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

Institut für Virologie (FB Veterinärmedizin), Justus-Liebig-Universität Giessen, Frankfurter Strasse 107, 35392 Giessen, Germany.
Journal of Virology (Impact Factor: 4.44). 11/2004; 78(19):10765-75. DOI: 10.1128/JVI.78.19.10765-10775.2004
Source: PubMed


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.

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Available from: Norbert Tautz, Jan 29, 2015
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    • "Cells infected with cytopathic (cp) viruses develop cytopathological changes including cytoplasmic vacuolization and cell death through apoptosis. More of the NS3 product is formed by cp BVDV while very little NS3 cleavage product is formed by noncytopathic (ncp) BVDV infection (Lackner et al., 2004) and there are no cytopathological changes in ncp BVDV infected cultured cells (Donis and Dubovi, 1987). When ncp BVDV infects the fetus during the first 40–120 days of gestation, the fetus may be born persistently infected (PI). "
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    ABSTRACT: Bovine viral diarrhea virus (BVDV) is a positive single stranded RNA virus belonging to the Pestivirus genus of the Flaviviridae family. BVDV has a wide host range that includes most ruminants. Noncytopathic (ncp) BVDV may establish lifelong persistent infections in calves following infection of the fetus between 40-120 days of gestation. Cytopathic (cp) BVDV strains arise from ncp strains via mutations. The most common cp mutations are insertions of RNA derived from either host or a duplication of viral sequences into the region of the genome coding for the NS2/3 protein. Superinfection of a persistently infected animal with a cp virus can give rise to mucosal disease, a condition that is invariably fatal. A herd of 136 bred 3-year old cows was studied. These cows gave birth to 36 PI animals of which 13 succumbed to mucosal disease. In this study, we characterized the ncp and cp viruses isolated from these 13 animals. All viruses belonged to the BVDV type 2a genotype and were highly similar. All the cp viruses contained an insertion in the NS2/3 coding region consisting of the sequences derived from the transcript encoding a DnaJ protein named Jiv90. Comparison of the inserted DnaJ regions along with the flanking viral sequences in the insertion 3' end of the 13 cp isolates revealed sequence identities ranging from 96%-99% with common borders. This suggested that one animal likely developed a cp virus that then progressively spread to the other 12 animals. Interestingly, when the inserted mammalian gene replicated within viral genome, it showed conservation of the same conserved motifs between the different species, which may indicate a role for these motifs in the insertion function within the virus genome. This is the first characterization of multiple cp bovine viral diarrhea virus isolates that spread in a herd under natural conditions.
    Virus Research 10/2014; 195. DOI:10.1016/j.virusres.2014.09.015 · 2.32 Impact Factor
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    • "NS2, together with the amino terminus of NS3, functions as an autoprotease that cleaves the NS2-NS3 junction of the polyprotein. This cleavage is required for RNA replication and is linked to BVDV cytopathogenicity and pathogenesis [14], [15]. NS3 is a multifunctional protein with a helicase/nucleoside triphosphatase and serine protease activity responsible for all downstream polyprotein cleavages [16]–[19]. "
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    ABSTRACT: Bovine viral diarrhea virus (BVDV) is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1-5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of BVDV-TSCr T1-5 in MDBK cells in the absence of TSC to evaluate the stability of the resistance. The viral populations obtained (BVDV R1-5) remained resistant to the antiviral compound and conserved the mutations in NS5B associated with this phenotype. Along the passages, BVDV R2, R3 and R5 presented a delay in the production of cytopathic effect that correlated with a decrease in cell apoptosis and intracellular accumulation of viral RNA. The complete genome sequences that encode for NS2 to NS5B, Npro and Erns were analyzed. Additional mutations were detected in the NS5B of BVDV R1, R3 and R4. In both BVDV R2 and R3, most of the mutations found were localized in NS5A, whereas in BVDV R5, the only mutation fixed was NS5A V177A. These results suggest that mutations in NS5A could alter BVDV cytopathogenicity. In conclusion, the stability of the resistance to TSC may be due to the fixation of different compensatory mutations in each BVDV-TSCr. During their replication in a TSC-free medium, some virus populations presented a kind of interaction with the host cell that resembled a persistent infection: decreased cytopathogenicity and viral genome synthesis. This is the first report on the stability of antiviral resistance and on the evolution of NNI-resistant BVDV variants. The results obtained for BVDV-TSCr could also be applied for other NNIs.
    PLoS ONE 06/2014; 9(6):e100528. DOI:10.1371/journal.pone.0100528 · 3.23 Impact Factor
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    • "Classical swine fever (CSF) is a highly contagious and often fatal disease of pigs and is classified by the World Organization for Animal Health (OIE) as a notifiable (previously List A) disease. The causative agent of CSF is classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family of viruses, which also contains the genera Flavivirus and Hepacivirus (hepatitis C viruses, HCV)[1]. CSFV harbors a 12.3 kb positive-sense, single-stranded RNA genome that consists of a large open reading frame that encodes a polyprotein which is processed into 12 mature proteins, namely, Npro, C, Erns, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B [2-4]. "
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    ABSTRACT: The membrane topology and molecular mechanisms for endoplasmic reticulum (ER) localization of classical swine fever virus (CSFV) non-structural 2 (NS2) protien is unclear. We attempted to elucidate the subcellular localization, and the molecular mechanisms responsible for the localization of this protein in our study. The NS2 gene was amplified by reverse transcription polymerase chain reaction, with the transmembrane region and hydrophilicity of the NS2 protein was predicted by bioinformatics analysis. Twelve cDNAs of the NS2 gene were amplified by the PCR deletion method and cloned into a eukaryotic expression vector, which was transfected into a swine umbilical vein endothelial cell line (SUVEC). Subcellular localization of the NS2 protein was characterized by confocal microscopy, and western blots were carried out to analyze protein expression. Our results showed that the -NH2 terminal of the CSFV NS2 protein was highly hydrophobic and the protein localized in the ER. At least four transmembrane regions and two internal signal peptide sequences (amino acids103-138 and 220-262) were identified and thought to be critical for its trans-localization to the ER. This is the first study to identify the internal signal peptide sequences of the CSFV NS2 protein and its subcellular localization, providing the foundation for further exploration of this protein's function of this protein and its role in CSFV pathogenesis.
    Virology Journal 05/2011; 8(1):236. DOI:10.1186/1743-422X-8-236 · 2.18 Impact Factor
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