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

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; The genera Flavivirus, Hepacivirus, and Pestivirus constitute the family Flaviviridae (Rice, 1996). Members of Elbers et al., 1996; N. Tautz, unpublished data). the genus Pestivirus include important pathogens of live- The degree of processing at the NS2/3 site shows stock, such as classical swine fever virus (CSFV), bovine remarkable variation among different pestivirus species viral diarrhea virus (BVDV-1 and BVDV-2), and border and strains (reviewed in Meyers and Thiel, 1996). NS2-3 disease virus (BDV). The single-stranded RNA genomes and NS3 represent multifunctional proteins with pro- of these enveloped viruses are of positive polarity and tease, RNA binding, NTPase, and helicase activity have in general a length of 12.3 kb (Becher et al., 1998; (Grassmann et al., 1999; Tamura et al., 1993; Warrener Collett et al., 1988b; Deng and Brock, 1992; Meyers et al., and Collett, 1995; Wiskerchen and Collett, 1991). The 1989; Renard et al., 1987). The genome contains one long protease located in the N-terminal domain of NS3 leads open reading frame (ORF) flanked by untranslated re- to the release of NS4A, NS4B, NS5A, and NS5B gions (UTRs). The order of the viral proteins in the hypo- (Wiskerchen, 1991; see later). NS4A has recently been thetical polyprotein is as follows: Npro,C,Erns, E1, E2, p7, established as cofactor of the NS3 protease (Xu et al., NS2-3 (NS2, NS3), NS4A, NS4B, NS5A, NS5B (Collett et 1997). The functions of NS4B and NS5A are not known. al., 1988a, 1991; Elbers et al., 1996; Meyers et al., 1992; NS5B represents the viral RNA-dependent RNA polymer- Ru¨menapf et al., 1993; Stark et al., 1993; Tautz et al., 1997; ase (Kao et al., 1999; Lai et al., 1999; Steffens et al., 1999; Xu et al., 1997). Npro represents a nonstructural (NS) Zhong et al., 1998). autoprotease, which releases itself from the polyprotein Hepaciviruses represent the closest relatives of pes- and thereby generates the N-terminus of the capsid tiviruses, particularly with regard to the genome organi- protein (C) (Stark et al., 1993; Wiskerchen et al., 1991). zation of the NS protein region and apparently also the rns The envelope glycoproteins E , E1, and E2, as well as function or functions of individual NS proteins (Rice, 1996). In addition, for HCV NS3 represents a serine

1 protease that catalyzes all cleavages required for the To whom reprint requests should be addressed at Institut fu¨r generation of NS4A, NS4B, NS5A, and NS5B (Barten- 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: schlager et al., 1993; Grakoui et al., 1993; Hijikata et al., [email protected]. 1993; Tomei et al., 1993; reviewed in Bartenschlager,

1997). Although processing at the 3/4A site is catalyzed sequence of BVDV NS3. Both proteases are stimulated in exclusively in cis, all other cleavages can be mediated in their activity by NS4A. NS3 of BVDV strictly depends on trans. The observed consensus sequence for the cleav- NS4A in its activity at the NS4B/5A and NS5A/5B sites age sites is: E/D-X-X-X-X-C/T 2 S/A (where X is any (Xu et al., 1997). The role of NS4A in the other NS3- amino acid and 2 symbolizes the cleavage site). The dependent cleavages, as well as the cofactor domain of catalytic triad of the protease is composed of histidine, NS4A, has not been studied so far. Our aim in the aspartic acid, and serine. Furthermore, a structurally present study was a comprehensive characterization of important Zn-binding motif was identified in the protease the pestiviral NS3 protease, its NS4A cofactor, and pro- domain (de Francesco et al., 1996; Kim et al., 1996; Love tease–cofactor interactions. et al., 1996; Stempniak et al., 1997). The minimal fragment of NS3 still capable of cleaving at all sites was shown to RESULTS consist of a 169-amino-acid peptide located in the N- Active center of the NS3 serine protease terminal region of the protein (Bartenschlager et al., 1994; Failla et al., 1995; Tanji et al., 1994). The NS3 protease is The models proposed for the NS3 protease predict an assisted by NS4A as a cofactor that stimulates the cleav- active role for serine at amino acid position 1752 (S1752) ages at the four sites to significantly different extents and for histidine at position 1658 (H1658) (Bazan and (Bartenschlager et al., 1994; Failla et al., 1994; Lin et al., Fletterick, 1989; Gorbalenya et al., 1989). Interestingly, 1994; Steinku¨hler et al., 1996; Tanji et al., 1995a). In NS4A, the models differed with respect to the third member of a central 12-amino-acid peptide was identified that pre- the catalytic triad by predicting aspartic acid at either serves cofactor activity (Bartenschlager et al., 1995b; Lin position 1686 (D1686) (Gorbalenya et al., 1989) or 1695 et al., 1995; Steinku¨hler et al., 1996; Tanji et al., 1995b). (D1695) (Bazan and Fletterick, 1989). From the proposed Coprecipitation analyses indicated a stable interaction residues only S1752 was experimentally verified by an between NS4A and the extreme N-terminus of NS3 (Bar- exchange to alanine (Mendez et al., 1998; Tautz et al., tenschlager et al., 1995a; Failla et al., 1995; Lin et al., 1996; Wiskerchen and Collett, 1991). 1994; Satoh et al., 1995). This interaction was confirmed We started our analysis of the NS3 protease of BVDV by the resolution of the three-dimensional structure of strain CP7 with the characterization of its active center in the crystallized HCV NS3 protease domain complexed a transient expression study by applying the T7 vaccinia with a synthetic NS4A peptide (Kim et al., 1996). virus system (Fuerst et al., 1986; Sutter et al., 1995). The In contrast to the NS3 protease of HCV, knowledge first set of mutants aimed at the identification of residues about the corresponding enzyme of pestiviruses is very that constitute the catalytic triad as well as at their limited. Two computer models, established for the pes- functional characterization. Mutations of S1752 to cys- tiviral enzyme (Bazan and Fletterick, 1989; Gorbalenya et teine (S1752C) or threonine (S1752T) were introduced to al., 1989), have been experimentally challenged so far determine whether either of these amino acids could only by the exchange of the putative active site serine; functionally replace S1752, which serves as nucleophile. mutation to alanine abolished the release of NS4A, The exchange of H1658 and aspartic acids at positions NS4B, NS5A, and NS5B from the BVDV polyprotein 1686/1695 by small nonpolar amino acids (glycine or (Wiskerchen and Collett, 1991). Identification of the other alanine, respectively) served to identify the other mem- members of the catalytic triad has not been approached bers of the catalytic triad. The mutations were initially experimentally. Mapping of the protease domain was tested in the context of a polyprotein fragment, extending attempted (Wiskerchen and Collett, 1991) but did not lead from the C-terminal region of E2 to the N-terminal part of to valid data because the activity of the NS2-3 fragments NS4B encoded by plasmid pE2*-4B* (Fig. 1 and Tautz et was determined at a site that is not processed by NS3 al., 1996). The release of authentic NS2, NS3, NS2-3, and (Petric et al., 1992; Xu et al., 1997). At the NS3/4A site, a NS4A from the polyprotein encoded by E2*-4B* (* indi- restriction to cis cleavage was postulated because pro- cates truncated proteins) has been demonstrated before cessing was not observed in trans; all other cleavages in the T7 vaccinia system (Tautz et al., 1996). Moreover, can be mediated by NS3 in trans (Wiskerchen and Col- the use of this short polyprotein fragment facilitated the lett, 1991). At the NS3-dependent cleavage sites of BVDV, detection of noncleaved protein species. In the S1752A a leucine residue is strictly conserved in the P1 position mutant, included as a control, the partial cleavage of (nomenclature according to Berger and Schlechter, NS2-3, which is independent of the NS3 protease, still 1970), and serine or alanine predominantly resides at the occurred (Tautz et al., 1996), whereas the NS3 protease- P1Ј position (Tautz et al., 1997; Xu et al., 1997). Thus the dependent cleavages at the NS3/4A, NS2-3/4A and sequences at the cleavage sites used by the NS3 pro- NS4A-4B* sites were abrogated. As a consequence, teases of BVDV and HCV differ significantly. Moreover, NS3-4B* and NS2-4B* with apparent molecular masses the overall degree of similarity between these enzymes of 92 and 150 kDa, respectively, appeared. These pro- is very low on amino acid level. In contrast to HCV, no teins could be precipitated by antisera specific for NS3 Zn-binding motif can be delineated from the primary or NS4 (Tautz et al., 1996, and Fig. 2, lanes 3 and 4). In the BVDV NS3 SERINE PROTEASE 353

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 mutant. Unexpectedly, a substantial amount of NS3 was used as a parameter to monitor the impairment of the still generated (Fig. 2, lane 7). The H1658A exchange in serine protease. contrast completely abrogated the generation of mature The S1752C exchange in construct pE2*-4B* led to an NS proteins (Fig. 2, lanes 9 and 10); this result was in line efficient release of NS3 and to the generation of a small with the postulated catalytic role of H1658 in the NS3 amount of NS3-4B* (Fig. 2, lane 5). By comparison, ex- protease. change S1752T caused an accumulation of NS3-4B*, Surprisingly, neither mutation D1686G (Fig. 2, lanes 11 which illustrates the severely reduced activity of this 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 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* S1752C and S1752T on the trans cleavage activity of the region of the BVDV polyprotein from transfected plasmids was protease. After the transient expression of pE2*-5B/ achieved by the use of a recombinant vaccinia virus expressing the T7 S1752C or pE2*-5B/S1752T, minor amounts of NS2-3 and RNA polymerase (MVA-T7pol; Sutter et al., 1995). The metabolically NS3 (data not shown), but neither NS4A, NS4B, NS5A, labeled proteins were immunoprecipitated with antisera against NS3 or nor NS5B, could be detected (Figs. 3A and 3B, lanes 3 NS4, respectively, and further analyzed by SDS–PAGE (Doucet and Trifaro, 1988) and fluorography. The transfected expression plasmids and 4, respectively). According to this assay system, the as well as the antisera used are indicated above the corresponding respective NS3 protease mutants exhibited only cleav- lanes. Mutations in the encoded CP7 polypeptides are indicated in the age activity at the NS3/4A site. names of the corresponding plasmids. Precipitates derived from cells Taken together, our results are in agreement with the infected with MVA-T7pol without plasmid transfection are separated in lane 13 (Mock). The positions of NS2-3, NS3, and NS3-4B* are indicated model of Gorbalenya et al. (1989) that predicted for the by arrowheads on the left. The positions of the mass standards NS3 protease a catalytic triad composed of H1658, ([14C]MW markers) are shown on the right; k ϭ kDa. D1686, and S1752. 354 TAUTZ, KAISER, AND THIEL

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 glutathi- one-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-NS3- 1760 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 demon- strates that the fusion partner had no influence on the obtained results (data not shown). Accordingly, the C- terminal 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 FIG. 3. NS3 protease mutants and processing of E2*-5B. (A) Detec- domain, we chose a strategy that allowed the expression tion of NS4A and NS4B. Immunoprecipitates obtained by the use of of N-terminally truncated NS3 molecules without any NS4 specific antiserum were separated by Tricine–SDS–PAGE (Scha¨g- ger and Jagow, 1987). Samples derived from cells infected with MVA- vector-derived amino acid at their N-termini. For this T7pol without plasmid transfection were separated in lane 7 (Mock). purpose, one ubiquitin monomer was inserted upstream Arrowheads indicate the position of NS4A and NS4B. The 38-kDa of the BVDV polyprotein. On expression, cellular ubiq- protein (lower band of the doublet) in lanes 2 and 6 most likely uitin-specific proteases (Jonnalagadda et al., 1989; represents noncleaved NS4A-4B (see also Fig. 5C). The amount of NS2 Mayer and Wilkinson, 1989; Rose, 1988) will cleave after detected on transfection of the different plasmids was used to stan- dardize transfection efficiencies (data not shown). The S1752A ex- the C-terminal amino acid of ubiquitin (glycine 76) and change was included as a control to show inhibition of NS3-dependent thereby generate the N-terminus of NS3. It has previ- cleavages. (B) Detection of NS5A-5B, NS5A, and NS5B. The transfected ously been demonstrated that this cleavage also occurs expression plasmids are depicted in the figure above the correspond- efficiently in the context of the BVDV polyprotein (Tautz et ing lanes. For immunoprecipitation, NS5-specific antisera were used. al., 1993). The BVDV polyprotein encoded by plasmid Arrowheads at the left indicate positions of NS5A-5B, NS5A, and NS5B. 1590 Precipitates derived from cells infected with MVA-T7pol without plas- pUb1590-5B starts with the first amino acid of NS3 (G ) mid transfection were separated in lane 7 (Mock). 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 rela- Characterization of the NS3 protease domain tive position of the first amino acid of the encoded BVDV For several BVDV strains, it has been experimentally polypeptide with respect to the viral polyprotein. After the demonstrated (Ku¨mmerer et al., 1998; Meyers et al., transfection of pUb1590-5B and pUb1592-5B, viral pro- 1998) or suggested (Mendez et al., 1998; Meyers and teins NS3, NS4A, NS4B, NS5A, and NS5B could be de- Thiel, 1996; Tautz et al., 1993) that the first amino acid of tected (Figs. 5B and 5C). The deletion of the 6 N-terminal NS3 is glycine 1590. Accordingly, authentic NS3 encom- amino acids resulted in the appearance of an additional passes amino acids 1590–2272 of the BVDV polyprotein protein of about 38 kDa, which could be precipitated by (Tautz et al., 1997; Xu et al., 1997). Our next aim was to antibodies against NS4 (Fig. 5C, lane 3). Due to its further define the protease domain of NS3. We started apparent molecular mass and the strong decline in the the corresponding analysis by the construction of ex- amount of NS4A and NS4B, this protein most likely rep- pression plasmids encoding C-terminally truncated NS3 resents noncleaved NS4A-4B. On the deletion of 20 N- molecules, which encompass 251, 211, or 171 amino terminal amino acids, only two mature viral proteins were BVDV NS3 SERINE PROTEASE 355

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 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 FIG. 4. C-terminal border of the NS3 protease domain. (A) Scheme of a suitable substrate. On the transfection of plasmid the used cDNA constructs. The C-terminally truncated NS3 derivatives pNS2*-3* (Fig. 6A), an NS3-specific antibody detected were expressed from plasmids pGST-NS3-1840, pGST-NS3-1800, or proteins NS2*-3* and NS3* (data not shown). The NS4A- 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 derived peptides were expressed as fusions to GST to C-terminus. The numbers in brackets indicate the amino acids of the allow their detection by Western blotting (Fig. 6C). As BVDV polyprotein encoded by the respective construct. (B) Western blot expected, the cotransfection of pNS2*-3* and pNS5A-5B- analysis of the GST-NS3* fusion proteins. Plasmids pGST-NS3-1840, myc in the absence of the cofactor NS4A did not lead to pGST-NS3-1800, or pGST-NS3-1760 were transfected into cells previously infected by MVA-T7pol virus. The respective cell lysates were detectable cleavage at the NS5A/5B site (Fig. 6B, lane 1). separated by Tricine–SDS–PAGE (Scha¨gger and Jagow, 1987) and an- After the cotransfection of plasmids pNS2*-3* and alyzed by Western blotting with a mouse mAb directed against GST pNS5A-5B-myc with pGST-NS4A fusion proteins encom- (Santa Cruz Biotechnology). The lanes combined in the figure were passing either complete NS4A together with a small derived from the same gel. Lysates of cells only infected with MVA- part of the NS4B coding region (pGST-NS4A-2349, T7pol virus were separated in lane 4 (Mock). The positions of the mass standards (prestained MW markers) are shown on the right; k ϭ 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 NS4A- 5B-myc substrate, the respective lysates were analyzed by Tricine– indicated at the left. In lane 5 (Mock), lysates of cells infected only with SDS–PAGE (Scha¨gger and Jagow, 1987) and Western blotting applying MVA-T7pol virus were separated. The lanes combined in the figure a c-myc–specific mAb (Invitrogen). The positions of unprocessed were derived from the same gel. On the right, the positions of the mass NS4A-5B-myc, NS5B-myc, and the cellular myc protein (c-myc) are standards (prestained MW markers) are shown; k ϭ kDa. 356 TAUTZ, KAISER, AND THIEL

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: ϩ, efficient cleavage; Ϫ, no cleavage; (ϩ), 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 ϭ 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 ϭ 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 NS4A truncated N-terminally by 9 amino acids still fragment with a 7-amino-acid deletion at its C-termi- assisted in NS5A-5B-myc cleavage (Fig. 6B, lane 8). As nus (pGST-NS4A-2329) (Fig. 6B, lane 4), NS5B-myc indicated by the very weak appearance of NS5B-myc was released. Larger C-terminal truncations (17 amino NS4A with a deletion of 18 amino acids from the acids or more) of NS4A abrogated its cofactor activity. N-terminus still retained residual cofactor activity (Fig. BVDV NS3 SERINE PROTEASE 357

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 ϭ 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 ϭ kDa.

6B, lane 9). The minimal NS4A fragment that preserves Interaction between NS4A and the N-terminal domain the cofactor function in cleavage at the NS5A/5B site of NS3 thus is probably composed of not more than amino In the HCV system, a stable interaction between the acids 2291–2329 even though a direct test with a NS4A cofactor domain and the N-terminal region of NS3 corresponding peptide was not performed. Compara- has been established (Bartenschlager et al., 1995a; Failla ble results were also obtained with NS4A fragments et al., 1995; Hijikata et al., 1993a,b; Kim et al., 1996; Lin et without GST-tag (data not shown). al., 1995; Satoh et al., 1995). It was intended to test 358 TAUTZ, KAISER, AND THIEL

ages can be mediated in trans (Wiskerchen and Col- lett, 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 FIG. 7. Coprecipitation of NS3 and NS4A. Proteins encoded by assume that an overall reduction in the enzymatic pUbNS3-4A and its derivatives were transiently expressed in the pres- activity due to the introduced mutations can result in ence of [35S]methionine/cysteine by using MVA-T7pol virus. Cell lysates the observed selective loss of trans cleavage activity. were prepared under nondenaturing conditions and subjected to im- The finding that serine and cysteine are interchange- munoprecipitation by an NS3-specific mAb. The obtained aggregates were denatured, separated by Tricine–SDS–PAGE, and processed for able as nucleophiles, at least to some extent, has al- fluorography. The positions of NS3 and NS4A are indicated. ready 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 whether a similar NS3/NS4A interaction occurs in the of picornaviruses and the 27-kDa NIa protease of the BVDV system. Accordingly, plasmids pUb1590-4A and its potyvirus tobacco etch virus (Dougherty et al., 1989; Law- derivatives encoding N-terminally truncated NS3-4A mol- son and Semler, 1991). It was surprising, however, that ecules were generated by the deletion of the NS4B-5B NS3 with the S1752T substitution exhibited a significant coding region from the series of plasmids based on residual protease activity at the NS3/4A site, which indi- pUb1590-5B. Proteins expressed in the presence of cates that the hydroxyl group of threonine serves in the [35S]methionine/cysteine by pUb1590-4A and its deriva- nucleophilic attack during proteolysis. There is only one tives were immunoprecipitated under nondenaturing other example of a serine protease where the exchange conditions with a monoclonal antibody (mAb) directed of the active site serine by threonine gave rise to an against NS3. SDS–PAGE analysis and fluorography of enzyme with substantial protease activity (10% of wild the precipitated proteins revealed that a 7-kDa protein type), namely, the capsid protease of Sindbis virus (Hahn coprecipitated with full-length NS3 (Fig. 7, lane 1). A and Strauss, 1990). This enzyme is an autoprotease that Western blot analysis carried out in parallel identified the catalyzes its release from the viral polyprotein, thereby 7-kDa protein as NS4A (data not shown). Although NS4A generating its own C-terminus. It was supposed that the could also be coprecipitated with the NS3 derivative, activity of the capsid protease with a threonine in the which was N-terminally truncated by 2 amino acids (Fig. active site is due to the intramolecular nature of the 7, lane 2), all NS3 species with larger N-terminal trunca- reaction and the very fast reaction kinetics of the wild- tions did not allow pulling down NS4A under the chosen type protease (Hahn and Strauss, 1990). Furthermore, for conditions (Fig. 7). These data are in line with an essen- the Sindbis virus capsid protease as well as the BVDV tial role of the N-terminal region of NS3 in the complex enzyme, the exchange of the active site aspartic acid did formation between NS3 and NS4A. not significantly reduce their cis cleavage activities. In addition to the similarities observed in this study with DISCUSSION regard to the biochemistry of these enzymes, it turned out recently that the three-dimensional structure of the The aim of the present study was the characterization NS3 protease of HCV, a close relative of pestiviruses, of the pestiviral NS3 protease. As a first step, the resi- shares its greatest structural similarity with the Sindbis dues constituting the catalytic triad were determined. virus capsid protease (Kim et al., 1996). These findings The obtained results are in agreement with the model of nicely corroborate the relationship between the de- Gorbalenya et al. (1989), in which the catalytic triad is scribed proteases, which remarkably was postulated composed of H1658, D1686, and S1752. There was no several years ago on the basis of computer-assisted indication for an essential role of D1695 in the protease sequence comparisons of flaviviruses, pestiviruses, and domain as proposed in an alternative model (Bazan and alphaviruses (Gorbalenya et al., 1989). Fletterick, 1990). According to our study, a 209-amino-acid fragment of The NS3 serine protease catalyzes two types of the BVDV NS3 serine protease is still capable of exerting cleavages. Although processing at the NS3/NS4A site the NS5A-NS5B cleavage. A protease domain of this size is believed to occur exclusively in cis, all other cleav- is in agreement with the data obtained for the NS3 BVDV NS3 SERINE PROTEASE 359

protease domains of HCV (169 amino acids) (Barten- MATERIALS AND METHODS schlager et al., 1994; Failla et al., 1995; Tanji et al., 1994) Cells and viruses and members of the genus Flavivirus (e.g., yellow fever virus, 181 amino acids; Chambers et al., 1990). Because BHK-21 cells were kindly provided by J. Cox (Federal fusions of GST to the N-terminus of NS3 had no delete- Research Center for Virus Diseases of Animals, Tu¨bingen, rious effect on the activity of the enzyme, the folding of Germany). Cells were grown in Dulbecco’s modified Eagle’s the NS3 protease domain can apparently occur indepen- medium (DMEM) supplemented with 10% fetal calf serum dent of the preceding peptide sequence. (FCS). The recombinant vaccinia virus expressing the T7 The sensitivity of the protease domain to N-terminal RNA polymerase [modified virus Ankara (MVA)-T7pol] was truncations and the stepwise loss of activity are remark- generously provided by G. Sutter (Institut fu¨r Molekulare able: a deletion of 6 N-terminal amino acids resulted in a Virologie, GSF-Forschungszentrum fu¨r Umwelt und Ge- ␤ strongly reduced activity at the NS4A/4B site (Figs. 5B sundheit GmbH, Oberschlei heim, Germany). and 5C). Larger N-terminal truncations of NS3 further Transient expression with the T7 vaccinia virus reduced the activity of the enzyme at the individual cleav- system age sites to different extents. The greatest tolerance to N-terminal truncation of the protease was observed for BHK-21 cells (5 ϫ 10 5, 3.5-cm dish) were infected with the cis cleavage and processing at the NS5A/5B site. MVA-T7pol at a multiplicity of infection of 5 in medium Interestingly, similar results have been obtained in the lacking FCS. After1hofincubation at 37°C, cells were HCV system for N-terminally truncated NS3 protease washed twice with medium lacking FCS followed by derivatives. Short deletions selectively interfered with DNA transfection with 2 ␮g plasmid DNA by the use of NS4A-NS4B and NS4B-NS5A cleavage, whereas more Superfect (Qiagen, Hilden, Germany) according to the extensive truncations of NS3 also inhibited cleavage at protocol of the manufacturer. For Western blot analysis, the NS5A/5B site and ultimately the cis cleavage at the the transfected cell cultures were harvested about 6 h NS3/4A site (reviewed in Bartenschlager, 1997). posttransfection (p.t.). Our coprecipitation study revealed an interaction be- Radioactive labeling of cells. At 2 h p.t., cells were tween BVDV proteins NS3 and NS4A, which was abro- washed two times with PBS and incubated with 1 ml of label medium lacking cysteine and methionine for 0.5 h gated by a deletion of the first 6 amino acids of NS3 (Fig. at 37°C. After the addition of 500 ␮Ci of [35S]methionine/ 7). This finding strongly suggests a role of the N-terminal cysteine ([35S]-Pro Mix; Amersham, Freiburg, Germany), domain of NS3 in NS4A binding. Interestingly, such a protein expression was allowed to proceed for an addi- truncated protease was proteolytically active at all sites. tional4hat37°C. After washing with PBS, cells were For HCV, binding of NS4A to the N-terminal region of NS3 stored at Ϫ70°C until they were further processed. has also been demonstrated by coprecipitation and was confirmed at the structural level (Kim et al., 1996). This Radioimmunoprecipitation and SDS–PAGE interaction could be abrogated by N-terminal truncations of NS3 (Bartenschlager et al., 1995a; Failla et al., 1995; Immunoprecipitation under denaturing conditions. Cell Lin et al., 1994; Satoh et al., 1995). N-terminally truncated extracts prepared in the presence of 2% SDS were di- ␮ as well as mutagenized NS3 derivatives could be iden- luted and incubated with 5 l of undiluted antiserum. For tified that show proteolytic activity despite impaired the formation of precipitates, protein A–agarose (Calbio- chem, La Jolla, CA) was used. SDS–PAGE analysis of NS4A binding. These data and the results obtained in proteins was performed by the use of Tricine gels this study suggest that a weak transient protease–cofac- (Scha¨gger and Jagow, 1987) or according to Doucet and tor interaction seems to be sufficient for activation of the Trifaro (1988). Gels were processed for fluorography us- NS3 proteases of HCV (for review, see de Francesco and 3 ing En Hance (New England Nuclear, Boston, MA). Steinku¨hler, 2000) and BVDV. Immunoprecipitation under nondenaturing conditions. Taken together, our study revealed striking parallels Cells were lysed with buffer IBP150 [20 mM Tris–HCl, pH between the NS3 proteases of BVDV and HCV, especially 8.0, 150 mM KCl, 1% Nonidet P-40, 1 mM EDTA, pH 8.0, 1 with respect to active site residues, size of protease mM EDTA, 0.1 mM Pefablock SC (Roche Diagnostics, domain, NS4A cofactor dependency, and NS3–NS4A in- Mannheim, Germany)] kept on ice for 5 min and slowly teraction. These findings again underline the fascinat- rotated for 30 min at 4°C. Insoluble parts were removed ingly close relationship of pestiviruses and hepacivi- by two consecutive centrifugations at 300 and 10,000 g. ruses. In addition, the similarities observed between the NS3-specific mAb 8.12.7 (Corapi et al., 1990) was bound capsid protease of Sindbis virus, a member of the genus via Fc-specific anti-mouse IgG (Sigma Chemical Co., St. Alphavirus, and the NS3 protease of a member of the Louis, MO) to protein A–agarose (Calbiochem) and Flaviviridae suggest a common ancestor for the serine cross-linked by diaminopimelic acid according to the proteases of these positive-strand RNA viruses. protocol of Harlow and Lane (1988). The cross-linked 360 TAUTZ, KAISER, AND THIEL antibody was incubated with the cleared cell lysates for pE2*-5B. Elongation of the BVDV CP7 cDNA in pC7.1 at 1 h at 4°C. Complexes were collected by centrifugation its 3Ј end by a fragment derived from construct pC7.3898 at 4000 g and washed once with IBP150 and twice with (Tautz et al., 1997) resulted in plasmid pE2*-5B. Thus the IBP500 (20 mM Tris–HCl, pH 8.0, 500 mM KCl, 1% Non- BVDV ORF encoded by this plasmid starts with codon idet P-40, 1 mM EDTA, pH 8.0, 1 mM EDTA, 0.1 mM 967 and ends with the authentic stop codon encom- Pefablock SC). After a final washing step with IBP150, the passed in the BVDV genome. precipitate was diluted in gel loading buffer containing pNS4A-5B-myc. This plasmid is based on pCITE-2A 2% SDS and 5% mercaptoethanol and boiled for 5 min. (Novagene, La Jolla, CA), which downstream of a T7 RNA polymerase promoter encompasses an internal ribo- Polymerase chain reaction (PCR) somal entry site (IRES) of the encephalomyocarditis virus (EMCV). The 5Ј end of the cDNA insert in this plasmid For PCR, a GENIUS thermocycler (Techne/thermo- was generated by PCR and starts downstream of the DUX, Wertheim, Germany) was used. Standard condi- vector-derived AUG codon with an alanine codon fol- tions used for DNA-based amplifications have been de- lowed by the BVDV ORF beginning with the codon for scribed previously (Tautz et al., 1999). serine 2274, the second amino acid of NS4A; the cDNA proceeds downstream to the NS5B gene. Into a PvuII site Nucleotide sequencing (nucleotides 12260–12265) in the 3Ј region of the 5B Sequencing was carried out with the Thermo Seque- gene, an EcoRV–PvuII fragment derived from pSecTag nase Cycle Sequencing Kit (Amersham Buchler, Braun- (Invitrogen, Groningen, Netherlands) was cloned; the re- schweig, Germany). The primers used for sequencing spective fragment encompassed part of the polylinker were labeled with infrared dye IRD41 (MWG-Biotech, and the coding sequence for an myc- and a His-tag Ebersberg, Germany). Analyses of sequencing gels were followed by a stop codon. In the polyprotein encoded by carried out with an automatic sequencer LI-COR 4000L pNS4A-5B-myc, accordingly the 6 C-terminal amino ac- (MWG-Biotech, Ebersberg, Germany). ids of NS5B are substituted by 34 pSecTag-derived amino acids, including the myc-epitope. Western blotting pGST-NS3-1760/1800/1840. The GST gene from pGEX-2T (Pharmacia) was amplified by PCR and inte- After SDS–PAGE proteins were transferred onto nitro- grated into the NcoI site of pCITE-2A by standard cloning cellulose membranes (Optitran BA-S83 Reinforced NC; techniques. Downstream of the GST gene, the resulting Schleicher & Schuell, Du¨ren, Germany). Blocking was plasmid, pCITE-GST, contains a singular NcoI site en- carried out with 2% dry milk in PBS for 1 h. The proteins compassing an in-frame AUG codon. After restriction were visualized by chemiluminescence (SuperSignal; with NcoI–XbaI, this vector was ligated to an NcoI–XbaI Pierce Chemical, Rockford, IL). fragment of pARC7 (Tautz et al., 1997). The resulting plasmid, pGST-NS3-2351, encoded for the CP7 polypro- Construction of the expression plasmids tein from amino acids 1587–2351. To generate the final All BVDV cDNAs used for the construction of the ex- constructs, PCR-derived fragments of the NS3 gene of pression plasmids throughout this study are derived from CP7 that encode stop codons downstream of serine BVDV strain CP7 (Meyers et al., 1996; Tautz et al., 1996). 1760, lysine 1800, or leucine 1840, respectively, were For PCR amplifications used in the construction of the used to substitute an HpaI–XbaI (nucleotides 5434–5439/ expression plasmids, the already described BVDV CP7 polylinker) fragment in pGST-NS3-2351. cDNA clones were used as templates (Meyers et al., pUb1590-5B. From plasmid FsubiNS3 (Tautz et al., 1996; Tautz et al., 1996, 1997). Numbering of nucleotides 1999), which encompasses the mouse ubiquitin gene or amino acids occurs throughout this report with re- (ubi), an NcoI (first codon of ubiquitin)–AgeI (BVDV nu- spect to the BVDV SD-1 sequence because the complete cleotides 5312–5316) fragment was used to replace a sequence of BVDV CP7 has not been published yet and corresponding NcoI–AgeI fragment of pC7.3898 (Tautz et SD-1 represents the only complete BVDV sequence de- al., 1997), which resulted in the final construct pUb1590- void of a strain-specific insertion (Deng and Brock, 1992). 5B. In this plasmid, the NS3 gene, starting with the codon pE2*-4B*. This plasmid has already been described for glycine 1590, is preceded by the 3Ј end of the ubiq- under the name pC7.1 (Tautz et al., 1996); in the same uitin gene, including an SacII site. To generate the de- publication, the generation of construct pC7PM1 was rivatives that encode N-terminally truncated NS3 mole- described. The latter construct is termed pE2*-4B*/ cules downstream of the ubiquitin, the SacII (3Ј end of S1752A in this report to obtain a uniform nomenclature. ubiquitin gene)–AgeI (BVDV nucleotides 5312–5316) frag- The other mutations in the active center of the NS3 ment was replaced by corresponding PCR-derived frag- protease were generated by standard PCR techniques ments; the sense primers used for the generation of the using oligonucleotide primers that encompass the indi- respective PCR products encompassed the 3Ј end of ubi, vidual nucleotide exchanges. including an SacII site and the 5Ј region of the truncated BVDV NS3 SERINE PROTEASE 361

NS3 gene. According to this scheme, plasmids pUb1592- teins carrying the myc-tag was achieved by a tag-spe- 5B, pUb1596-5B, pUb1596-5B, pUb1610-5B, pUb1625-5B, cific mAb purchased from Invitrogen. and pUb1635-5B were established. The plasmid names indicate the first amino acid of the BVDV polyprotein that ACKNOWLEDGMENTS is located downstream of ubiquitin. For cloning of pUb1590-4A, an NcoI–MscI (ubi start The authors thank Alexander E. Gorbalenya for critical reading of the codon/BVDV nucleotides 7136–7141) fragment of manuscript and Gregor Meyers and Karin Kegreiss for their support in pUb1590-5B was inserted into pCITE-2A restricted with the initial phase of this project. The authors are grateful to M. S. Collett NcoI–HincII. Into this plasmid, the CP7 cDNA down to for supplying the NS5-specific antiserum. This study was supported by Ј the SFB535 “Invasionsmechanismen und Replikationsstrategien von the 3 end of the NS4A gene (codon 2336) was inserted Krankheitserregern” from the “Deutsche Forschungsgemeinschaft.” together with a stop codon by PCR and standard cloning techniques. Constructs pUb1592-4A, pUb1596-4A, pUb1610-4A, pUb1625-4A, and pUb1635-4A were gener- REFERENCES ated as described for the derivatives of pUb1590-5B by Bartenschlager, R. (1997). Molecular targets in inhibition of hepatitis C the use of SacII and AgeI fragments. virus replication. Antiviral Chem. Chemother. 8, 281–301. pNS5A-5B-myc. For PCR amplification of the NS5A-B Bartenschlager, R., Ahlborn-Laake, L., Mous, J., and Jacobsen, H. (1993). region, pE2*-5B-myc served as DNA template. The re- Nonstructural protein 3 of the hepatitis C virus encodes a serine-type sulting PCR product encompassed a primer derived NcoI proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J. 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