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

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-Ju¨rgen Thiel Institut fu¨r Virologie (FB Veterina¨rmedizin), Justus-Liebig-Universita¨t Gie␤en, D-35392 Gie␤en, 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 polyprotein that is processed cotranslationally and posttranslationally by viral and cellular enzymes. A protease located in the N-terminal region 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 constitute together 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 residual activity. This finding links the NS3 protease of pestiviruses to the capsid protease of Sindbis virus. Furthermore, we observed that the minimal protease domain of NS3 encompasses about 209 amino acids. The NS3 protease was found to be sensitive to N-terminal truncation because a deletion of 6 amino acids significantly reduced the cleavage efficiency at the NS4A/4B site. Larger N-terminal deletions also impaired the activity of the enzyme with respect to the other cleavage sites but to a different degree at each site. The NS3 protease of BVDV has previously been shown to depend on NS4A as cofactor. We demonstrate here that the central region of NS4A represents the cofactor domain. Furthermore, coprecipitation studies strongly 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 suggest a common ancestry between these enzymes and the capsid protease of Sindbis virus. © 2000 Academic Press

INTRODUCTION

the NS protein p7, are presumably released from the polyprotein via cellular proteases (Ru¨menapf et al., 1993; Elbers et al., 1996; N. Tautz, unpublished data). The degree of processing at the NS2/3 site shows remarkable variation among different pestivirus species and strains (reviewed in Meyers and Thiel, 1996). NS2-3 and NS3 represent multifunctional proteins with protease, RNA binding, NTPase, and helicase activity (Grassmann et al., 1999; Tamura et al., 1993; Warrener and Collett, 1995; Wiskerchen and Collett, 1991). The protease located in the N-terminal domain of NS3 leads to the release of NS4A, NS4B, NS5A, and NS5B (Wiskerchen, 1991; see later). NS4A has recently been established as cofactor of the NS3 protease (Xu et al., 1997). The functions of NS4B and NS5A are not known. NS5B represents the viral RNA-dependent RNA polymerase (Kao et al., 1999; Lai et al., 1999; Steffens et al., 1999; Zhong et al., 1998). Hepaciviruses represent the closest relatives of pestiviruses, particularly with regard to the genome organization of the NS protein region and apparently also the function or functions of individual NS proteins (Rice, 1996). In addition, for HCV NS3 represents a serine protease that catalyzes all cleavages required for the generation of NS4A, NS4B, NS5A, and NS5B (Bartenschlager et al., 1993; Grakoui et al., 1993; Hijikata et al., 1993; Tomei et al., 1993; reviewed in Bartenschlager,

The genera Flavivirus, Hepacivirus, and Pestivirus constitute the family Flaviviridae (Rice, 1996). Members of the genus Pestivirus include important pathogens of livestock, such as classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV-1 and BVDV-2), and border disease virus (BDV). The single-stranded RNA genomes of these enveloped viruses are of positive polarity and have 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 long open reading frame (ORF) flanked by untranslated regions (UTRs). The order of the viral proteins in the hypothetical polyprotein is as follows: N pro, C, E rns, E1, E2, p7, NS2-3 (NS2, NS3), NS4A, NS4B, NS5A, NS5B (Collett et al., 1988a, 1991; Elbers et al., 1996; Meyers et al., 1992; Ru¨menapf et al., 1993; Stark et al., 1993; Tautz et al., 1997; Xu et al., 1997). N pro represents a nonstructural (NS) autoprotease, which releases itself from the polyprotein and thereby generates the N-terminus of the capsid protein (C) (Stark et al., 1993; Wiskerchen et al., 1991). The envelope glycoproteins E rns, E1, and E2, as well as 1 To whom reprint requests should be addressed at Institut fu¨r Virologie (FB 10). Justus-Liebig-Universita¨t Gie␤en, Frankfurter Strasse 107, D-35392 Gie␤en, Germany. Fax: 49-(641)-99-3-83-59. E-mail: [email protected].

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1997). Although processing at the 3/4A site is catalyzed exclusively in cis, all other cleavages can be mediated in trans. The observed consensus sequence for the cleavage sites is: E/D-X-X-X-X-C/T 2 S/A (where X is any amino acid and 2 symbolizes the cleavage site). The catalytic triad of the protease is composed of histidine, aspartic acid, and serine. Furthermore, a structurally important Zn-binding motif was identified in the protease domain (de Francesco et al., 1996; Kim et al., 1996; Love et al., 1996; Stempniak et al., 1997). The minimal fragment of NS3 still capable of cleaving at all sites was shown to consist of a 169-amino-acid peptide located in the Nterminal region of the protein (Bartenschlager et al., 1994; Failla et al., 1995; Tanji et al., 1994). The NS3 protease is assisted by NS4A as a cofactor that stimulates the cleavages at the four sites to significantly different extents (Bartenschlager et al., 1994; Failla et al., 1994; Lin et al., 1994; Steinku¨hler et al., 1996; Tanji et al., 1995a). In NS4A, a central 12-amino-acid peptide was identified that preserves cofactor activity (Bartenschlager et al., 1995b; Lin et al., 1995; Steinku¨hler et al., 1996; Tanji et al., 1995b). Coprecipitation analyses indicated a stable interaction between NS4A and the extreme N-terminus of NS3 (Bartenschlager et al., 1995a; Failla et al., 1995; Lin et al., 1994; Satoh et al., 1995). This interaction was confirmed by the resolution of the three-dimensional structure of the crystallized HCV NS3 protease domain complexed with a synthetic NS4A peptide (Kim et al., 1996). In contrast to the NS3 protease of HCV, knowledge about the corresponding enzyme of pestiviruses is very limited. Two computer models, established for the pestiviral enzyme (Bazan and Fletterick, 1989; Gorbalenya et al., 1989), have been experimentally challenged so far only 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 other members of the catalytic triad has not been approached experimentally. Mapping of the protease domain was attempted (Wiskerchen and Collett, 1991) but did not lead to valid data because the activity of the NS2-3 fragments was determined at a site that is not processed by NS3 (Petric et al., 1992; Xu et al., 1997). At the NS3/4A site, a restriction to cis cleavage was postulated because processing was not observed in trans; all other cleavages can be mediated by NS3 in trans (Wiskerchen and Collett, 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 the P1⬘ position (Tautz et al., 1997; Xu et al., 1997). Thus the sequences at the cleavage sites used by the NS3 proteases of BVDV and HCV differ significantly. Moreover, the overall degree of similarity between these enzymes is very low on amino acid level. In contrast to HCV, no Zn-binding motif can be delineated from the primary

sequence of BVDV NS3. Both proteases are stimulated in their activity by NS4A. NS3 of BVDV strictly depends on NS4A in its activity at the NS4B/5A and NS5A/5B sites (Xu et al., 1997). The role of NS4A in the other NS3dependent cleavages, as well as the cofactor domain of NS4A, has not been studied so far. Our aim in the present study was a comprehensive characterization of the pestiviral NS3 protease, its NS4A cofactor, and protease–cofactor interactions. RESULTS Active center of the NS3 serine protease The models proposed for the NS3 protease predict an active role for serine at amino acid position 1752 (S1752) and for histidine at position 1658 (H1658) (Bazan and Fletterick, 1989; Gorbalenya et al., 1989). Interestingly, the models differed with respect to the third member of the catalytic triad by predicting aspartic acid at either position 1686 (D1686) (Gorbalenya et al., 1989) or 1695 (D1695) (Bazan and Fletterick, 1989). From the proposed residues only S1752 was experimentally verified by an exchange to alanine (Mendez et al., 1998; Tautz et al., 1996; Wiskerchen and Collett, 1991). We started our analysis of the NS3 protease of BVDV strain CP7 with the characterization of its active center in a transient expression study by applying the T7 vaccinia virus system (Fuerst et al., 1986; Sutter et al., 1995). The first set of mutants aimed at the identification of residues that constitute the catalytic triad as well as at their functional characterization. Mutations of S1752 to cysteine (S1752C) or threonine (S1752T) were introduced to determine whether either of these amino acids could functionally replace S1752, which serves as nucleophile. The exchange of H1658 and aspartic acids at positions 1686/1695 by small nonpolar amino acids (glycine or alanine, respectively) served to identify the other members of the catalytic triad. The mutations were initially tested in the context of a polyprotein fragment, extending from the C-terminal region of E2 to the N-terminal part of NS4B encoded by plasmid pE2*-4B* (Fig. 1 and Tautz et al., 1996). The release of authentic NS2, NS3, NS2-3, and NS4A from the polyprotein encoded by E2*-4B* (* indicates truncated proteins) has been demonstrated before in the T7 vaccinia system (Tautz et al., 1996). Moreover, the use of this short polyprotein fragment facilitated the detection of noncleaved protein species. In the S1752A mutant, included as a control, the partial cleavage of NS2-3, which is independent of the NS3 protease, still occurred (Tautz et al., 1996), whereas the NS3 proteasedependent cleavages at the NS3/4A, NS2-3/4A and NS4A-4B* sites were abrogated. As a consequence, NS3-4B* and NS2-4B* with apparent molecular masses of 92 and 150 kDa, respectively, appeared. These proteins could be precipitated by antisera specific for NS3 or NS4 (Tautz et al., 1996, and Fig. 2, lanes 3 and 4). In the

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FIG. 1. Schematic diagram of the BVDV CP7 polyprotein (upper part) and the parts encoded by T7 expression plasmids pE2*-4B* and pE2*-5B; truncated proteins are marked by *. The shadowed bar INSERTION symbolizes the 9-amino-acid insertion present in NS2 of BVDV CP7. This sequence has been shown to induce NS2-3 cleavage independent of the NS3 serine protease.

analyses shown in Fig. 2, the amount of NS3-4B* was used as a parameter to monitor the impairment of the serine protease. The S1752C exchange in construct pE2*-4B* led to an efficient release of NS3 and to the generation of a small amount of NS3-4B* (Fig. 2, lane 5). By comparison, exchange S1752T caused an accumulation of NS3-4B*, which illustrates the severely reduced activity of this

FIG. 2. Single amino acid substitutions in the serine protease domain of NS3 and cleavage at the NS3/4A site. Expression of the E2*-4B* region of the BVDV polyprotein from transfected plasmids was achieved by the use of a recombinant vaccinia virus expressing the T7 RNA polymerase (MVA-T7pol; Sutter et al., 1995). The metabolically labeled proteins were immunoprecipitated with antisera against NS3 or NS4, respectively, and further analyzed by SDS–PAGE (Doucet and Trifaro, 1988) and fluorography. The transfected expression plasmids as well as the antisera used are indicated above the corresponding lanes. Mutations in the encoded CP7 polypeptides are indicated in the names of the corresponding plasmids. Precipitates derived from cells infected with MVA-T7pol without plasmid transfection are separated in lane 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 ⫽ kDa.

mutant. Unexpectedly, a substantial amount of NS3 was still generated (Fig. 2, lane 7). The H1658A exchange in contrast completely abrogated the generation of mature NS proteins (Fig. 2, lanes 9 and 10); this result was in line with the postulated catalytic role of H1658 in the NS3 protease. Surprisingly, neither mutation D1686G (Fig. 2, lanes 11 and 12) nor exchange D1695A (data not shown) induced obvious changes in processing. Only for mutant D1686G did a minor band corresponding to NS3-4B* appear. This result, which was obtained mainly by monitoring the cleavage activity at the NS3/4A site, thus did not allow establishment of the third member of the catalytic triad. To identify the respective residue, we introduced exchanges D1686G and D1695A into a polyprotein that extended from E2* to NS5B encoded by plasmid pE2*-5B (Fig. 1). This setup allows to monitor the effect of mutations on the trans cleavages at sites NS4A/4B, NS4B/5A, and NS5A/5B. The transfection of plasmids pE2*-5B and pE2*-5B/D1695A led to the generation of all viral NS proteins (Figs. 3A and 3B, lanes 2 and 6, and data not shown), whereas the D1686G exchange completely inhibited the trans cleavages of the NS3 protease (Figs. 3A and 3B, lane 5); as expected, the cleavage at the NS3/4A site was almost unaffected (data not shown). Furthermore, we examined the effect of mutations S1752C and S1752T on the trans cleavage activity of the protease. After the transient expression of pE2*-5B/ S1752C or pE2*-5B/S1752T, minor amounts of NS2-3 and NS3 (data not shown), but neither NS4A, NS4B, NS5A, nor NS5B, could be detected (Figs. 3A and 3B, lanes 3 and 4, respectively). According to this assay system, the respective NS3 protease mutants exhibited only cleavage activity at the NS3/4A site. Taken together, our results are in agreement with the model of Gorbalenya et al. (1989) that predicted for the NS3 protease a catalytic triad composed of H1658, D1686, and S1752.

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FIG. 3. NS3 protease mutants and processing of E2*-5B. (A) Detection of NS4A and NS4B. Immunoprecipitates obtained by the use of NS4 specific antiserum were separated by Tricine–SDS–PAGE (Scha¨gger and Jagow, 1987). Samples derived from cells infected with MVAT7pol without plasmid transfection were separated in lane 7 (Mock). Arrowheads indicate the position of NS4A and NS4B. The 38-kDa protein (lower band of the doublet) in lanes 2 and 6 most likely represents noncleaved NS4A-4B (see also Fig. 5C). The amount of NS2 detected on transfection of the different plasmids was used to standardize transfection efficiencies (data not shown). The S1752A exchange was included as a control to show inhibition of NS3-dependent cleavages. (B) Detection of NS5A-5B, NS5A, and NS5B. The transfected expression plasmids are depicted in the figure above the corresponding 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 plasmid transfection were separated in lane 7 (Mock).

Characterization of the NS3 protease domain For several BVDV strains, it has been experimentally demonstrated (Ku¨mmerer et al., 1998; Meyers et al., 1998) or suggested (Mendez et al., 1998; Meyers and Thiel, 1996; Tautz et al., 1993) that the first amino acid of NS3 is glycine 1590. Accordingly, authentic NS3 encompasses amino acids 1590–2272 of the BVDV polyprotein (Tautz et al., 1997; Xu et al., 1997). Our next aim was to further define the protease domain of NS3. We started the corresponding analysis by the construction of expression plasmids encoding C-terminally truncated NS3 molecules, which encompass 251, 211, or 171 amino

acids from the NS3 region; the C-terminal amino acid of these NS3 derivatives encoded by pGST-NS3-1840, pGST-NS3-1800, or pGST-NS3-1760 (Fig. 4A) is located in the polyprotein at the positions indicated by the plasmid names. The NS3 fragments were expressed as glutathione-S-transferase (GST) fusion proteins to ease their detection (Figs. 4A and 4B). The substrate for the protease was expressed from pNS4A-5B-myc (Fig. 4A) encoding an NS4A-5B polyprotein fragment with a C-terminal myc-tag; the latter facilitates protein detection. On the coexpression of NS4A-5B-myc with GST-NS3-1800 or GST-NS3-1840, a polypeptide with an apparent molecular mass expected for NS5B-myc (about 75 kDa) could be detected with the myc-tag-specific antibody (Fig. 4C, lanes 3 and 4). In contrast, cotransfection of pGST-NS31760 and pNS4A-5B-myc did not result in the cleavage of NS4A-5B-myc (Fig. 4C, lane 2). The expression of the GST-NS3 fusion proteins in the respective cell lysates was also verified by Western blotting (data not shown). The use of identically truncated NS3 molecules devoid of the GST fusion led to comparable results, which demonstrates that the fusion partner had no influence on the obtained results (data not shown). Accordingly, the Cterminal border of the NS3 serine protease domain resides between amino acid positions 1760 and 1800 at least with respect to cleavage at the NS5A/5B site. To determine the N-terminal border of the protease domain, we chose a strategy that allowed the expression of N-terminally truncated NS3 molecules without any vector-derived amino acid at their N-termini. For this purpose, one ubiquitin monomer was inserted upstream of the BVDV polyprotein. On expression, cellular ubiquitin-specific proteases (Jonnalagadda et al., 1989; Mayer and Wilkinson, 1989; Rose, 1988) will cleave after the C-terminal amino acid of ubiquitin (glycine 76) and thereby generate the N-terminus of NS3. It has previously been demonstrated that this cleavage also occurs efficiently in the context of the BVDV polyprotein (Tautz et al., 1993). The BVDV polyprotein encoded by plasmid pUb1590-5B starts with the first amino acid of NS3 (G 1590) and proceeds to the end of NS5B (Fig. 5A). In a series of derivatives of pUb1590-5B, the N-terminus of NS3 was truncated by 2, 6, 20, 35, and 45 amino acids (Fig. 5A); the numbers in the names of the constructs reflect the relative position of the first amino acid of the encoded BVDV polypeptide with respect to the viral polyprotein. After the transfection of pUb1590-5B and pUb1592-5B, viral proteins NS3, NS4A, NS4B, NS5A, and NS5B could be detected (Figs. 5B and 5C). The deletion of the 6 N-terminal amino acids resulted in the appearance of an additional protein of about 38 kDa, which could be precipitated by antibodies against NS4 (Fig. 5C, lane 3). Due to its apparent molecular mass and the strong decline in the amount of NS4A and NS4B, this protein most likely represents noncleaved NS4A-4B. On the deletion of 20 Nterminal amino acids, only two mature viral proteins were

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released from the polyprotein, namely NS3 (Fig. 5B, lane 7) and a minor amount of NS5B (Fig. 5B, lane 8). The additional proteins with apparent molecular masses of about 95 and 160 kDa, respectively, could be precipitated with an NS5-specific antiserum (Fig. 5B, lane 8) and by the serum directed against NS4 (data not shown) but not with antibodies specific for NS3 (Fig. 5B, lane 7). In accordance with their apparent molecular masses and reactivity with defined antibodies, these proteins most likely represent NS4A-5A and NS4A-5B. The transfection of pUb1625-5B (deletion of 35 N-terminal amino acids) resulted in the generation of the truncated NS3 as the only mature viral protein; this NS3 derivative migrates due to its truncation significantly faster than authentic NS3 (Fig. 5B, lane 9). In addition; NS4A-5A and NS4A-5B were precipitated by the antisera specific for NS5 (Fig. 5B, lane 10). The appearance of these proteins indicates that cleavage still takes place at the NS3/4A and NS5A/5B sites, albeit at a very low level. The failure to detect NS5B might be due to the low cleavage efficiency and the described inherent instability of NS5B (Collett et al., 1991). The deletion of the first 45 amino acids of NS3 abolished the activity of the protease because neither one of the mature BVDV proteins nor NS4A-5A and NS4A-5B could be identified (Figs. 5B, lanes 11 and 12, and 5C, lane 6). Cofactor domain of NS4A

FIG. 4. C-terminal border of the NS3 protease domain. (A) Scheme of the used cDNA constructs. The C-terminally truncated NS3 derivatives were expressed from plasmids pGST-NS3-1840, pGST-NS3-1800, or pGST-NS3-1760 as fusions to GST. Plasmid pNS4A-5B-myc encodes the NS protein region from NS4A-5B with an myc-tag fused to the C-terminus. The numbers in brackets indicate the amino acids of the BVDV polyprotein encoded by the respective construct. (B) Western blot analysis of the GST-NS3* fusion proteins. Plasmids pGST-NS3-1840, pGST-NS3-1800, or pGST-NS3-1760 were transfected into cells previously infected by MVA-T7pol virus. The respective cell lysates were separated by Tricine–SDS–PAGE (Scha¨gger and Jagow, 1987) and analyzed by Western blotting with a mouse mAb directed against GST (Santa Cruz Biotechnology). The lanes combined in the figure were derived from the same gel. Lysates of cells only infected with MVAT7pol virus were separated in lane 4 (Mock). The positions of the mass standards (prestained MW markers) are shown on the right; k ⫽ kDa. (C) Proteolytic activity of the GST-NS3* fusion proteins. On coexpression of GST-NS3-1840, GST-NS3-1800, or GST-NS3-1760 and the NS4A5B-myc substrate, the respective lysates were analyzed by Tricine– SDS–PAGE (Scha¨gger and Jagow, 1987) and Western blotting applying a c-myc–specific mAb (Invitrogen). The positions of unprocessed NS4A-5B-myc, NS5B-myc, and the cellular myc protein (c-myc) are

NS4A, which encompasses amino acids 2273–2336 of the BVDV polyprotein (Tautz et al., 1997; Xu et al., 1997), acts as an essential cofactor of the NS3 protease, at least for the cleavages at sites NS4B/5A and the NS5A/5B sites (Xu et al., 1997). To determine whether the complete NS4A protein is necessary to activate the protease, we established an appropriate cleavage assay. pNS5A-5B-myc (Fig. 6A) was used for the expression of a suitable substrate. On the transfection of plasmid pNS2*-3* (Fig. 6A), an NS3-specific antibody detected proteins NS2*-3* and NS3* (data not shown). The NS4Aderived peptides were expressed as fusions to GST to allow their detection by Western blotting (Fig. 6C). As expected, the cotransfection of pNS2*-3* and pNS5A-5Bmyc in the absence of the cofactor NS4A did not lead to detectable cleavage at the NS5A/5B site (Fig. 6B, lane 1). After the cotransfection of plasmids pNS2*-3* and pNS5A-5B-myc with pGST-NS4A fusion proteins encompassing either complete NS4A together with a small part of the NS4B coding region (pGST-NS4A-2349,

indicated at the left. In lane 5 (Mock), lysates of cells infected only with MVA-T7pol virus were separated. The lanes combined in the figure were derived from the same gel. On the right, the positions of the mass standards (prestained MW markers) are shown; k ⫽ kDa.

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FIG. 5. N-terminal border of the NS3 protease domain. (A) Schematic drawing of the cDNA constructs. In the upper part of the scheme, the organization of the polyprotein encoded by the expression plasmids is depicted. In all clones, ubiquitin resides upstream of NS3. In the lower left part of the figure, the amino acid sequence at the cleavage site between ubiquitin and NS3 is given in the one-letter code. The relative position of the first amino acid of the encoded NS3 derivative is indicated by the plasmid name. The C-terminal tripeptide of ubiquitin, namely (RGG), is indicated. Cellular ubiquitin specific proteases are known to cleave downstream of this sequence. This cleavage is strongly impaired only if a proline residue follows the C-terminal amino acid of ubiquitin. We did therefore not generate an NS3 derivative in which just 1 amino acid was deleted from the N-terminus. The right part of the figure summarizes the obtained results: ⫹, efficient cleavage; ⫺, no cleavage; (⫹), inefficient cleavage. (B) Transient expression of NS3–5B and N-terminally truncated derivatives. After transient expression in the presence of [ 35S]methionine/cysteine by use of the T7 vaccinia virus system, the cell lysates were subjected to a radioimmunoprecipitation analysis. The transfected plasmids and the utilized antisera are indicated. In lanes 12 and 13 (Mock), precipitates generated from lysates of MVA-T7pol-infected cells were separated. The positions of NS3, NS5A, NS5B, NS4A-5A, and NS4A-5B are indicated by arrowheads. The positions of the mass standards ([ 14C]MW markers) are shown on the right; k ⫽ kDa. (C) Processing at NS3/4A and NS4A/4B sites. After transient expression, cell lysates were further processed by precipitation using NS4-specific antibodies. Precipitates were separated by Tricine–SDS–PAGE (Scha¨gger and Jagow, 1987). Lane 7 (Mock) represents an analysis of cells only infected with MVA-T7pol virus. The positions of NS4A, NS4B, and NS4A-4B are indicated on the left. The positions of the mass standards ([ 14C]MW markers) are shown on the right; k ⫽ kDa. The results concerning processing at the NS3/4A site were confirmed by the use of derivatives of the plasmids described above; the corresponding constructs encoded NS3-4B* (data not shown).

pGSTNS4A-2339) (Fig. 6B, lanes 2 and 3) or an NS4A fragment with a 7-amino-acid deletion at its C-terminus (pGST-NS4A-2329) (Fig. 6B, lane 4), NS5B-myc was released. Larger C-terminal truncations (17 amino acids or more) of NS4A abrogated its cofactor activity.

NS4A truncated N-terminally by 9 amino acids still assisted in NS5A-5B-myc cleavage (Fig. 6B, lane 8). As indicated by the very weak appearance of NS5B-myc NS4A with a deletion of 18 amino acids from the N-terminus still retained residual cofactor activity (Fig.

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FIG. 6. Cofactor domain of NS4A. (A) Scheme of expressed proteins. pNS2*-3* was used to produce NS2*-3* and NS3* in the absence of NS4A. pNS5A-5B-myc served for generation of a suitable substrate for cofactor dependent proteolysis. The myc-tag at the C-terminus of NS5B as well as GST fused to NS4A served for detection of individual proteins. Numbers in parentheses indicate the part of the BVDV polyprotein encoded by the respective plasmids. (B) NS4A-dependent cleavage assay. After coexpression of NS2*-3*, NS5A-5B-myc, and the different NS4A derivatives, the cell lysates were subjected to Western blot analysis. Proteins were separated by Tricine–SDS–PAGE and further analyzed by Western blotting using an myc-tag-specific mAb. In lane 11 (Mock), lysates of cells infected only with MVA-T7pol were analyzed. The positions of NS5A-5B-myc, NS5B-myc, and the cellular myc protein (c-myc) are indicated. On the right, the positions of the mass standards (prestained MW markers) are shown; k ⫽ kDa. (C) Detection of the GST-NS4A fusion proteins. The cell lysates described above (see legend for Fig. 6B) were analyzed by Tricine–SDS–PAGE and Western blot analysis applying an mAb directed against GST. In lane 11 (Mock), lysates of cells infected only with MVA-T7pol were analyzed. The positions of the mass standards (prestained MW markers) are shown on the right; k ⫽ kDa.

6B, lane 9). The minimal NS4A fragment that preserves the cofactor function in cleavage at the NS5A/5B site thus is probably composed of not more than amino acids 2291–2329 even though a direct test with a corresponding peptide was not performed. Comparable results were also obtained with NS4A fragments without GST-tag (data not shown).

Interaction between NS4A and the N-terminal domain of NS3 In the HCV system, a stable interaction between the NS4A cofactor domain and the N-terminal region of NS3 has been established (Bartenschlager et al., 1995a; Failla et al., 1995; Hijikata et al., 1993a,b; Kim et al., 1996; Lin et al., 1995; Satoh et al., 1995). It was intended to test

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FIG. 7. Coprecipitation of NS3 and NS4A. Proteins encoded by pUbNS3-4A and its derivatives were transiently expressed in the presence of [ 35S]methionine/cysteine by using MVA-T7pol virus. Cell lysates were prepared under nondenaturing conditions and subjected to immunoprecipitation by an NS3-specific mAb. The obtained aggregates were denatured, separated by Tricine–SDS–PAGE, and processed for fluorography. The positions of NS3 and NS4A are indicated.

whether a similar NS3/NS4A interaction occurs in the BVDV system. Accordingly, plasmids pUb1590-4A and its derivatives encoding N-terminally truncated NS3-4A molecules were generated by the deletion of the NS4B-5B coding region from the series of plasmids based on pUb1590-5B. Proteins expressed in the presence of [ 35S]methionine/cysteine by pUb1590-4A and its derivatives were immunoprecipitated under nondenaturing conditions with a monoclonal antibody (mAb) directed against NS3. SDS–PAGE analysis and fluorography of the precipitated proteins revealed that a 7-kDa protein coprecipitated with full-length NS3 (Fig. 7, lane 1). A Western blot analysis carried out in parallel identified the 7-kDa protein as NS4A (data not shown). Although NS4A could also be coprecipitated with the NS3 derivative, which was N-terminally truncated by 2 amino acids (Fig. 7, lane 2), all NS3 species with larger N-terminal truncations did not allow pulling down NS4A under the chosen conditions (Fig. 7). These data are in line with an essential role of the N-terminal region of NS3 in the complex formation between NS3 and NS4A. DISCUSSION The aim of the present study was the characterization of the pestiviral NS3 protease. As a first step, the residues constituting the catalytic triad were determined. The obtained results are in agreement with the model of Gorbalenya et al. (1989), in which the catalytic triad is composed of H1658, D1686, and S1752. There was no indication for an essential role of D1695 in the protease domain as proposed in an alternative model (Bazan and Fletterick, 1990). The NS3 serine protease catalyzes two types of cleavages. Although processing at the NS3/NS4A site is believed to occur exclusively in cis, all other cleav-

ages can be mediated in trans (Wiskerchen and Collett, 1991). Studies on the kinetics of proteolysis of the NS region of the BVDV polyprotein in virus-infected cells indicated that the cleavage at the NS3/4A site takes place with significantly higher efficiency and faster kinetics than the other NS3-mediated cleavages (Collett et al., 1991). Our study revealed that the NS3 mutants carrying exchanges S1752C, S1752T, or D1686G, respectively, still catalyzed the NS3-4A cleavage, whereas they showed no activity at the other cleavage sites in our assay system. With respect to the higher activity of the protease at the NS3/4A site, we assume that an overall reduction in the enzymatic activity due to the introduced mutations can result in the observed selective loss of trans cleavage activity. The finding that serine and cysteine are interchangeable as nucleophiles, at least to some extent, has already been described for other viral serine proteases (Hahn and Strauss, 1990; see later) as well as viral serine-like cysteine proteases such as the 3C protease of picornaviruses and the 27-kDa NIa protease of the potyvirus tobacco etch virus (Dougherty et al., 1989; Lawson and Semler, 1991). It was surprising, however, that NS3 with the S1752T substitution exhibited a significant residual protease activity at the NS3/4A site, which indicates that the hydroxyl group of threonine serves in the nucleophilic attack during proteolysis. There is only one other example of a serine protease where the exchange of the active site serine by threonine gave rise to an enzyme with substantial protease activity (10% of wild type), namely, the capsid protease of Sindbis virus (Hahn and Strauss, 1990). This enzyme is an autoprotease that catalyzes its release from the viral polyprotein, thereby generating its own C-terminus. It was supposed that the activity of the capsid protease with a threonine in the active site is due to the intramolecular nature of the reaction and the very fast reaction kinetics of the wildtype protease (Hahn and Strauss, 1990). Furthermore, for the Sindbis virus capsid protease as well as the BVDV enzyme, the exchange of the active site aspartic acid did not significantly reduce their cis cleavage activities. In addition to the similarities observed in this study with regard to the biochemistry of these enzymes, it turned out recently that the three-dimensional structure of the NS3 protease of HCV, a close relative of pestiviruses, shares its greatest structural similarity with the Sindbis virus capsid protease (Kim et al., 1996). These findings nicely corroborate the relationship between the described proteases, which remarkably was postulated several years ago on the basis of computer-assisted sequence comparisons of flaviviruses, pestiviruses, and alphaviruses (Gorbalenya et al., 1989). According to our study, a 209-amino-acid fragment of the BVDV NS3 serine protease is still capable of exerting the NS5A-NS5B cleavage. A protease domain of this size is in agreement with the data obtained for the NS3

BVDV NS3 SERINE PROTEASE

protease domains of HCV (169 amino acids) (Bartenschlager et al., 1994; Failla et al., 1995; Tanji et al., 1994) and members of the genus Flavivirus (e.g., yellow fever virus, 181 amino acids; Chambers et al., 1990). Because fusions of GST to the N-terminus of NS3 had no deleterious effect on the activity of the enzyme, the folding of the NS3 protease domain can apparently occur independent of the preceding peptide sequence. The sensitivity of the protease domain to N-terminal truncations and the stepwise loss of activity are remarkable: a deletion of 6 N-terminal amino acids resulted in a strongly reduced activity at the NS4A/4B site (Figs. 5B and 5C). Larger N-terminal truncations of NS3 further reduced the activity of the enzyme at the individual cleavage sites to different extents. The greatest tolerance to N-terminal truncation of the protease was observed for the cis cleavage and processing at the NS5A/5B site. Interestingly, similar results have been obtained in the HCV system for N-terminally truncated NS3 protease derivatives. Short deletions selectively interfered with NS4A-NS4B and NS4B-NS5A cleavage, whereas more extensive truncations of NS3 also inhibited cleavage at the NS5A/5B site and ultimately the cis cleavage at the NS3/4A site (reviewed in Bartenschlager, 1997). Our coprecipitation study revealed an interaction between BVDV proteins NS3 and NS4A, which was abrogated by a deletion of the first 6 amino acids of NS3 (Fig. 7). This finding strongly suggests a role of the N-terminal domain of NS3 in NS4A binding. Interestingly, such a truncated protease was proteolytically active at all sites. For HCV, binding of NS4A to the N-terminal region of NS3 has also been demonstrated by coprecipitation and was confirmed at the structural level (Kim et al., 1996). This interaction could be abrogated by N-terminal truncations of NS3 (Bartenschlager et al., 1995a; Failla et al., 1995; Lin et al., 1994; Satoh et al., 1995). N-terminally truncated as well as mutagenized NS3 derivatives could be identified that show proteolytic activity despite impaired NS4A binding. These data and the results obtained in this study suggest that a weak transient protease–cofactor interaction seems to be sufficient for activation of the NS3 proteases of HCV (for review, see de Francesco and Steinku¨hler, 2000) and BVDV. Taken together, our study revealed striking parallels between the NS3 proteases of BVDV and HCV, especially with respect to active site residues, size of protease domain, NS4A cofactor dependency, and NS3–NS4A interaction. These findings again underline the fascinatingly close relationship of pestiviruses and hepaciviruses. In addition, the similarities observed between the capsid protease of Sindbis virus, a member of the genus Alphavirus, and the NS3 protease of a member of the Flaviviridae suggest a common ancestor for the serine proteases of these positive-strand RNA viruses.

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MATERIALS AND METHODS Cells and viruses BHK-21 cells were kindly provided by J. Cox (Federal Research Center for Virus Diseases of Animals, Tu¨bingen, Germany). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). The recombinant vaccinia virus expressing the T7 RNA polymerase [modified virus Ankara (MVA)-T7pol] was generously provided by G. Sutter (Institut fu¨r Molekulare Virologie, GSF-Forschungszentrum fu¨r Umwelt und Gesundheit GmbH, Oberschlei␤heim, Germany). Transient expression with the T7 vaccinia virus system BHK-21 cells (5 ⫻ 10 5, 3.5-cm dish) were infected with MVA-T7pol at a multiplicity of infection of 5 in medium lacking FCS. After 1 h of incubation at 37°C, cells were washed twice with medium lacking FCS followed by DNA transfection with 2 ␮g plasmid DNA by the use of Superfect (Qiagen, Hilden, Germany) according to the protocol of the manufacturer. For Western blot analysis, the transfected cell cultures were harvested about 6 h posttransfection (p.t.). Radioactive labeling of cells. At 2 h p.t., cells were washed two times with PBS and incubated with 1 ml of label medium lacking cysteine and methionine for 0.5 h at 37°C. After the addition of 500 ␮Ci of [ 35S]methionine/ cysteine ([ 35S]-Pro Mix; Amersham, Freiburg, Germany), protein expression was allowed to proceed for an additional 4 h at 37°C. After washing with PBS, cells were stored at ⫺70°C until they were further processed. Radioimmunoprecipitation and SDS–PAGE Immunoprecipitation under denaturing conditions. Cell extracts prepared in the presence of 2% SDS were diluted and incubated with 5 ␮l of undiluted antiserum. For the formation of precipitates, protein A–agarose (Calbiochem, La Jolla, CA) was used. SDS–PAGE analysis of proteins was performed by the use of Tricine gels (Scha¨gger and Jagow, 1987) or according to Doucet and Trifaro (1988). Gels were processed for fluorography using En 3Hance (New England Nuclear, Boston, MA). Immunoprecipitation under nondenaturing conditions. Cells were lysed with buffer IBP150 [20 mM Tris–HCl, pH 8.0, 150 mM KCl, 1% Nonidet P-40, 1 mM EDTA, pH 8.0, 1 mM EDTA, 0.1 mM Pefablock SC (Roche Diagnostics, Mannheim, Germany)] kept on ice for 5 min and slowly rotated for 30 min at 4°C. Insoluble parts were removed by two consecutive centrifugations at 300 and 10,000 g. NS3-specific mAb 8.12.7 (Corapi et al., 1990) was bound via Fc-specific anti-mouse IgG (Sigma Chemical Co., St. Louis, MO) to protein A–agarose (Calbiochem) and cross-linked by diaminopimelic acid according to the protocol of Harlow and Lane (1988). The cross-linked

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antibody was incubated with the cleared cell lysates for 1 h at 4°C. Complexes were collected by centrifugation at 4000 g and washed once with IBP150 and twice with IBP500 (20 mM Tris–HCl, pH 8.0, 500 mM KCl, 1% Nonidet P-40, 1 mM EDTA, pH 8.0, 1 mM EDTA, 0.1 mM Pefablock SC). After a final washing step with IBP150, the precipitate was diluted in gel loading buffer containing 2% SDS and 5% mercaptoethanol and boiled for 5 min. Polymerase chain reaction (PCR) For PCR, a GENIUS thermocycler (Techne/thermoDUX, Wertheim, Germany) was used. Standard conditions used for DNA-based amplifications have been described previously (Tautz et al., 1999). Nucleotide sequencing Sequencing was carried out with the Thermo Sequenase Cycle Sequencing Kit (Amersham Buchler, Braunschweig, Germany). The primers used for sequencing were labeled with infrared dye IRD41 (MWG-Biotech, Ebersberg, Germany). Analyses of sequencing gels were carried out with an automatic sequencer LI-COR 4000L (MWG-Biotech, Ebersberg, Germany). Western blotting After SDS–PAGE proteins were transferred onto nitrocellulose membranes (Optitran BA-S83 Reinforced NC; Schleicher & Schuell, Du¨ren, Germany). Blocking was carried out with 2% dry milk in PBS for 1 h. The proteins were visualized by chemiluminescence (SuperSignal; Pierce Chemical, Rockford, IL). Construction of the expression plasmids All BVDV cDNAs used for the construction of the expression plasmids throughout this study are derived from BVDV strain CP7 (Meyers et al., 1996; Tautz et al., 1996). For PCR amplifications used in the construction of the expression plasmids, the already described BVDV CP7 cDNA clones were used as templates (Meyers et al., 1996; Tautz et al., 1996, 1997). Numbering of nucleotides or amino acids occurs throughout this report with respect to the BVDV SD-1 sequence because the complete sequence of BVDV CP7 has not been published yet and SD-1 represents the only complete BVDV sequence devoid of a strain-specific insertion (Deng and Brock, 1992). pE2*-4B*. This plasmid has already been described under the name pC7.1 (Tautz et al., 1996); in the same publication, the generation of construct pC7PM1 was described. 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 NS3 protease were generated by standard PCR techniques using oligonucleotide primers that encompass the individual nucleotide exchanges.

pE2*-5B. Elongation of the BVDV CP7 cDNA in pC7.1 at its 3⬘ end by a fragment derived from construct pC7.3898 (Tautz et al., 1997) resulted in plasmid pE2*-5B. Thus the BVDV ORF encoded by this plasmid starts with codon 967 and ends with the authentic stop codon encompassed in the BVDV genome. pNS4A-5B-myc. This plasmid is based on pCITE-2A (Novagene, La Jolla, CA), which downstream of a T7 RNA polymerase promoter encompasses an internal ribosomal entry site (IRES) of the encephalomyocarditis virus (EMCV). The 5⬘ end of the cDNA insert in this plasmid was generated by PCR and starts downstream of the vector-derived AUG codon with an alanine codon followed by the BVDV ORF beginning with the codon for serine 2274, the second amino acid of NS4A; the cDNA proceeds downstream to the NS5B gene. Into a PvuII site (nucleotides 12260–12265) in the 3⬘ region of the 5B gene, an EcoRV–PvuII fragment derived from pSecTag (Invitrogen, Groningen, Netherlands) was cloned; the respective fragment encompassed part of the polylinker and the coding sequence for an myc- and a His-tag followed by a stop codon. In the polyprotein encoded by pNS4A-5B-myc, accordingly the 6 C-terminal amino acids of NS5B are substituted by 34 pSecTag-derived amino acids, including the myc-epitope. pGST-NS3-1760/1800/1840. The GST gene from pGEX-2T (Pharmacia) was amplified by PCR and integrated into the NcoI site of pCITE-2A by standard cloning techniques. Downstream of the GST gene, the resulting plasmid, pCITE-GST, contains a singular NcoI site encompassing an in-frame AUG codon. After restriction with NcoI–XbaI, this vector was ligated to an NcoI–XbaI fragment of pARC7 (Tautz et al., 1997). The resulting plasmid, pGST-NS3-2351, encoded for the CP7 polyprotein from amino acids 1587–2351. To generate the final constructs, PCR-derived fragments of the NS3 gene of CP7 that encode stop codons downstream of serine 1760, lysine 1800, or leucine 1840, respectively, were used 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 nucleotides 5312–5316) fragment was used to replace a corresponding NcoI–AgeI fragment of pC7.3898 (Tautz et al., 1997), which resulted in the final construct pUb15905B. In this plasmid, the NS3 gene, starting with the codon for glycine 1590, is preceded by the 3⬘ end of the ubiquitin gene, including an SacII site. To generate the derivatives that encode N-terminally truncated NS3 molecules downstream of the ubiquitin, the SacII (3⬘ end of ubiquitin gene)–AgeI (BVDV nucleotides 5312–5316) fragment was replaced by corresponding PCR-derived fragments; the sense primers used for the generation of the respective PCR products encompassed the 3⬘ end of ubi, including an SacII site and the 5⬘ region of the truncated

BVDV NS3 SERINE PROTEASE

NS3 gene. According to this scheme, plasmids pUb15925B, pUb1596-5B, pUb1596-5B, pUb1610-5B, pUb1625-5B, and pUb1635-5B were established. The plasmid names indicate the first amino acid of the BVDV polyprotein that is located downstream of ubiquitin. For cloning of pUb1590-4A, an NcoI–MscI (ubi start codon/BVDV nucleotides 7136–7141) fragment of pUb1590-5B was inserted into pCITE-2A restricted with NcoI–HincII. Into this plasmid, the CP7 cDNA down to the 3⬘ end of the NS4A gene (codon 2336) was inserted together with a stop codon by PCR and standard cloning techniques. Constructs pUb1592-4A, pUb1596-4A, pUb1610-4A, pUb1625-4A, and pUb1635-4A were generated as described for the derivatives of pUb1590-5B by the use of SacII and AgeI fragments. pNS5A-5B-myc. For PCR amplification of the NS5A-B region, pE2*-5B-myc served as DNA template. The resulting PCR product encompassed a primer derived NcoI site (including the start AUG) that is followed by a glycine codon and the BVDV ORF beginning with codon 2586. The final construct, pNS5A-5B-mcy, was created by the replacement of an NcoI (start AUG)–PstI (8682–8686) fragment of pNS4A-5B-myc by an identically restricted fragment of the PCR fragment described earlier. pGST-NS4-2350. The basic plasmid for this construct and its derivatives is pCITE-GST (see ear.ier). Into the ORF of this vector, PCR-derived fragments that contain parts of the NS4A-NS4B region were integrated by standard cloning techniques. In clones pGST-NS4A-2349/2339/-2329/-2319/-2309/-2299, the 5⬘ end of the BVDV cDNA is identical to that of pNS4A-5B-myc (see earlier). The last amino acid of the BVDV polyprotein encoded by the respective construct is illustrated by the plasmid name. For pGST-2281-2349, pGST-2291-2349, and pGST2300-2349, the names of the constructs indicate the encoded fragments of the BVDV polyprotein downstream of the GST gene. Antisera and mAbs Rabbit antiserum A3 generated against a bacterial fusion protein containing part of NS3 of CSFV strain Alfort Tu¨bingen was used for immunoprecipitation of NS3 under denaturing conditions (Thiel et al., 1991); for detection of NS3 in Western blot analysis, hybridoma supernatant of mouse mAb 8.12.7 (Corapi et al., 1990) was applied. Rabbit antiserum P1 was used for immunoprecipitations of NS4A and NS4B (Meyers et al., 1992) and for the detection of NS4A by Western blotting. The NS5-specific serum used in immunoprecipitation analysis was a 1:1 mixture of rabbit antisera 62D and B3B directed against NS5A or NS5B, respectively (Collett et al., 1988a); the latter two sera were generously provided by M. S. Collett (ViroPharma Incorporated, Exton, PA). The mAb directed against GST is distributed by Santa Cruz Biotechnology (Santa Cruz, CA). Detection of pro-

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teins carrying the myc-tag was achieved by a tag-specific mAb purchased from Invitrogen. ACKNOWLEDGMENTS The authors thank Alexander E. Gorbalenya for critical reading of the manuscript and Gregor Meyers and Karin Kegreiss for their support in the initial phase of this project. The authors are grateful to M. S. Collett for supplying the NS5-specific antiserum. This study was supported by the SFB535 “Invasionsmechanismen und Replikationsstrategien von Krankheitserregern” from the “Deutsche Forschungsgemeinschaft.”

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