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Inhibition of sphingosine kinase by bovine viral diarrhea virus NS3 is crucial for efficient viral replication and cytopathogenesis.

ABSTRACT: Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in diverse cellular functions including survival, proliferation, tumorigenesis, inflammation, and immunity. Sphingosine kinase (SphK) contributes to these functions by converting sphingosine to S1P. We report here that the nonstructural protein NS3 from bovine viral diarrhea virus (BVDV), a close relative of hepatitis C virus (HCV), binds to and inhibits the catalytic activity of SphK1 independently of its serine protease activity, whereas HCV NS3 does not affect SphK1 activity. Uncleaved NS2-3 from BVDV was also found to interact with and inhibit SphK1. We suspect that inhibition of SphK1 activity by BVDV NS3 and NS2-3 may benefit viral replication, because SphK1 inhibition by small interfering RNA, chemical inhibitor, or overexpression of catalytically inactive SphK1 results in enhanced viral replication, although the mechanisms by which SphK1 inhibition leads to enhanced viral replication remain unknown. A role of SphK1 inhibition in viral cytopathogenesis is also suggested as overexpression of SphK1 significantly attenuates the induction of apoptosis in cells infected with cytopathogenic BVDV. These findings suggest that SphK is targeted by this virus to regulate its catalytic activity.

SUBMITTER: Yamane D

PROVIDER: S-EPMC2679466 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Inhibition of sphingosine kinase by bovine viral diarrhea virus NS3 is crucial for efficient viral replication and cytopathogenesis.

Yamane Daisuke D Zahoor Muhammad A MA Mohamed Yassir M YM Azab Walid W Kato Kentaro K Tohya Yukinobu Y Akashi Hiroomi H

The Journal of biological chemistry 20090317 20

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in diverse cellular functions including survival, proliferation, tumorigenesis, inflammation, and immunity. Sphingosine kinase (SphK) contributes to these functions by converting sphingosine to S1P. We report here that the nonstructural protein NS3 from bovine viral diarrhea virus (BVDV), a close relative of hepatitis C virus (HCV), binds to and inhibits the catalytic activity of SphK1 independently of its serine protease activ ...[more]

PMID: 19293152

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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.

Project description:DNA damage-inducible transcript 3 (DDIT3) plays important roles in endoplasmic reticulum (ER) stress-induced apoptosis and autophagy, but its role in innate immunity is not clear. Here, we report that DDIT3 inhibits the antiviral immune response during bovine viral diarrhea virus (BVDV) infection by targeting mitochondrial antiviral signaling (MAVS) in Madin-Darby bovine kidney (MDBK) cells and in mice. BVDV infection induced high DDIT3 mRNA and protein expression. DDIT3 overexpression inhibited type I interferon (IFN-I) and IFN-stimulated gene production, thereby promoting BVDV replication, while DDIT3 knockdown promoted the antiviral innate immune response to suppress viral replication. DDIT3 promoted NF-κB-dependent ovarian tumor (OTU) deubiquitinase 1 (OTUD1) expression. Furthermore, OTUD1 induced upregulation of the E3 ubiquitin ligase Smurf1 by deubiquitinating Smurf1, and Smurf1 degraded MAVS in MDBK cells in a ubiquitination-dependent manner, ultimately inhibiting IFN-I production. Moreover, knocking out DDIT3 promoted the antiviral innate immune response to reduce BVDV replication and pathological changes in mice. These findings provide direct insights into the molecular mechanisms by which DDIT3 inhibits IFN-I production by regulating MAVS degradation.IMPORTANCE Extensive studies have demonstrated roles of DDIT3 in apoptosis and autophagy during viral infection. However, the role of DDIT3 in innate immunity remains largely unknown. Here, we show that DDIT3 is positively regulated in bovine viral diarrhea virus (BVDV)-infected Madin-Darby bovine kidney (MDBK) cells and could significantly enhance BVDV replication. Importantly, DDIT3 induced OTU deubiquitinase 1 (OTUD1) expression by activating the NF-κB signaling pathway, thus increasing intracellular Smurf1 protein levels to degrade MAVS and inhibit IFN-I production during BVDV infection. Together, these results indicate that DDIT3 plays critical roles in host innate immunity repression and viral infection facilitation.

Dataset Information

Inhibition of sphingosine kinase by bovine viral diarrhea virus NS3 is crucial for efficient viral replication and cytopathogenesis.

Publications

Inhibition of sphingosine kinase by bovine viral diarrhea virus NS3 is crucial for efficient viral replication and cytopathogenesis.

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