Inhibition of Sphingosine Kinase by Bovine Viral Diarrhea Virus NS3 Is
Crucial for Efficient Viral Replication and
Cytopathogenesis |
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Authors: | Daisuke Yamane Muhammad A Zahoor Yassir M Mohamed Walid Azab Kentaro Kato Yukinobu Tohya and Hiroomi Akashi |
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Institution: | Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan |
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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 ~9 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
(7–9).
Because free NS3, but not NS2-3, can form an active viral replicase complex
with other NS proteins, increased viral RNA synthesis promoted through the
release of free NS3 has been suggested to be a determinant of the
characteristic lytic phenotype of CP BVDV infections
(10). However, little is known
about the regulation of cellular signaling by BVDV NS2-3, NS3, and NS3/4A,
which is crucial for the control of both viral replication and biotype.Recent studies on the mechanisms of viral replication revealed that HCV RNA
synthesis occurs on a lipid raft membrane structure where the active viral
replicase complex is found
(11,
12). The significance of the
lipid raft as a scaffold for viral replication is further demonstrated by the
identification of a novel HCV replication inhibitor, NA255, which prevents the
biosynthesis of sphingolipids, the major components of lipid rafts
(13). Administration of NA255
results in disruption of the HCV replicase complexes from the lipid rafts.
This report proposes that the interaction between HCV NS5B and sphingomyelin
on lipid rafts plays a crucial role for HCV RNA replication. Cellular
sphingolipid metabolism is regulated by a large number of converting enzymes
that maintain a homeostasis
(14) but viral mechanisms that
affect the sphingolipid metabolism to facilitate viral replication have yet to
be identified.In a search for potential host proteins that interact with BVDV NS3, we
identified sphingosine kinase 1 (SphK1) as a binding partner of NS3 using the
yeast two-hybrid system. SphK1 is a lipid kinase that catalyzes the
phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), a
bioactive sphingolipid implicated in diverse cellular functions, including
proliferation, survival, tumorigenesis, development, inflammation, and
immunity (14,
15). Here, we analyze the
biological significance of the SphK1 interaction with NS3, NS2-3, and NS3/4A.
Using purified recombinant SphK1 and NS3, SphK activity was inhibited by NS3
in a dose-dependent manner, independently of its serine protease activity. The
inhibition appears to be specific for BVDV NS3 because HCV NS3 had no effect
on SphK activity. Using specific chemical inhibitors, small interfering RNA
(siRNA), and a catalytically inactive mutant of SphK1, we investigated the
significance of SphK inhibition in the viral replication. The present study is
the first report demonstrating that SphK1 is targeted by a virus to inhibit
its catalytic activity, and this mechanism may contribute to the efficient
replication and pathogenesis of BVDV. |
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