Membrane Topology of Pestiviral Non-Structural Protein 2 and determination of the minimal autoprotease domain.

Pestiviruses like bovine viral diarrhea virus (BVDV) belong to the family Flaviviridae A distinctive feature of the Flaviviridae is the importance of non-structural (NS) proteins for RNA genome replication and virus morphogenesis. For pestiviruses, the NS2 protease-mediated release of NS3 is essential for RNA replication, whereas uncleaved NS2-3 is indispensable for producing viral progeny. Accordingly, in the pestiviral life cycle the switch from RNA replication to virion morphogenesis is temporally regulated by the extent of NS2-3 cleavage, which is catalyzed by the NS2 autoprotease. A detailed knowledge of the structural and functional properties of pestiviral NS2 and NS2-3 is mandatory for a better understanding of these processes.In the present study, we experimentally determined the membrane topology of NS2 of BVDV-1 strain NCP7 by the Substituted Cysteine Accessibility Method (SCAM) assay. According to the resulting model, the N terminus of NS2 resides in the ER lumen and is followed by three transmembrane segments (TM) and a cytoplasmic C-terminal protease domain. We used the resulting model for fine mapping of the minimal autoprotease domain. Only one TM segment was found to be essential for maintaining residual autoprotease activity. While the topology of pestiviral NS2 is overall comparable to the one of hepatitis C virus (HCV) NS2, our data also reveal potentially important differences between the two molecules. The improved knowledge about structural and functional properties of this protein will support future functional and structural studies on pestiviral NS2.ImportancePestiviral NS2 is central to the regulation of RNA replication and virion morphogenesis via its autoprotease activity. This activity is temporally regulated by the cellular DNAJC14 as a cofactor: while free NS3 is required for RNA replication as a component of the viral replicase, only uncleaved NS2-3 supports virion morphogenesis. For a better understanding of the underlying molecular interactions, topological and structural data are required. The topology-based determination of the minimal NS2-protease domain in the present study will facilitate future attempts to determine the structure of this unusual protease cofactor complex. In the hepatitis C virus system, NS2 functions as a hub in virion morphogenesis by interacting with structural as well as non-structural proteins. Our knowledge of the membrane topology will significantly support future detailed interaction studies for pestiviral NS2.

CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses.

Pestiviruses like bovine viral diarrhea virus (BVDV) are a threat to livestock. For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections, which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2, the activity of which depends on its cellular cofactor, DNAJC14. Since this chaperone is available in small amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cp BVDV strains arising mostly by RNA recombination show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses has been established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14 dependent. By generating bovine and porcine DNAJC14 knockout cells, we could show that (i) replication of 6 distinct noncp pestivirus species (A to D, F, and G) depends on DNAJC14, (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14, and (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as a pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses.IMPORTANCE Only noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.

Determination of Critical Requirements for Classical Swine Fever Virus NS2-3-Independent Virion Formation.

For members of the Flaviviridae, it is known that, besides the structural proteins, nonstructural (NS) proteins also play a critical role in virion formation. Pestiviruses, such as bovine viral diarrhea virus (BVDV), rely on uncleaved NS2-3 for virion formation, while its cleavage product, NS3, is selectively active in RNA replication. This dogma was recently challenged by the selection of gain-of-function mutations in NS2 and NS3 which allowed virion formation in the absence of uncleaved NS2-3 in BVDV type 1 (BVDV-1) variants encoding either a ubiquitin (Ubi) (NS2-Ubi-NS3) or an internal ribosome entry site (IRES) (NS2-IRES-NS3) between NS2 and NS3. To determine whether the ability to adapt to NS2-3-independent virion morphogenesis is conserved among pestiviruses, we studied the corresponding NS2 and NS3 mutations (2/T444-V and 3/M132-A) in classical swine fever virus (CSFV). We observed that these mutations were capable of restoring low-level NS2-3-independent virion formation only for CSFV NS2-Ubi-NS3. Interestingly, a second NS2 mutation (V439-D), identified by selection, was essential for high-titer virion production. Similar to previous findings for BVDV-1, these mutations in NS2 and NS3 allowed for low-titer virion production only in CSFV NS2-IRES-NS3. For efficient virion morphogenesis, additional exchanges in NS4A (A48-T) and NS5B (D280-G) were required, indicating that these proteins cooperate in NS2-3-independent virion formation. Interestingly, both NS5B mutations, selected independently for NS2-IRES-NS3 variants of BVDV-1 and CSFV, are located in the fingertip region of the viral RNA-dependent RNA polymerase, classifying this structural element as a novel determinant for pestiviral NS2-3-independent virion formation. Together, these findings will stimulate further mechanistic studies on the genome packaging of pestiviruses.IMPORTANCE For Flaviviridae members, the nonstructural proteins are essential for virion formation and thus exert a dual role in RNA replication and virion morphogenesis. However, it remains unclear how these proteins are functionalized for either process. In wild-type pestiviruses, the NS3/4A complex is selectively active in RNA replication, while NS2-3/4A is essential for virion formation. Mutations recently identified in BVDV-1 rendered NS3/4A capable of supporting NS2-3-independent virion morphogenesis. A comparison of NS3/4A complexes incapable/capable of supporting virion morphogenesis revealed that changes in NS3/NS4A surface interactions are decisive for the gain of function. However, so far, the role of the NS2 mutations as well as the accessory mutations additionally required in the NS2-IRES-NS3 virus variant has not been clarified. To unravel the course of genome packaging, the additional sets of mutations obtained for a second pestivirus species (CSFV) are of significant importance to develop mechanistic models for this complex process.

Characterization of the Determinants of NS2-3-Independent Virion Morphogenesis of Pestiviruses.

UNLABELLED: A peculiarity of the Flaviviridae is the critical function of nonstructural (NS) proteins for virus particle formation. For pestiviruses, like bovine viral diarrhea virus (BVDV), uncleaved NS2-3 represents an essential factor for virion morphogenesis, while NS3 is an essential component of the viral replicase. Accordingly, in natural pestivirus isolates, processing at the NS2-3 cleavage site is not complete, to allow for virion morphogenesis. Virion morphogenesis of the related hepatitis C virus (HCV) shows a major deviation from that of pestiviruses: while RNA replication also requires free NS3, virion formation does not depend on uncleaved NS2-NS3. Recently, we described a BVDV-1 chimera based on strain NCP7 encompassing the NS2-4B*-coding region of strain Osloss (E. Lattwein, O. Klemens, S. Schwindt, P. Becher, and N. Tautz, J Virol 86:427-437, 2012, doi:10.1128/JVI.06133-11). This chimera allowed for the production of infectious virus particles in the absence of uncleaved NS2-3. The Osloss sequence deviates in the NS2-4B* part from NCP7 in 48 amino acids and also has a ubiquitin insertion between NS2 and NS3. The present study demonstrates that in the NCP7 backbone, only two amino acid exchanges in NS2 (E1576V) and NS3 (V1721A) are sufficient and necessary to allow for efficient NS2-3-independent virion morphogenesis. The adaptation of a bicistronic virus encompassing an internal ribosomal entry site element between the NS2 and NS3 coding sequences to efficient virion morphogenesis led to the identification of additional amino acids in E2, NS2, and NS5B that are critically involved in this process. The surprisingly small requirements for approximating the packaging schemes of pestiviruses and HCV with respect to the NS2-3 region is in favor of a common mechanism in an ancestral virus. IMPORTANCE: For positive-strand RNA viruses, the processing products of the viral polyprotein serve in RNA replication as well as virion morphogenesis. For bovine viral diarrhea virus, nonstructural protein NS2-3 is of critical importance to switch between these processes. While free NS3 is essential for RNA replication, uncleaved NS2-3, which accumulates over time in the infected cell, is required for virion morphogenesis. In contrast, the virion morphogenesis of the related hepatitis C virus is independent from uncleaved NS2-NS3. Here, we demonstrate that pestiviruses can adapt to virion morphogenesis in the absence of uncleaved NS2-3 by just two amino acid exchanges. While the mechanism behind this gain of function remains elusive, the fact that it can be achieved by such minor changes is in line with the assumption that an ancestral virus already used this mechanism but lost it in the course of adapting to a new host/infection strategy.

Pestivirus virion morphogenesis in the absence of uncleaved nonstructural protein 2-3.

The family Flaviviridae contains three genera of positive-strand RNA viruses, namely, Flavivirus, Hepacivirus (e.g., hepatitis C virus [HCV]), and Pestivirus. Pestiviruses, like bovine viral diarrhea virus (BVDV), bear a striking degree of similarity to HCV concerning polyprotein organization, processing, and function. Along this line, in both systems, release of nonstructural protein 3 (NS3) is essential for viral RNA replication. However, both viruses differ significantly with respect to processing efficiency at the NS2/3 cleavage site and abundance as well as functional relevance of uncleaved NS2-3. In BVDV-infected cells, significant amounts of NS2-3 accumulate at late time points postinfection and play an essential but ill-defined role in the production of infectious virions. In contrast, complete cleavage of the HCV NS2-3 counterpart has been reported, and unprocessed NS2-3 is not required throughout the life cycle of HCV, at least in cell culture. Here we describe the selection and characterization of the first pestiviral genome with the capability to complete productive infection in the absence of uncleaved NS2-3. Despite the insertion of a ubiquitin gene or an internal ribosomal entry site between the NS2 and NS3 coding sequences, the selected chimeric BVDV-1 genomes gave rise to infectious virus progeny. In this context, a mutation in the N-terminal third of NS2 was identified as a critical determinant for efficient production of infectious virions in the absence of uncleaved NS2-3. These findings challenge a previously accepted dogma for pestivirus replication and provide new implications for virion morphogenesis of pestiviruses and HCV.

Hepatitis C virus NS2 is a protease stimulated by cofactor domains in NS3.

Chronic infection with hepatitis C virus (HCV) affects 130 million people worldwide and is a major cause of liver cirrhosis and liver cancer. After translation of the HCV RNA genome into a polyprotein, 2 viral proteases process its non-structural protein (NS) region. While the essential chymotrypsin-like serine protease NS3-4A mediates all cleavages downstream of NS3, the NS2-3 cysteine protease catalyzes a vital cleavage at the NS2/3 site. Protease activity of NS2-3 has been described to require, besides NS2, the N-terminal 181 aa of NS3. The latter domain corresponds to the NS3 serine protease domain and contains a structural Zn(2+)-binding site with functional importance for both viral proteases. The catalytic triad of the NS2-3 protease resides in NS2; the role of the NS3 part in proteolysis remained largely undefined. Here we report a basal proteolytic activity for NS2 followed by only 2 amino acids of NS3. Basal activity could be dramatically enhanced by the NS3 Zn(2+)-binding domain (NS3 amino acids 81-213) not only in cis but also in trans which, however, required a more extended N-terminal part of NS3 downstream of NS2 in cis. Thus, this study defines for the first time (i) NS2 as a bona fide protease, (ii) NS3 as its regulatory cofactor, and (iii) functional subdomains in NS3 that cooperate in NS2 protease activation. These findings give new mechanistic insights into function and regulation of the NS2 protease and have important implications for the development of anti-HCV therapeutics.

Molecular characterization of positive-strand RNA viruses: pestiviruses and the porcine reproductive and respiratory syndrome virus (PRRSV).

Molecular characterization has become an important tool for the analysis of viruses including their classification. The manuscript focuses on the molecular analysis of two members of the genus pestivirus (hog cholera virus, HCV and bovine viral diarrhea virus, BVDV) and of the recently discovered porcine reproductive and respiratory syndrome virus (PRRSV). The first protein encoded within the single large pestivirus ORF is a nonstructural protein with autoproteolytic activity. The cleavage site between the protease and the capsid protein p14 has been predicted previously, but recent experimental data indicate that processing occurs at a different site. The capsid protein is followed by a putative internal signal sequence and three glycoproteins which are part of the virion envelope. According to a new proposal for the nomenclature of the structural proteins of pestiviruses they are termed C, E0, E1 and E2. The genomes of BVDV pairs isolated from animals which came down with mucosal disease were analyzed. The genomes from cytopathogenic (cp) BVD viruses may contain insertions highly homologous to cellular sequences. In addition, cp BVDV may differ from its non cytopathogenic (noncp) counterpart by mere rearrangement of viral sequences. The disease PRRS, which emerged a few years ago, is caused by a single strand RNA virus; the viral genome is of positive polarity and has a size of 15 kb. Data concerning morphology, morphogenesis and virion composition suggested already that PRRSV belongs to a group of so-called arteriviruses which comprises equine arteritis virus (EAV), lactate dehydrogenase elevating virus (LDV) and simian hemorrhagic fever virus (SHFV). This conclusion has now been confirmed by analysis of genome organization, gene expression strategy and by comparison of deduced protein sequences.

Insertion of cellular sequences in the genome of bovine viral diarrhea virus.

The genomic sequences of four pestiviruses, two BVDV strains (Osloss and NADL, both of which are cytopathogenic) and two HCV strains, were analyzed. Comparative studies revealed the presence of small insertions of cellular sequences in the genomes of both BVDV strains; the insertions are located in a region coding for a nonstructural protein. Such insertions are not present in the HCV sequences. The insertion identified in BVDV Osloss encodes a complete ubiquitin-like element. The sequence inserted in the BVDV NADL genome shows no homology to a ubiquitin gene but is almost identical with another bovine mRNA sequence. Molecular characterization of a BVDV "pair", isolated from an animal with mucosal disease, led to the detection of a ubiquitin-like sequence in the genome of the cytopathogenic strain, but not of the noncytopathogenic strain. It is proposed that recombination between viral and cellular RNA leads to formation of cpBVDV genomes. This hypothesis has direct implications for the pathogenesis of mucosal disease.

The genetic basis for cytopathogenicity of pestiviruses.

Two biotypes of pestiviruses, cytopathogenic (cp) and noncp viruses, can be distinguished by their effects on tissue culture cells. Identification of cp bovine viral diarrhea virus (BVDV) has been frequently reported since antigenically closely related noncp and cp BVDV can be isolated from cattle with fatal mucosal disease (MD) and are called a virus pair. In contrast to the BVDV system, only few cp border disease virus (BDV) and cp classical swine fever virus (CSFV) strains have been described. Serological analyses and sequence comparison studies showed that cp pestiviruses arise from noncp viruses by mutation. Elaborate studies during the last 10 years revealed that in most cases RNA recombination is responsible for the generation of the cp viruses. Recent results showed a second way for the development of a cp pestivirus which is based on the introduction of a set of point mutations within the NS2 gene.

Translation from the internal ribosome entry site of bovine viral diarrhea virus is independent of the interaction with polypyrimidine tract-binding protein.

Translation of the pestiviral polyprotein is initiated cap independently at an internal site of the viral RNA, the internal ribosome entry site (IRES). We investigated the translation from the IRES of bovine viral diarrhea virus (BVDV) and the possible interaction of the unconventional cellular RNA-binding proteins, particularly of polypyrimidine tract-binding protein (PTB). The BVDV IRES is translationally active in rabbit reticulocyte lysate (RRL), and it is translated most efficiently at low concentrations of Mg(2+)- and K(+)-ions. In the UV cross-link assay, several proteins from RRL bind to the BVDV IRES, including proteins of 50, 65 and 72kDa, but no protein of 57kDa possibly corresponding to PTB, although PTB is endogenously present in RRL. However, the BVDV IRES can bind PTB weakly under certain conditions. Interestingly, in a functional depletion and add-back translation system, PTB does not enhance translation of BVDV, although PTB enhances translation of a picornavirus in this translation stimulation assay. These results indicate that PTB can bind the BVDV IRES RNA, but translation is independent of the action of PTB.

Dissection of a viral autoprotease elucidates a function of a cellular chaperone in proteolysis.

Replication of positive-strand RNA viruses involves translation of polyproteins which are proteolytically processed into functional peptides. These maturation steps often involve virus-encoded autoproteases specialized in generating their own N or C termini. Nonstructural protein 2 (NS2) of the pestivirus bovine viral diarrhea virus represents such an enzyme. Bovine viral diarrhea virus NS2 creates in cis its own C terminus and thereby releases an essential viral replication factor. As a unique feature, this enzyme requires for proteolytic activity stoichiometric amounts of a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) or its fragment Jiv90. To obtain insight into the structural organization of the NS2 autoprotease, the basis for its restriction to cis cleavage, as well as its activation by Jiv, we dissected NS2 into functional domains. Interestingly, an N-terminal NS2 fragment covering the active center of the protease, cleaved in trans an artificial substrate composed of a C-terminal NS2 fragment and two downstream amino acids. In the authentic NS2, the 4 C-terminal amino acids interfered with binding and cleavage of substrates offered in trans. These findings strongly suggest an intramolecular product inhibition for the NS2 autoprotease. Remarkably, the chaperone fragment Jiv90 independently interacted with protease and substrate domain and turned out to be essential for the formation of a protease/substrate complex that is required for cleavage. Thus, the function of the cell-derived protease cofactor Jiv in proteolysis is regulation of protease/substrate interaction, which ultimately results in positioning of active site and substrate peptide into a cleavage-competent conformation.

Persistence of bovine viral diarrhea virus is determined by a cellular cofactor of a viral autoprotease.

Polyprotein processing control is a crucial step in the life cycle of positive-strand RNA viruses. Recently, a vital autoprotease generating an essential viral replication factor was identified in such a virus, namely, the pestivirus bovine viral diarrhea virus. Surprisingly, the activity of this protease, which resides in nonstructural protein 2 (NS2), diminishes early after infection, resulting in the limitation of viral RNA replication. Here, we describe that a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) acts as a cofactor of the NS2 protease. Consumption of the intracellular Jiv pool is responsible for temporal regulation of protease activity: overexpression of Jiv interfered with regulation and correlated with increased accumulation of viral RNA; downregulation of the cellular Jiv level accelerated the decline of protease activity and reduced intracellular viral RNA levels and virion production. Accordingly, the amount of a cellular protein controls pestiviral replication by limiting the generation of active viral protease molecules and replication complexes. Importantly, this unique mechanism of replication control is essential for maintenance of the noncytopathogenic phenotype of the virus and thereby for its ability to establish persistent infections. These results add an entirely novel aspect to the understanding of the molecular basis of viral persistence.

Cytopathogenicity of pestiviruses: cleavage of bovine viral diarrhea virus NS2-3 has to occur at a defined position to allow viral replication.

Despite of highly divergent genome organizations, the N terminus of nonstructural protein 3 (NS3) is highly conserved between cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strains. Generation of NS3, often by NS2-3 cleavage, is a marker of cp BVDV. The significance of the cleavage site within NS2-3 for viral replication was addressed by the use of BVDV replicons. Our results demonstrate that elongation as well as truncation of NS3 strongly interfere with viral RNA replication. This finding strongly suggests that the observed conservation of the N terminus of NS3 between cp BVDV is caused by functional selection and not by the presence of a hotspot of recombination.

E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies.

The genes encoding pestivirus E2 and NS2-3 are separated by a sequence that encodes a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa (p7). It has been shown that cleavage between E2 and p7 is incomplete, resulting in proteins E2-p7, E2, and p7. We found no precursor-product relationship between E2-p7 and E2, which indicates a stable nature of E2-p7. To study the function of the E2-p7 region of the polyprotein, mutations were introduced into an infectious cDNA of bovine viral diarrhea virus (BVDV). When cleavage between E2 and p7 was abolished, viral RNA replication occurred; however, no infectious virus could be recovered. A corresponding result was obtained with a construct encompassing a large in-frame deletion of p7. To prevent synthesis of E2-p7, a translational stop codon was introduced after the last codon of the E2 gene and an internal ribosome entry site element followed by a signal peptide coding sequence was inserted upstream of the p7 gene. Transfection of RNA transcribed from the bicistronic construct led to the release of infectious virus particles. Thus, synthesis of E2-p7 is not essential for the generation of infectious virions. Cell lines constitutively expressing BVDV p7 and/or E2 were generated for complementation studies. Transfection of BVDV RNAs with point mutations or a deletion in the E2-p7 region into the complementing cell lines led to the generation of infectious virions. According to our studies, p7 as well as E2 can be complemented in trans.

NS3 serine protease of bovine viral diarrhea virus: characterization of active site residues, NS4A cofactor domain, and protease-cofactor interactions.

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.

Processing of poly-ubiquitin in the polyprotein of an RNA virus.

The RNA genome of several cytopathogenic (cp) strains of the pestivirus bovine viral diarrhea virus (BVDV) contains ubiquitin coding sequences (ucs). In noncytopathogenic BVDV strains, such insertions are missing. Gene expression of BVDV occurs via synthesis of a polyprotein which is subsequently processed by virus-encoded and cellular proteases. The insertion of ucs in the genomes of cpBVDV strains CP14 and Osloss leads to additional cleavages in the viral polyprotein. The respective processing events are mediated by cellular ubiquitin C-terminal hydrolases (UCHs). Release of monomeric ubiquitin (ubi) from the poly-ubi fragment encoded by CP14 is achieved by cleavage at the C-terminus as well as at the N-terminus of a complete ubi monomer. This result extends the current knowledge about poly-ubi processing. Processing of the polyprotein of CP14 and Osloss by UCHs generates an 80-kDa protein (p80), the marker protein of cpBVD viruses. Thus, the cp phenotype of both strains is apparently caused by the uptake of the ucs in the viral genome. Since cpBVDV strains arise in cattle in the course of a fatal disease, a direct linkage exists between the insertion of ucs and a lethal disease.

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.

Pathogenesis of mucosal disease, a deadly disease of cattle caused by a pestivirus.

BACKGROUND: Two biotypes of pestiviruses, cytopathogenic (cp) and non-cytopathogenic (noncp) viruses, are distinguished by their effects on tissue culture cells. In contrast to the bovine viral diarrhoea virus (BVDV) system, only a few cp border disease virus (BDV) and cp classical swine fever virus (CSFV) strains have been described. Antigenically closely related noncp and cp BVDV can be isolated from cattle with fatal mucosal disease (MD) and are called a virus pair. The generation of cp BVDV in an animal persistently infected with noncp BVDV is regarded as causative for the development of MD. OBJECTIVES: To analyse viral pairs of BVDV at the molecular level and thereby identify differences between the viruses of each pair. STUDY DESIGN: BVDV pairs were isolated from several animals coming down with MD; the genomes of the respective BVD viruses were sequenced on cDNA level. Studies concerning the polyprotein processing of each strain were carried out. RESULTS: Molecular analysis of BVDV pairs demonstrated a linkage between RNA recombination, generation of NS3 and the onset of fatal MD. CONCLUSION: The molecular analysis of BVDV pairs revealed that the respective cp strains arise by RNA recombination from noncp viruses.

Serine protease of pestiviruses: determination of cleavage sites.

The single-stranded genomic RNA of pestiviruses is of positive polarity and encompasses one large open reading frame of about 4,000 codons. The resulting polyprotein is processed co- and posttranslationally by virus-encoded and host cell proteases to give rise to the mature viral proteins. A serine protease residing in the nonstructural (NS) protein NS3 (p80) has been shown to be essential for the release of the NS proteins located downstream of NS3. In this report the NS3 serine protease-dependent cleavage sites for bovine viral diarrhea virus (BVDV) strain CP7 are described. Proteins used for analysis were generated in Escherichia coli or in eukaryotic cells by the use of the T7 vaccinia virus system. The N termini of NS4A, NS4B, NS5A, and NS5B were determined by protein sequencing. Analysis of the data obtained showed that leucine at P1 is the only position conserved for all cleavage sites. At P1' alanine is found at the NS4A-NS4B site, whereas serine resides at this position at the NS3-NS4A, NS4B-NS5A, and NS5A-NS5B cleavage sites. For all cleavage sites the amino acids found at P1 and P1' are conserved for different genotypes of pestiviruses, despite the high degree of sequence variation found between these viruses. It is therefore assumed that the cleavage sites determined for BVDV CP7 are representative of those for all pestiviruses.

Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs.

After cDNA cloning of the genome of bovine viral diarrhea virus (BVDV) isolate CP7, a full-length cDNA clone was constructed. RNA transcribed in vitro from this construct was shown to direct the generation of infectious BVDV upon transfection into bovine cells. To confirm the de novo generation of infectious BVDV from cloned cDNA a genetically tagged virus was constructed. In comparison with parental BVDV, the recombinant virus was slightly retarded in growth. The NS2 coding region of the CP7 genome contains a duplication of 27 nucleotides which is not present in the genome of its noncytopathogenic counterpart, NCP7. Exchange of a small fragment harboring this insertion against the corresponding part of the NCP7 sequence led to recovery of noncytopathogenic BVDV. Alteration of the construct by introduction of a fragment derived from a cytopathogenic BVDV defective interfering particle resulted in a chimeric defective interfering particle which exhibits a cytopathogenic phenotype. These findings confirm the hypothesis that the recombination-induced alterations in the genomes of cytopathogenic BVDV are responsible for the induction of cell lysis.