Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.

Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication.     

Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.

Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position.     

Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins.

The proteolytic cleavages at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions of hepatitis C virus (HCV) polyprotein are effected by the virus-encoded serine protease contained within NS3. Using transient expression in HeLa cells of cDNA fragments that code for regions of the HCV polyprotein, we studied whether viral functions other than NS3 are required for proteolytic processing at these sites. We found that, in addition to NS3, a C-terminal 33-amino-acid sequence of the NS4A protein is required for cleavage at the NS3-NS4A and NS4B-NS5A sites and that it accelerates the rate of cleavage at the NS5A-NS5B junction. In addition, we show that NS4A can activate the NS3 protease when supplied in trans. Our data suggest that HCV NS4A may be the functional analog of flavivirus NS2B and pestivirus p10 proteins.     

Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.

Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B region.     

The hepatitis C virus NS4A protein: interactions with the NS4B and NS5A proteins.

Hepatitis C virus encodes a large polyprotein precursor that is proteolytically processed into at least 10 distinct products, in the order NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. A serine proteinase encoded in the N-terminal 181 residues of the NS3 nonstructural protein is responsible for cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonstructural region. NS4A, a 54-residue nonstructural protein which forms a stable complex with the NS3 proteinase, is required as a cofactor for cleavage at the 3/4A and 4B/5A sites and enhances processing at the 4A/4B and 5A/5B sites. Recently reported crystal structures demonstrated that NS4A forms an integral part of the NS3 serine proteinase. In this report, we present evidence that NS4A forms a nonionic-detergent-stable complex with the NS4B5A polyprotein substrate, which may explain the requirement of NS4A for the 4B/5A cleavage. Isoleucine-29 of NS4A, which has been previously shown to be essential for its proteinase cofactor activity and formation of the NS3 complex, was found to be important for the interaction between NS4A and the NS4B5A substrate. In addition, two more hydrophobic residues in the NS4A central region (valine-23 and isoleucine-25) were also shown to be essential for the cofactor activity and for the interaction with either the NS3 proteinase or the NS4B5A polyprotein substrate. Finally, the possible mechanisms by which these viral proteins interact with each other are discussed.     

Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics.

The hepatitis C virus H strain (HCV-H) polyprotein is cleaved to produce at least 10 distinct products, in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. An HCV-encoded serine proteinase activity in NS3 is required for cleavage at four sites in the nonstructural region (3/4A, 4A/4B, 4B/5A, and 5A/5B). In this report, the HCV-H serine proteinase domain (the N-terminal 181 residues of NS3) was tested for its ability to mediate trans-processing at these four sites. By using an NS3-5B substrate with an inactivated serine proteinase domain, trans-cleavage was observed at all sites except for the 3/4A site. Deletion of the inactive proteinase domain led to efficient trans-processing at the 3/4A site. Smaller NS4A-4B and NS5A-5B substrates were processed efficiently in trans; however, cleavage of an NS4B-5A substrate occurred only when the serine proteinase domain was coexpressed with NS4A. Only the N-terminal 35 amino acids of NS4A were required for this activity. Thus, while NS4A appears to be absolutely required for trans-cleavage at the 4B/5A site, it is not an essential cofactor for serine proteinase activity. To begin to examine the conservation (or divergence) of serine proteinase-substrate interactions during HCV evolution, we demonstrated that similar trans-processing occurred when the proteinase domains and substrates were derived from two different HCV subtypes. These results are encouraging for the development of broadly effective HCV serine proteinase inhibitors as antiviral agents. Finally, the kinetics of processing in the nonstructural region was examined by pulse-chase analysis. NS3-containing precursors were absent, indicating that the 2/3 and 3/4A cleavages occur rapidly. In contrast, processing of the NS4A-5B region appeared to involve multiple pathways, and significant quantities of various polyprotein intermediates were observed. NS5B, the putative RNA polymerase, was found to be significantly less stable than the other mature cleavage products. This instability appeared to be an inherent property of NS5B and did not depend on expression of other viral polypeptides, including the HCV-encoded proteinases.     

Cellular sequences in pestivirus genomes encoding gamma-aminobutyric acid (A) receptor-associated protein and Golgi-associated ATPase enhancer of 16 kilodaltons

The presence of cellular protein coding sequences within viral RNA genomes is a unique and particularly interesting feature of cytopathogenic (cp) pestiviruses. Here we report the identification and characterization of two novel cellular sequences in the genomes of cp bovine viral diarrhea virus (BVDV) strains. In BVDV strain CP X604, we detected a duplication of the genomic region encoding NS3, NS4A, and part of NS4B, together with an insertion of sequences that code for cellular gamma-aminobutyric acid (A) receptor-associated protein [GABA(A)-RAP]. Transient-expression studies showed that the GABA(A)-RAP sequence leads to additional processing of the viral polyprotein and thereby to the expression of nonstructural protein NS3. Transfection of bovine cells with RNA transcribed from an infectious cDNA clone revealed that the GABA(A)-RAP-encoding insertion together with the duplicated viral sequences constitutes the genetic basis for the cytopathogenicity of strain CP X604. Surprisingly, molecular analysis of another cp BVDV strain (CP 721) resulted in the identification of a cellular Golgi-associated ATPase enhancer of 16 kDa (GATE-16)-encoding insertion together with duplicated viral sequences. To our knowledge, the genomes of CP X604 and CP 721 are the first viral RNAs found with cellular sequences encoding GABA(A)-RAP and GATE-16, respectively. Interestingly, the two cellular proteins belong to a family of eukaryotic proteins involved in various intracellular trafficking processes. Processing after the C-terminal glycine residue of GABA(A)-RAP and GATE-16 by cellular proteases is essential for covalent attachment to target molecules. Accordingly, it can be assumed that these cellular proteases also recognize the cleavage sites in the context of the respective viral polyproteins and thereby lead to the generation of NS3, the marker protein of cp BVDV.     

Noncytopathogenic pestivirus strains generated by nonhomologous RNA recombination: alterations in the NS4A/NS4B coding region

Several studies have demonstrated that cytopathogenic (cp) pestivirus strains evolve from noncytopathogenic (noncp) viruses by nonhomologous RNA recombination. In addition, two recent reports showed the rapid emergence of noncp Bovine viral diarrhea virus (BVDV) after a few cell culture passages of cp BVDV strains by homologous recombination between identical duplicated viral sequences. To allow the identification of recombination sites from noncp BVDV strains that evolve from cp viruses, we constructed the cp BVDV strains CP442 and CP552. Both harbor duplicated viral sequences of different origin flanking the cellular insertion Nedd8*; the latter is a prerequisite for their cytopathogenicity. In contrast to the previous studies, isolation of noncp strains was possible only after extensive cell culture passages of CP442 and CP552. Sequence analysis of 15 isolated noncp BVDVs confirmed that all recombinant strains lack at least most of Nedd8*. Interestingly, only one strain resulted from homologous recombination while the other 14 strains were generated by nonhomologous recombination. Accordingly, our data suggest that the extent of sequence identity between participating sequences influences both frequency and mode (homologous versus nonhomologous) of RNA recombination in pestiviruses. Further analyses of the noncp recombinant strains revealed that a duplication of 14 codons in the BVDV nonstructural protein 4B (NS4B) gene does not interfere with efficient viral replication. Moreover, an insertion of viral sequences between the NS4A and NS4B genes was well tolerated. These findings thus led to the identification of two genomic loci which appear to be suited for the insertion of heterologous sequences into the genomes of pestiviruses and related viruses.     

Activity of purified hepatitis C virus protease NS3 on peptide substrates.

The protease domain of the hepatitis C virus (HCV) protein NS3 was expressed in Escherichia coli, purified to homogeneity, and shown to be active on peptides derived from the sequence of the NS4A-NS4B junction. Experiments were carried out to optimize protease activity. Buffer requirements included the presence of detergent, glycerol, and dithiothreitol, pH between 7.5 and 8.5, and low ionic strength. C- and N-terminal deletion experiments defined a peptide spanning from the P6 to the P4' residue as a suitable substrate. Cleavage kinetics were subsequently measured by using decamer P6-P4' peptides corresponding to all intermolecular cleavage sites of the HCV polyprotein. The following order of cleavage efficiency, in terms of kcat/Km, was determined: NS5A-NS5B > NS4A-NS4B >> NS4B-NS5A. A 14-mer peptide containing residues 21 to 34 of the protease cofactor NS4A (Pep4A 21-34), when added in stoichiometric amounts, was shown to increase cleavage rates of all peptides, the largest effect (100-fold) being observed on the hydrolysis of the NS4B-NS5A decamer. From the kinetic analysis of cleavage data, we conclude that (i) primary structure is an important determinant of the efficiency with which each site is cleaved during polyprotein processing, (ii) slow cleavage of the NS4B-NS5A site in the absence of NS4A is due to low binding affinity of the enzyme for this site, and (iii) formation of a 1:1 complex between the protease and Pep4A 21-34 is sufficient and required for maximum activation.     

Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A

The hepatitis C virus nonstructural 3 protein (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. The serine protease activity is required for proteolytic processing at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B polyprotein cleavage sites. NS3 forms a complex with NS4A, a 54-residue polypeptide that was shown to act as an essential cofactor of the NS3 protease. We have expressed in Escherichia coli the NS3-NS4A precursor; cleavage at the junction between NS3 and NS4A occurs during expression in the bacteria cells, resulting in the formation of a soluble noncovalent complex with a sub-nanomolar dissociation constant. We have assessed the minimal ionic strength and detergent and glycerol concentrations required for maximal proteolytic activity and stability of the purified NS3-NS4A complex. Using a peptide substrate derived from the NS5A-NS5B junction, the catalytic efficiency (kcat/Km) of NS3-NS4A-associated protease under optimized conditions was 55 000 s-1 M-1, very similar to that measured with a recombinant complex purified from eukaryotic cells. Dissociation of the NS3-NS4A complex was found to be fully reversible. No helicase activity was exhibited by the purified NS3-NS4A complex, but NS3 was fully active as a helicase upon dissociation of NS4A. On the other hand, both basal and poly(U)-induced NTPase activity and ssRNA binding activity associated with the NS3-NS4A complex were very similar to those exhibited by NS3 alone. Therefore, NS4A appears to uncouple the ATPase/ssRNA binding and RNA unwinding activities associated with NS3.     

Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease.

The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation. Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produced uncleaved polyproteins. These findings indicate that NS2B is required for processing of the downstream nonstructural proteins, including the NS3/NS4A and NS4B/NS5 junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavage site follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 was cleaved at the NS4A/NS4B junction in the absence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5 expressed only the polyprotein, earlier results demonstrated that cleavage at the NS4A/NS4B junction occurred when an analogous recombinant, vNS3-NS4A-84%NS4B, was expressed. Thus, it appears that uncleaved NS3 plus NS5 inhibit NS4A/NS4B cleavage presumably because the putative signal sequence is not accessible for recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with vNS2B-30%NS3 or with vNS2B plus v30%NS3. These results indicate that cleavage at the NS4B/NS5 junction can be mediated by NS2B and NS3 in trans.     

Cell-derived sequences in the N-terminal region of the polyprotein of a cytopathogenic pestivirus

Efficient proteolytic release of nonstructural protein 3 (NS3) from the viral polyprotein is considered to be crucial for the cytopathogenicity of pestiviruses. Here we describe a novel cytopathogenic (cp) bovine viral diarrhea virus strain (BVDV CP8) with a complex insertion composed of viral and cell-derived sequences, including two fragments of the cellular J-domain protein Jiv (J-domain protein interacting with viral protein) located in the N-terminal region of the polyprotein. BVDV CP8 expresses a Jiv fusion protein of 513 amino acids in addition to a complete set of viral proteins. This protein has the capacity to induce NS2-3 cleavage in trans. Accordingly, CP8 is a representative of a novel type of cp pestivirus with a cp-specific mutation located outside of the NS2-3 gene.     

Genetic analysis of the pestivirus nonstructural coding region: defects in the NS5A unit can be complemented in trans

The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5' and 3' untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in cis or in trans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in trans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to be cis dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery.     

Inhibition of Sphingosine Kinase by Bovine Viral Diarrhea Virus NS3 Is Crucial for Efficient Viral Replication and Cytopathogenesis

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in diverse cellular functions including survival, proliferation, tumorigenesis, inflammation, and immunity. Sphingosine kinase (SphK) contributes to these functions by converting sphingosine to S1P. We report here that the nonstructural protein NS3 from bovine viral diarrhea virus (BVDV), a close relative of hepatitis C virus (HCV), binds to and inhibits the catalytic activity of SphK1 independently of its serine protease activity, whereas HCV NS3 does not affect SphK1 activity. Uncleaved NS2-3 from BVDV was also found to interact with and inhibit SphK1. We suspect that inhibition of SphK1 activity by BVDV NS3 and NS2-3 may benefit viral replication, because SphK1 inhibition by small interfering RNA, chemical inhibitor, or overexpression of catalytically inactive SphK1 results in enhanced viral replication, although the mechanisms by which SphK1 inhibition leads to enhanced viral replication remain unknown. A role of SphK1 inhibition in viral cytopathogenesis is also suggested as overexpression of SphK1 significantly attenuates the induction of apoptosis in cells infected with cytopathogenic BVDV. These findings suggest that SphK is targeted by this virus to regulate its catalytic activity.Bovine viral diarrhea virus (BVDV)² is an enveloped, positive-sense single-stranded RNA virus classified in the genus Pestivirus of the family Flaviviridae. BVDV establishes persistent infections in cattle populations worldwide. Because BVDV shares virological and molecular properties with the Flaviviridae family member hepatitis C virus (HCV), which chronically infects an estimated 200 million patients worldwide (1), BVDV is regarded as a surrogate model for HCV (2). Both HCV and BVDV encode a single large precursor polyprotein that is processed by cellular and viral proteases into mature structural and nonstructural (NS) proteins.BVDV NS3 exhibits serine protease and helicase/ATPase activities that require its cofactor NS4A (3). NS3/4A protease is essential for generating mature NS proteins that are required for viral replication. HCV NS3/4A is well characterized and has been shown to suppress type-I interferons by cleaving the cellular interferon mediators IPS-1 and TRIF (4, 5). However, neither interferon suppression nor cellular targets have been identified for the BVDV NS3/4A protease (6).Lytic and persistent BVDV infections depend on the virus biotype. Cytopathogenic (CP) BVDV causes cytopathic effects via apoptosis, whereas noncytopathogenic (NCP) BVDV does not induce obvious changes in cell morphology and viability. These features are distinguished by NS2-3 processing differences; free NS3 produced by NS2-3 cleavage is generated continuously following CP BVDV infections, whereas NS3 is detected only until ∼9 h postinfection (p.i.) for NCP BVDV due to down-regulation of NS2-3 cleavage by this biotype (7). The CP biotype is characterized by dramatic up-regulation of viral RNA synthesis that could be correlated with the induction of cytopathic effect (7–9). Because free NS3, but not NS2-3, can form an active viral replicase complex with other NS proteins, increased viral RNA synthesis promoted through the release of free NS3 has been suggested to be a determinant of the characteristic lytic phenotype of CP BVDV infections (10). However, little is known about the regulation of cellular signaling by BVDV NS2-3, NS3, and NS3/4A, which is crucial for the control of both viral replication and biotype.Recent studies on the mechanisms of viral replication revealed that HCV RNA synthesis occurs on a lipid raft membrane structure where the active viral replicase complex is found (11, 12). The significance of the lipid raft as a scaffold for viral replication is further demonstrated by the identification of a novel HCV replication inhibitor, NA255, which prevents the biosynthesis of sphingolipids, the major components of lipid rafts (13). Administration of NA255 results in disruption of the HCV replicase complexes from the lipid rafts. This report proposes that the interaction between HCV NS5B and sphingomyelin on lipid rafts plays a crucial role for HCV RNA replication. Cellular sphingolipid metabolism is regulated by a large number of converting enzymes that maintain a homeostasis (14) but viral mechanisms that affect the sphingolipid metabolism to facilitate viral replication have yet to be identified.In a search for potential host proteins that interact with BVDV NS3, we identified sphingosine kinase 1 (SphK1) as a binding partner of NS3 using the yeast two-hybrid system. SphK1 is a lipid kinase that catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), a bioactive sphingolipid implicated in diverse cellular functions, including proliferation, survival, tumorigenesis, development, inflammation, and immunity (14, 15). Here, we analyze the biological significance of the SphK1 interaction with NS3, NS2-3, and NS3/4A. Using purified recombinant SphK1 and NS3, SphK activity was inhibited by NS3 in a dose-dependent manner, independently of its serine protease activity. The inhibition appears to be specific for BVDV NS3 because HCV NS3 had no effect on SphK activity. Using specific chemical inhibitors, small interfering RNA (siRNA), and a catalytically inactive mutant of SphK1, we investigated the significance of SphK inhibition in the viral replication. The present study is the first report demonstrating that SphK1 is targeted by a virus to inhibit its catalytic activity, and this mechanism may contribute to the efficient replication and pathogenesis of BVDV.     

Azapeptides as inhibitors of the hepatitis C virus NS3 serine protease

Truncation and substitution SAR studies of azapeptide-based inhibitors of the Hepatitis C virus (HCV) NS3 serine protease have been performed. These azapeptides were designed from the HCV polyprotein's NS5A-NS5B trans cleavage junction and contained an azaamino acid residue at the P1 position. These azapeptides exhibited predominantly non-acylating, competitive inhibition, contrary to classical azapeptides.     

Secondary structure of the 5'' nontranslated regions of hepatitis C virus and pestivirus genomic RNAs.

The RNA genomes of human hepatitis C virus (HCV) and the animal pestiviruses responsible for bovine viral diarrhea (BVDV) and hog cholera (HChV) have relatively lengthy 5' nontranslated regions (5'NTRs) sharing short segments of conserved primary nucleotide sequence. The functions of these 5'NTRs are poorly understood. By comparative sequence analysis and thermodynamic modeling of the 5'NTRs of multiple BVDV and HChV strains, we developed models of the secondary structures of these RNAs. These pestiviral 5'NTRs are highly conserved structurally, despite substantial differences in their primary nucleotide sequences. The assignment of similar structures to conserved segments of primary nucleotide sequence present in the 5'NTR of HCV resulted in a model of the secondary structure of the HCV 5'NTR which was refined by determining sites at which synthetic HCV RNA was cleaved by double- and single-strand specific RNases. These studies indicate the existence of a large conserved stem-loop structure within the 3' 200 bases of the 5'NTRs of both HCV and pestiviruses which corresponds to the ribosomal landing pad (internal ribosomal entry site) of HCV. This structure shows little relatedness to the ribosomal landing pad of hepatitis A virus, suggesting that these functionally similar structures may have evolved independently.     

Isolation and characterization of noncytopathic pestivirus mutants reveals a role for nonstructural protein NS4B in viral cytopathogenicity

Isolates of bovine viral diarrhea virus (BVDV), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. The cp viruses also differ from ncp viruses by the production of viral nonstructural protein NS3. However, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. Here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. A bicistronic BVDV (cp strain NADL) was created that expressed puromycin acetyltransferase as a dominant selectable marker. This bicistronic virus exhibited slightly slower growth kinetics and smaller plaques than NADL but remained cp. A number of independent ncp variants were isolated by puromycin selection. Remarkably, these ncp variants produced NS3 and viral RNA at levels comparable to those of the cp parent. Sequence analyses uncovered no change in NS3, but for all ncp variants a Y2441C substitution at residue 15 of NS4B was found. Introduction of the Y2441C substitution into the NADL or bicistronic cp viruses reconstituted the ncp phenotype. Y2441 is highly conserved among pestiviruses and is located in a region of NS4B predicted to be on the cytosolic side of the endoplasmic reticulum membrane. Other engineered substitutions for Y2441 also affected viral cytopathogenicity and viability, with Y2441V being cp, Y2441A being ncp, and Y2441D rendering the virus unable to replicate. The ncp substitutions for Y2441 resulted in slightly increased levels of NS2-3 relative to NS3. We also showed that NS3, NS4B, and NS5A could be chemically cross-linked in NADL-infected cells, indicating that they are associated as components of a multiprotein complex. Although the mechanism remains to be elucidated, these results demonstrate that mutations in NS4B can attenuate BVDV cytopathogenicity despite NS3 production.     

Establishment and Characterization of Cytopathogenic and Noncytopathogenic Pestivirus Replicons

Defective interfering particles (DIs) of bovine viral diarrhea virus (BVDV) have been identified and shown to be cytopathogenic (cp) in the presence of noncytopathogenic (noncp) helper virus. Moreover, a subgenomic (sg) RNA corresponding in its genome structure to one of those BVDV DIs (DI9) was replication competent in the absence of helper virus. We report here that an sg BVDV replicon which encodes from the viral proteins only the first three amino acids of the autoprotease N(pro) in addition to nonstructural (NS) proteins NS3 to NS5B replicates autonomously and also induces lysis of its host cells. This demonstrates that the presence of a helper virus is not required for the lysis of the host cell. On the basis of two infectious BVDV cDNA clones, namely, BVDV CP7 (cp) and CP7ins- (noncp), bicistronic replicons expressing proteins NS2-3 to NS5B were established. These replicons express, in addition to the viral proteins, the reporter gene encoding beta-glucuronidase; the release of this enzyme from transfected culture cells was used to monitor cell lysis. Applying these tools, we were able to show that the replicon derived from CP7ins- does not induce cell lysis. Accordingly, neither N(pro) nor any of the structural proteins are necessary to maintain the noncp phenotype. Furthermore, these sg RNAs represent the first pair of cp and noncp replicons which mimic complete BVDV CP7 and CP7ins- with respect to cytopathogenicity. These replicons will facilitate future studies aimed at the determination of the molecular basis for the cytopathogenicity of BVDV.