Norbert Tautz

Viral cytopathogenicity correlated with integration of ubiquitin-coding sequences☆

Abstract The RNA genomes of cytopathogenic bovine viral diarrhea virus (BVDV) isolates contain insertions highly homologous to cellular sequences. For two of them the insert was identified as ubiquitin coding sequence. The genome of BVDV Osloss contains exactly one ubiquitin gene monomer. In the case of BVDV CP1 the cellular insertion comprises one complete ubiquitin gene and part of a second monomer. The host cell-derived element in the CP1 genome is embedded in a large duplication of about 2.4 kb of viral sequences. Cellular insertion and duplication were not found in the genome of NCP1, the noncytopathogenic counterpart of CP1. These results strongly suggest that recombination between viral and cellular RNA is responsible for development of the cytopathogenic viruses, which is linked to pathogenesis of a lethal disease in cattle.

E2-p7 Region of the Bovine Viral Diarrhea Virus Polyprotein: Processing and Functional Studies

ABSTRACT 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.

Temporal Modulation of an Autoprotease Is Crucial for Replication and Pathogenicity of an RNA Virus

ABSTRACT Pestiviruses belong to the family Flaviviridae, and their genome is a single-stranded RNA of positive polarity encoding one large polyprotein which is further processed into mature proteins. Noncytopathogenic (noncp) strains of the pestivirus bovine viral diarrhea virus (BVDV) can establish persistent infection. In persistently infected animals, noncp BVDVs occasionally acquire mutations in viral nonstructural protein 2 (NS2) that give rise to cytopathogenic (cp) BVDV variants, and, eventually, lead to the onset of lethal disease. A molecular marker of cp BVDV infection is a high-level expression of the replicative NS3 protease/helicase that together with NS2 is derived from NS2-3. Here, we present evidence for NS2-3 autoprocessing by a newly identified cysteine protease in NS2 that is distantly related to the NS2-3 autoprotease of hepatitis C and GB viruses. The vital role of this autoprotease in BVDV infection was established, implying an essential function for NS3 in pestiviral RNA replication which cannot be supplied by its NS2-3 precursor. Accordingly, and contrary to a current paradigm, we detected almost complete cleavage of NS2-3 in noncp BVDV at early hours of infection. At 6 to 9 h postinfection, NS2-3 autoprocessing diminished to barely detectable levels for noncp BVDV but decreased only moderately for cp BVDV. Viral RNA synthesis rates strictly correlated with different NS3 levels in noncp and cp BVDV-infected cells, implicating the NS2 autoprotease in RNA replication control. The biotype-specific modulation of NS2-3 autoprocessing indicates a crucial role of the NS2 autoprotease in the pathogenicity of BVDV.

Cyclosporine A inhibits hepatitis C virus nonstructural protein 2 through cyclophilin A

Numerous anti‐hepatitis C virus (HCV) drugs targeting either the viral nonstructural 3 (NS3) protease or NS5B polymerase are currently in clinical testing. However, rapid resistance development is a major problem and optimal therapy will clearly require a combination of multiple mechanisms of action. Cyclosporine A (CsA) and its nonimmunosuppressant derivatives are among the more promising drugs under development. Based on work with subgenomic HCV replicons it has been thought that they act as NS5B‐inhibitors. In this study we show that CsA inhibits replication of full‐length HCV Japanese Fulminant Hepatitis (JFH1) genomes about 10‐fold more efficiently than subgenomic replicons. This effect is dependent on the presence of NS2 in the viral polyprotein and mediated through cellular cyclophilin A. NS2 is either an additional target for CsA‐dependent inhibition or modulates the antiviral activity against NS3 to NS5B proteins. CsA is thus the first anti‐HCV drug shown to act through NS2. Conclusion: CsA inhibits replication of JFH1 full‐length genomes much more efficiently than subgenomic replicons by targeting cleavage at the NS2/NS3 junction and possibly other nonreplication lifecycle steps. (HEPATOLOGY 2009.)

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.

Genetic Analysis of the Pestivirus Nonstructural Coding Region: Defects in the NS5A Unit Can Be Complemented in trans

ABSTRACT 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 cisor 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 becis 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.

A cellular J-domain protein modulates polyprotein processing and cytopathogenicity of a pestivirus.

ABSTRACT Pestiviruses are positive-strand RNA viruses closely related to human hepatitis C virus. Gene expression of these viruses occurs via translation of a polyprotein, which is further processed by cellular and viral proteases. Here we report the formation of a stable complex between an as-yet-undescribed cellular J-domain protein, a member of the DnaJ-chaperone family, and pestiviral nonstructural protein NS2. Accordingly, we termed the cellular protein Jiv, for J-domain protein interacting with viral protein. Jiv has the potential to induce intrans one specific processing step in the viral polyprotein, namely, cleavage of NS2-3. Efficient generation of its cleavage product NS3 has previously been shown to be obligatory for the cytopathogenicity of the pestiviruses. Regulated expression of Jiv in cells infected with noncytopathogenic bovine viral diarrhea virus disclosed a direct correlation between the intracellular level of Jiv, the extent of NS2-3 cleavage, and pestiviral cytopathogenicity.

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.

Persistence of Bovine Viral Diarrhea Virus Is Determined by a Cellular Cofactor of a Viral Autoprotease

ABSTRACT 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.

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.