Cytopathogenicity of classical Swine Fever virus correlates with attenuation in the natural host

For the important livestock pathogens classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV), cytopathogenic (cp) and non-cp viruses are distinguished according to the induction of apoptosis in infected tissue culture cells. However, it is currently unknown whether cp CSFV differs from non-cp CSFV with regard to virulence in the acutely infected host. In this study, we generated helper virus-independent CSFV Alfort-Jiv, which encompasses sequences encoding domain Jiv-90 of cellular J-domain protein interacting with viral protein (Jiv). Expanding the knowledge of BVDV, our results suggest that Jiv acts as a regulating cofactor for the nonstructural (NS) protein NS2 autoprotease of CSFV and initiates NS2-3 cleavage in trans. For Alfort-Jiv, the resulting expression of large amounts of NS3 correlated with increased viral RNA synthesis and viral cytopathogenicity. Moreover, both cp Alfort-Jiv and the parental non-cp CSFV strain Alfort-p447 efficiently replicate in cell culture. Animal experiments demonstrated that in contrast to parental non-cp Alfort-p447, infection with cp Alfort-Jiv did not cause disease in pigs but induced high levels of neutralizing antibodies, thus elucidating that cp CSFV is highly attenuated in its natural host. In contrast to virulent Alfort-p447, the attenuated CSFV strain Alfort-Jiv induces the expression of cellular Mx protein in porcine PK-15 cells. Accordingly, the remarkable difference between cp and non-cp CSFV with regard to the ability to cause classical swine fever in pigs correlates with different effects of cp and non-cp CSFV on cellular antiviral defense mechanisms.     

Insertion of a Sequence Encoding Light Chain 3 of Microtubule-Associated Proteins 1A and 1B in a Pestivirus Genome: Connection with Virus Cytopathogenicity and Induction of Lethal Disease in Cattle

Pestiviruses represent the first RNA viruses for which recombination with cellular protein-coding sequences has been reported. As a result of such recombinations cytopathogenic (cp) pestiviruses can develop from noncytopathogenic (noncp) viruses. In the case of bovine viral diarrhea virus (BVDV), the generation of cp mutants is linked to the induction of the lethal syndrome mucosal disease (MD) in cattle. The cp BVDV JaCP was isolated from an animal which had come down with MD. The genome of JaCP contains a novel kind of cellular insertion (LC3*) which is flanked by duplicated pestivirus sequences. Neither insertion nor duplication is present in the genome of the accompanying noncp virus JaNCP. As part of the viral polyprotein, the insertion in the JaCP genome is translated into a polypeptide almost identical to a fragment of light chain 3, a subunit of the microtubule-associated proteins 1A and 1B from the rat. Transient-expression studies revealed that the LC3* sequence is able to induce an additional cleavage of the viral polyprotein. The respective cleavage occurs directly downstream of the LC3*-encoded sequence and is not dependent on the NS3 serine protease. Insertion of LC3* into an infectious noncp pestivirus cDNA clone without duplicated viral sequences resulted in recovery of a defective cp virus able to replicate only in the presence of a noncp helper virus. In contrast, introduction of both insertion and duplication led to an autonomously replicating cp virus.     

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.     

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.     

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.     

Ribosomal S27a Coding Sequences Upstream of Ubiquitin Coding Sequences in the Genome of a Pestivirus

Molecular characterization of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP Rit, a temperature-sensitive strain widely used for vaccination, revealed that the viral genomic RNA is about 15.2 kb long, which is about 2.9 kb longer than the one of noncytopathogenic (noncp) BVDV strains. Molecular cloning and nucleotide sequencing of parts of the genome resulted in the identification of a duplication of the genomic region encoding nonstructural proteins NS3, NS4A, and part of NS4B. In addition, a nonviral sequence was found directly upstream of the second copy of the NS3 gene. The 3′ part of this inserted sequence encodes an N-terminally truncated ubiquitin monomer. This is remarkable since all described cp BVDV strains with ubiquitin coding sequences contain at least one complete ubiquitin monomer. The 5′ region of the nonviral sequence did not show any homology to cellular sequences identified thus far in cp BVDV strains. Databank searches revealed that this second cellular insertion encodes part of ribosomal protein S27a. Further analyses included molecular cloning and nucleotide sequencing of the cellular recombination partner. Sequence comparisons strongly suggest that the S27a and the ubiquitin coding sequences found in the genome of CP Rit were both derived from a bovine mRNA encoding a hybrid protein with the structure NH₂-ubiquitin-S27a-COOH. Polyprotein processing in the genomic region encoding the N-terminal part of NS4B, the two cellular insertions, and NS3 was studied by a transient-expression assay. The respective analyses showed that the S27a-derived polypeptide, together with the truncated ubiquitin, served as processing signal to yield NS3, whereas the truncated ubiquitin alone was not capable of mediating the cleavage. Since the expression of NS3 is strictly correlated with the cp phenotype of BVDV, the altered genome organization leading to expression of NS3 most probably represents the genetic basis of cytopathogenicity of CP Rit.     

Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus

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.     

Correlation between point mutations in NS2 and the viability and cytopathogenicity of Bovine viral diarrhea virus strain Oregon analyzed with an infectious cDNA clone

Cytopathogenicity of Bovine viral diarrhea virus (BVDV) is correlated with expression of the nonstructural protein NS3, which can be generated by processing of a fusion protein termed NS2-3. For the cytopathogenic (cp) BVDV strain Oregon, NS2-3 processing is based on a set of point mutations within NS2. To analyze the correlation between NS2-3 cleavage and cytopathogenicity, a full-length cDNA clone composed of cDNA from BVDV Oregon and the utmost 5'- and 3'-terminal sequences of a published infectious BVDV clone was established. After transfection of RNA transcribed from this cDNA clone, infectious virus with similar growth characteristics to wild-type BVDV Oregon could be recovered that also exhibited a cytopathic effect. Based on this cDNA construct and published cp and noncp infectious clones, chimeric full-length cDNA clones were constructed. Analysis of the recovered viruses demonstrated that the presence of the NS2 gene of BVDV Oregon in a chimeric construct is sufficient for NS2-3 processing and a cp phenotype. Since previous studies had revealed that the amino acid serine at position 1555 of BVDV Oregon plays an important role in efficient NS2-3 cleavage, mutants of BVDV Oregon with different amino acids at this position were constructed. Some of these mutants showed NS2-3 cleavage efficiencies in the range of the wild-type sequence and allowed the recovery of viruses that behaved similarly to wild-type virus with regard to growth characteristics and cytopathogenicity. In contrast, other mutants with considerably reduced NS2-3 cleavage efficiencies propagated much more slowly and reverted to viruses expressing polyproteins with sequences allowing efficient NS2-3 cleavage. These viruses apparently induced cytopathic effects only after reversion.     

Analysis of Borna Disease Virus Trafficking in Live Infected Cells by Using a Virus Encoding a Tetracysteine-Tagged P Protein

Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus characterized by noncytolytic persistent infection and replication in the nuclei of infected cells. To gain further insight on the intracellular trafficking of BDV components during infection, we sought to generate recombinant BDV (rBDV) encoding fluorescent fusion viral proteins. We successfully rescued a virus bearing a tetracysteine tag fused to BDV-P protein, which allowed assessment of the intracellular distribution and dynamics of BDV using real-time live imaging. In persistently infected cells, viral nuclear inclusions, representing viral factories tethered to chromatin, appeared to be extremely static and stable, contrasting with a very rapid and active trafficking of BDV components in the cytoplasm. Photobleaching (fluorescence recovery after photobleaching [FRAP] and fluorescence loss in photobleaching [FLIP]) imaging approaches revealed that BDV components were permanently and actively exchanged between cellular compartments, including within viral inclusions, albeit with a fraction of BDV-P protein not mobile in these structures, presumably due to its association with viral and/or cellular proteins. We also obtained evidence for transfer of viral material between persistently infected cells, with routing of the transferred components toward the cell nucleus. Finally, coculture experiments with noninfected cells allowed visualization of cell-to-cell BDV transmission and movement of the incoming viral material toward the nucleus. Our data demonstrate the potential of tetracysteine-tagged recombinant BDV for virus tracking during infection, which may provide novel information on the BDV life cycle and on the modalities of its interaction with the nuclear environment during viral persistence.     

Processing of a pestivirus protein by a cellular protease specific for light chain 3 of microtubule-associated proteins

The genome of the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) JaCP contains a cellular insertion coding for light chain 3 (LC3) of microtubule-associated proteins, the mammalian homologue of yeast Aut7p/Apg8p. The cellular insertion induces cp BVDV-specific processing of the viral polyprotein by a cellular cysteine protease homologous to the known yeast protease Aut2p/Apg4p. Three candidate bovine protease genes were identified on the basis of the sequence similarity of their products with the Saccharomyces cerevisiae enzyme. The search for a system for functional testing of these putative LC3-specific proteases revealed that the components involved in this processing have been highly conserved during evolution, so that the substrate derived from a mammalian virus is processed in cells of mammalian, avian, fish, and insect origin, as well as in rabbit reticulocyte lysate, but not in wheat germ extracts. Moreover, two of these proteases and a homologous protein from chickens were able to rescue the defect of a yeast AUT2 deletion mutant. In coexpression experiments with yeast and wheat germ extracts one of the bovine proteases and the corresponding enzyme from chickens were able to process the viral polyprotein containing LC3. Northern blots showed that bovine viral diarrhea virus infection of cells has no significant influence on the expression of either LC3 or its protease, bAut2B2. However, LC3-specific processing of the viral polyprotein containing the cellular insertion is essential for replication of the virus since mutants with changes in the LC3 insertion significantly affecting processing at the LC3/NS3 site were not viable.     

Bovine viral diarrhea virus: characterization of a cytopathogenic defective interfering particle with two internal deletions.

Molecular characterization of bovine viral diarrhea virus pair 13 revealed that isolate CP13 is composed of a cytopathogenic (cp) defective interfering particle (DI13) and a noncytopathogenic (noncp) helper virus. The DI13 genome possesses two internal deletions of 1,611 and 3,102 nucleotides. Except for a small fragment of the gene coding for glycoprotein E1, all structural protein genes are deleted together with most of the Npro gene, the region coding for nonstructural proteins p7 and NS2. While the amino terminus of NS3 seems to be strictly conserved for all other cp bovine viral diarrhea viruses, NS3 of DI13 is amino-terminally truncated and fused to 23 amino acids derived from Npro and E1. Characterization of the DI-helper virus system revealed a striking discrepancy between RNA production and generation of infectious viruses.     

Protein synthesis shut-off induced by influenza virus infection is independent of PKR activity

The role of PKR activity in influenza virus-induced cell shut-off was studied by infection of PKR(+) or PKR(-) cell cultures and metabolic labeling in vivo. No differences in the synthesis of viral proteins or the decay of cellular protein synthesis were observed. To investigate the relevance of the inhibition of cellular pre-mRNA polyadenylation and nucleocytoplasmic transport in virus-induced shut-off, we carried out similar experiments with mutant viruses lacking C-terminal sequences of NS1 protein. No differences in the shut-off induced by mutant versus wild-type viruses were observed, indicating that these nuclear events are not relevant for shut-off. The analysis of cytoplasmic mRNA stability indicated that the accumulation of viral mRNA during the infection correlated with the progressive decay of cellular mRNA, in both the wild type and an NS1 deletion mutant.     

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.     

RNA Recombination between Persisting Pestivirus and a Vaccine Strain: Generation of Cytopathogenic Virus and Induction of Lethal Disease

Molecular analysis of a cytopathogenic (cp) bovine viral diarrhea virus (BVDV) isolate (1741) obtained from a case of mucosal disease (MD) led to the identification of five different viral subgenomic RNAs in addition to a noncytopathogenic (noncp) strain (NCP 1741). For each of the subgenomes, a large internal deletion was found together with an inserted sequence encoding part of ribosomal protein S27a fused to an N-terminally truncated ubiquitin monomer. Surprisingly, the two cellular insertions together with flanking viral sequences encoding parts of NS3 and NS4B are >99% identical to the previously described sequence of BVDV vaccine strain RIT (P. Becher, M. Orlich, and H.-J. Thiel, J. Virol. 72:8697-8704, 1998), while the remainder of the subgenomes is derived from the genome of NCP 1741. Further analyses including molecular cloning and nucleotide sequencing of the recombination partners revealed that both homologous and nonhomologous RNA recombination contributed to the generation of the viral subgenomes. Interestingly, for another cp BVDV isolate (CP 4584) from an independent case of MD, again an insertion of a RIT-derived sequence element was detected. In contrast to CP 1741, for CP 4584 a duplication of the genomic region encoding NS3 and parts of NS4A and NS4B was found. Transfection of bovine cells with RNA transcribed from a chimeric cDNA construct showed that the RIT-derived insertion together with the CP 4584-specific duplication of viral sequences represents the genetic basis of cytopathogenicity of CP 4584. Remarkably, passages of the recovered cp virus in cell culture led to emergence of noncp BVDV and a number of viral subgenomes whose genome organization was similar to that in BVDV 1741.     

A Single Point Mutation in Nonstructural Protein NS2 of Bovine Viral Diarrhea Virus Results in Temperature-Sensitive Attenuation of Viral Cytopathogenicity

For Bovine viral diarrhea virus (BVDV), the type species of the genus Pestivirus in the family Flaviviridae, cytopathogenic (cp) and noncytopathogenic (ncp) viruses are distinguished according to their effect on cultured cells. It has been established that cytopathogenicity of BVDV correlates with efficient production of viral nonstructural protein NS3 and with enhanced viral RNA synthesis. Here, we describe generation and characterization of a temperature-sensitive (ts) mutant of cp BVDV strain CP7, termed TS2.7. Infection of bovine cells with TS2.7 and the parent CP7 at 33°C resulted in efficient viral replication and a cytopathic effect. In contrast, the ability of TS2.7 to cause cytopathogenicity at 39.5°C was drastically reduced despite production of high titers of infectious virus. Further experiments, including nucleotide sequencing of the TS2.7 genome and reverse genetics, showed that a Y1338H substitution at residue 193 of NS2 resulted in the temperature-dependent attenuation of cytopathogenicity despite high levels of infectious virus production. Interestingly, TS2.7 and the reconstructed mutant CP7-Y1338H produced NS3 in addition to NS2-3 throughout infection. Compared to the parent CP7, NS2-3 processing was slightly decreased at both temperatures. Quantification of viral RNAs that were accumulated at 10 h postinfection demonstrated that attenuation of the cytopathogenicity of the ts mutants at 39.5°C correlated with reduced amounts of viral RNA, while the efficiency of viral RNA synthesis at 33°C was not affected. Taken together, the results of this study show that a mutation in BVDV NS2 attenuates viral RNA replication and suppresses viral cytopathogenicity at high temperature without altering NS3 expression and infectious virus production in a temperature-dependent manner.The pestiviruses Bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, Classical swine fever virus (CSFV), and Border disease virus (BDV) are causative agents of economically important livestock diseases. Together with the genera Flavivirus, including several important human pathogens like Dengue fever virus, West Nile virus, Yellow fever virus, and Tick-borne encephalitis virus, and Hepacivirus (human Hepatitis C virus [HCV]), the genus Pestivirus constitutes the family Flaviviridae (8, 20). All members of this family are enveloped viruses with a single-stranded positive-sense RNA genome encompassing one large open reading frame (ORF) flanked by 5′ and 3′ nontranslated regions (NTR) (see references 8 and 28 for reviews). The ORF encodes a polyprotein which is co- and posttranslationally processed into the mature viral proteins by viral and cellular proteases. For BVDV, the RNA genome is about 12.3 kb in length and encodes a polyprotein of about 3,900 amino acids. The first third of the ORF encodes a nonstructural (NS) autoprotease and four structural proteins, while the remaining part of the genome encodes NS proteins which share many common characteristics and functions with the corresponding NS proteins encoded by the HCV genome (8, 28). NS2 of BVDV represents a cysteine autoprotease which is distantly related to the HCV NS2-3 protease (26). NS3, NS4A, NS4B, NS5A, and NS5B are essential components of the pestivirus replicase (7, 10, 49). NS3 possesses multiple enzymatic activities, namely serine protease (48, 52, 53), NTPase (46), and helicase activity (51). NS4A acts as an essential cofactor for the NS3 proteinase. NS5B represents the RNA-dependent RNA polymerase (RdRp) (22, 56). The functions of NS4B and NS5A remain to be determined. NS5A has been shown to be a phosphorylated protein that is associated with cellular serine/threonine kinases (44).According to their effects in tissue culture, two biotypes of pestiviruses are distinguished: cytopathogenic (cp) and noncytopathogenic (ncp) viruses (17, 27). The occurrence of cp BVDV in cattle persistently infected with ncp BVDV is directly linked to the induction of lethal mucosal disease in cattle (12, 13). Previous studies have shown that cp BVDV strains evolved from ncp BVDV strains by different kinds of mutations. These include RNA recombination with various cellular mRNAs, resulting in insertions of cellular protein-coding sequences into the viral genome, as well as insertions, duplications, and deletions of viral sequences, and point mutations (1, 2, 9, 24, 33, 36, 37, 42). A common consequence of all these genetic changes in cp BVDV genomes is the efficient production of NS3 at early and late phases of infection. In contrast, NS3 cannot be detected in cells at late time points after infection with ncp BVDV. An additional major difference is that the cp viruses produce amounts of viral RNA significantly larger than those of their ncp counterparts (7, 32, 50). While there is clear evidence that cell death induced by cp BVDV is mediated by apoptosis, the molecular mechanisms involved in pestiviral cytopathogenicity are poorly understood. In particular, the role of NS3 in triggering apoptosis remains unclear. It has been hypothesized that the NS3 serine proteinase might be involved in activation of the apoptotic proteolytic cascade (21, 55). Furthermore, it has been suggested that the NS3-mediated, enhanced viral RNA synthesis of cp BVDV and subsequently larger amounts of viral double-stranded RNAs may play a crucial role in triggering apoptosis (31, 54).In this study, we describe generation and characterization of a temperature-sensitive (ts) cp BVDV mutant whose ability to cause viral cytopathogenicity at high temperature is strongly attenuated. Our results demonstrate that a single amino acid substitution in NS2 attenuates BVDV cytopathogenicity at high temperature without affecting production of infectious viruses and expression of NS3 in a temperature-dependent manner.     

Comparative Analysis of Seven Viral Nuclear Export Signals (NESs) Reveals the Crucial Role of Nuclear Export Mediated by the Third NES Consensus Sequence of Nucleoprotein (NP) in Influenza A Virus Replication

The assembly of influenza virus progeny virions requires machinery that exports viral genomic ribonucleoproteins from the cell nucleus. Currently, seven nuclear export signal (NES) consensus sequences have been identified in different viral proteins, including NS1, NS2, M1, and NP. The present study examined the roles of viral NES consensus sequences and their significance in terms of viral replication and nuclear export. Mutation of the NP-NES3 consensus sequence resulted in a failure to rescue viruses using a reverse genetics approach, whereas mutation of the NS2-NES1 and NS2-NES2 sequences led to a strong reduction in viral replication kinetics compared with the wild-type sequence. While the viral replication kinetics for other NES mutant viruses were also lower than those of the wild-type, the difference was not so marked. Immunofluorescence analysis after transient expression of NP-NES3, NS2-NES1, or NS2-NES2 proteins in host cells showed that they accumulated in the cell nucleus. These results suggest that the NP-NES3 consensus sequence is mostly required for viral replication. Therefore, each of the hydrophobic (Φ) residues within this NES consensus sequence (Φ1, Φ2, Φ3, or Φ4) was mutated, and its viral replication and nuclear export function were analyzed. No viruses harboring NP-NES3 Φ2 or Φ3 mutants could be rescued. Consistent with this, the NP-NES3 Φ2 and Φ3 mutants showed reduced binding affinity with CRM1 in a pull-down assay, and both accumulated in the cell nucleus. Indeed, a nuclear export assay revealed that these mutant proteins showed lower nuclear export activity than the wild-type protein. Moreover, the Φ2 and Φ3 residues (along with other Φ residues) within the NP-NES3 consensus were highly conserved among different influenza A viruses, including human, avian, and swine. Taken together, these results suggest that the Φ2 and Φ3 residues within the NP-NES3 protein are important for its nuclear export function during viral replication.     

Monoclonal antibody analyses of cytopathic and noncytopathic viruses from fatal bovine viral diarrhea virus infections.

A panel of monoclonal antibodies that recognize the two major glycoproteins of bovine viral diarrhea virus (BDV) was used to evaluate the antigenic relationship between cytopathic (CP) and noncytopathic (NCP) viruses isolated from cattle dead or dying from fatal BDV infections. Various unrelated BDV isolates were initially screened by indirect immunofluorescence with monoclonal antibodies directed against the 56- to 58- and 48-kilodalton glycoproteins of the virus. A wide spectrum of reactivity that was independent of biotype was found. Biological clones of the same isolate showed only minor variations from the parental isolate, as did isolates taken from different animals located on the same farm. A similar analysis was repeated with pairs of CP and NCP viruses isolated from 16 unrelated clinical cases of BDV infection resulting in fatal disease. The reactivity patterns within individual pairs of isolates taken from the same animals were in most instances very similar and in some cases indistinguishable from one another. The results demonstrate that antigenic similarity between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree of variation in reactivity patterns between unrelated BDV isolates, the close antigenic similarity of CP BDV to the homologous NCP BDV of a given pair strongly suggests that CP BDV arises by mutation from NCP BDV.     

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.