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. Bovine viral diarrhea virus (BVDV)2 is an enveloped, positive-sense single-stranded RNA virus classified in the genus Pestivirus of the family Flaviviridae. BVDV establishes persistent infections in cattle populations worldwide. Because BVDV shares virological and molecular properties with the Flaviviridae family member hepatitis C virus (HCV), which chronically infects an estimated 200 million patients worldwide (1), BVDV is regarded as a surrogate model for HCV (2). Both HCV and BVDV encode a single large precursor polyprotein that is processed by cellular and viral proteases into mature structural and nonstructural (NS) proteins.BVDV NS3 exhibits serine protease and helicase/ATPase activities that require its cofactor NS4A (3). NS3/4A protease is essential for generating mature NS proteins that are required for viral replication. HCV NS3/4A is well characterized and has been shown to suppress type-I interferons by cleaving the cellular interferon mediators IPS-1 and TRIF (4, 5). However, neither interferon suppression nor cellular targets have been identified for the BVDV NS3/4A protease (6).Lytic and persistent BVDV infections depend on the virus biotype. Cytopathogenic (CP) BVDV causes cytopathic effects via apoptosis, whereas noncytopathogenic (NCP) BVDV does not induce obvious changes in cell morphology and viability. These features are distinguished by NS2-3 processing differences; free NS3 produced by NS2-3 cleavage is generated continuously following CP BVDV infections, whereas NS3 is detected only until ϳ9 h postinfection (p.i.) for NCP BVDV due to down-regulation of NS2-3 cleavage by this biotype (7). The CP biotype is characterized by dramatic up-regulation of viral RNA synthesis that could be correlated with the induction of cytopathic effect (...

Bovine viral diarrhea virus (BVDV) is a positive-sense, single-stranded RNA virus that causes an economically important livestock disease worldwide. Previous studies have suggested that nonstructural protein 5A (NS5A) from hepatitis C virus (HCV) and BVDV plays a similar role during virus infection. Extensive reports are available on HCV NS5A and its interactions with the host cellular proteins; however, the role of NS5A during BVDV infection remains largely unclear. To identify the cellular proteins that interact with the N terminus of NS5A and could be involved in its function, we conducted a yeast two-hybrid screening. As a result, we identified a cellular protein termed bovine NIK-and IKKb-binding protein (NIBP), which is involved in protein trafficking and nuclear factor kappa B (NF-kB) signalling in cells. The interaction of NS5A with NIBP was confirmed both in vitro and in vivo. Complementing our glutathione S-transferase pull-down and immunoprecipitation data are the confocal immunofluorescence results, which indicate that NS5A colocalized with NIBP on the endoplasmic reticulum in the cytoplasm of BVDV-infected cells. Moreover, the minimal residues of NIBP that interact with NS5A were mapped as aa 597-623. In addition, overexpression of NS5A inhibited NF-kB activation in HEK293 and LB9.K cells as determined by luciferase reporter-gene assay. We further showed that inhibition of endogenous NIBP by small interfering RNA molecules enhanced virus replication, indicating the importance of NIBP implications in BVDV pathogenesis. Being the first reported interaction between NIBP and a viral protein, this finding suggests a novel mechanism whereby viruses may subvert host-cell machinery for mediating trafficking as well as NF-kB signalling. INTRODUCTIONPestiviruses are important livestock pathogens that belong to the family Flaviviridae, which also includes the closely related genera Hepacivirus (Hepatitis C virus; HCV) and Flavivirus (Yellow fever virus and West Nile virus). The genus Pestivirus includes the species Bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, Border disease virus and Classical swine fever virus. As a well-characterized prototype member of the genus Pestivirus within the family Flaviviridae, BVDV sometimes serves as a surrogate model for HCV life-cycle studies. BVDV is an enveloped virus containing a single, positive-sense RNA of approximately 12.3 kb. The genomic RNA consists of one long open reading frame (ORF), which is flanked by untranslated regions at both ends. The ORF encodes a single polyprotein of about 4000 aa, which is processed co-and posttranslationally by both viral and cellular proteases into at least 11 mature viral proteins (N pro , C, E rns , E1, E2, p7, NS2-3, NS4A, NS4B, NS5A and NS5B) (Lindenbach et al., 2007).BVDV non-structural protein 5A (NS5A) is a phosphoprotein of 56-58 kDa that plays an essential role in BVDV replication (Tellinghuisen et al., 2006) and shares many features with its counterpart HCV NS5A, e.g. both proteins are phosphorylated by the same or simila...

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Bovine viral diarrhea virus (BVDV) nonstructural protein 5A (NS5A) is one the least studied of the BVDV proteins. Therefore, to develop a tool for unraveling the functions performed by BVDV NS5A, monoclonal antibodies (MAbs) were generated by fusion of myeloma cells with spleen cells from mice immunized with recombinant E. coli-expressed GST-NS5A protein. Two MAbs (1H12 and 2F9) were established on the basis of immunofluorescence and Western blot analysis. Both the MAbs were of IgG1 subclass and recognized an epitope clustered within the N-terminal region of NS5A. Furthermore, the MAb 1H12 was used successfully to detect NS5A protein in BVDV field isolates belonging to genotypes 1 and 2. Temporal expression pattern studies during an infectious cycle revealed that BVDV NS5A could be detected 12-60 h postinfection. Confocal microscopy studies showed a cytoplasmic staining pattern and revealed that NS5A is localized on the endoplasmic reticulum membrane in BVDV infected cells.