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. 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 (...

Reduction in sphingosine kinase 1 influences the susceptibility to dengue virus infection by altering antiviral responses

“…Recently, SK2 has been shown to increase the replication of some hepatitis C virus strains by interfering with oxidative stress and lipid peroxidation in infected cells (Yamane et al, 2014). The above studies linking high levels of SK with enhancement of viral infection are in contrast to our own observations of a reduction in SK1 activity in response to dengue virus (DENV) infection (Wati et al, 2011), as also reported for the related flavivirus bovine viral diarrhea virus (Yamane et al, 2009). Close analysis of the time-course of the reduction in SK1 in DENV-infected cells demonstrates a correlation with high levels of the DENV 39 UTR, which can compete for the SK1 activator eEF1A (Carr et al, 2013a;Leclercq et al, 2008Leclercq et al, , 2011, which is utilized by flaviviruses to assist replication (Blackwell & Brinton, 1997;Davis et al, 2007).…”

“…Close analysis of the time-course of the reduction in SK1 in DENV-infected cells demonstrates a correlation with high levels of the DENV 39 UTR, which can compete for the SK1 activator eEF1A (Carr et al, 2013a;Leclercq et al, 2008Leclercq et al, , 2011, which is utilized by flaviviruses to assist replication (Blackwell & Brinton, 1997;Davis et al, 2007). Additionally, our previous data (Carr et al, 2013a;Calvert et al, 2015), as well as that of Yamane et al (2009), suggest an increase in SK1 activity in the early hours following infection. Thus the roles of SK1 in viral infection may vary in relation to the time post-infection (p.i.…”

Understanding the Pathogenesis of Cytopathic and Noncytopathic Bovine Viral Diarrhea Virus Infection Using Proteomics

“…Studies used the yeast-two hybrid (Y2H) screening system to screen individual BVDV proteins for host binding partners ( demonstrated that NS3 from BVDV binds to and inhibits the catalytic activity of sphingosine kinase 1 (SphK1). NS3-SphK1 binding enhance BVDV replication and BVDVinduced apoptosis (Yamane et al 2009a). Using Y2H screening, the subunit of bovine translation elongation factor 1A (eEF1A) was shown to interact with the NS5A polypeptide of BVDV (Johnson et al 2001).…”

Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKβ-binding protein

“…Goat anti-mouse IgG1, IgG2a or anti-rabbit IgG, IgG1 antibodies conjugated with Alexa 488, Alexa 594, Alexa 568 or Alexa 488, respectively, were from Invitrogen. Protein concentrations in samples were determined with a Protein Quantification kitRapid using BSA as a standard as described previously (Yamane et al, 2009a;Zahoor et al, 2009). Recombinant NS5A was used as an antigen to generate mouse mAb and its specificity was verified by ELISA, immunoprecipitation, immunoblot and immunofluorescence as described previously (Zahoor et al, 2009).…”