionsmechanismen und Replikationsstrategien vonKrankheitserregern” from the “Deutsche Forschungsgemeinschaft.”

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ns3 serine protease of bovine viral diarrhea virus ... · ns4a, has not been studied so far. our...

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