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Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting Erns RNase and Npro protease.

ABSTRACT: Different genetically engineered mutants of bovine viral diarrhea virus (BVDV) were analyzed for the ability to establish infection in the fetuses of pregnant heifers. The virus mutants exhibited either a deletion of the overwhelming part of the genomic region coding for the N-terminal protease N(pro), a deletion of codon 349, which abrogates the RNase activity of the structural glycoprotein E(rns), or a combination of both mutations. Two months after infection of pregnant cattle with wild-type virus or either of the single mutants, the majority of the fetuses contained virus or were aborted or found dead in the uterus. In contrast, the double mutant was not recovered from fetal tissues after a similar challenge, and no dead fetuses were found. This result was verified with a nonrelated BVDV containing similar mutations. After intrauterine challenge with wild-type virus, mutated viruses, and cytopathogenic BVDV, all viruses could be detected in fetal tissue after 5, 7, and 14 days. Type 1 interferon (IFN) could be detected in fetal serum after challenge, except with wild-type noncytopathogenic BVDV. On days 7 and 14 after challenge, the largest quantities of IFN in fetal serum were induced by the N(pro) and RNase-negative double mutant virus. The longer duration of fetal infection with the double mutant resulted in abortion. Therefore, for the first time, we have demonstrated the essential role of both N(pro) and E(rns) RNase in blocking interferon induction and establishing persistent infection by a pestivirus in the natural host.

SUBMITTER: Meyers G

PROVIDER: S-EPMC1866084 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting Erns RNase and Npro protease.

Meyers Gregor G Ege Andreas A Fetzer Christiane C von Freyburg Martina M Elbers Knut K Carr Veronica V Prentice Helen H Charleston Bryan B Schürmann Eva-Maria EM

Journal of virology 20070110 7

Different genetically engineered mutants of bovine viral diarrhea virus (BVDV) were analyzed for the ability to establish infection in the fetuses of pregnant heifers. The virus mutants exhibited either a deletion of the overwhelming part of the genomic region coding for the N-terminal protease N(pro), a deletion of codon 349, which abrogates the RNase activity of the structural glycoprotein E(rns), or a combination of both mutations. Two months after infection of pregnant cattle with wild-type ...[more]

PMID: 17215285

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Project description:Unique to pestiviruses, the N-terminal protein encoded by the bovine viral diarrhea virus (BVDV) genome is a cysteine protease (Npro) responsible for a self-cleavage that releases the N terminus of the core protein (C). This unique protease is dispensable for viral replication, and its coding region can be replaced by a ubiquitin gene directly fused in frame to the core. To develop an antiviral assay that allows the assessment of anti-hepatitis C virus (HCV) NS3 protease inhibitors, a chimeric BVDV in which the coding region of Npro was replaced by that of an NS4A cofactor-tethered HCV NS3 protease domain was generated. This cofactor-tethered HCV protease domain was linked in frame to the core protein of BVDV through an HCV NS5A-NS5B junction site and mimicked the proteolytic function of Npro in the release of BVDV core for capsid assembly. A similar chimeric construct was built with an inactive HCV NS3 protease to serve as a control. Genomic RNA transcripts derived from both chimeric clones, P(H/B) (wild-type HCV NS3 protease) and P(H/B(S139A)) (mutant HCV NS3 protease) were then transfected into bovine cells (MDBK). Only the RNA transcripts from the P(H/B) clone yielded viable viruses, whereas the mutant clone, P(H/B(S139A)), failed to produce any signs of infection, suggesting that the unprocessed fusion protein rendered the BVDV core protein defective in capsid assembly. Like the wild-type BVDV (NADL), the chimeric virus was cytopathic and formed plaques on the cell monolayer. Sequence and biochemical analyses confirmed the identity of the chimeric virus and further revealed variant viruses due to growth adaptation. Growth analysis revealed comparable replication kinetics between the wild-type and the chimeric BVDVs. Finally, to assess the genetic stability of the chimeric virus, an Npro-null BVDV (BVDV-Npro in which the entire Npro coding region was deleted) was produced. Although cytopathic, BVDV-Npro was highly defective in viral replication and growth, a finding consistent with the observed stability of the chimeric virus after serial passages.

Dataset Information

Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting Erns RNase and Npro protease.

Publications

Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting Erns RNase and Npro protease.

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