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 (...
This paper presents a design of microelectromechanical systems (MEMS) accelerometers for sensing sub-1g (g = 9.8 m/s2) acceleration. The accelerometer has a high-density proof mass to suppress the Brownian noise that dominates the output noise of the sensor. The low-temperature (<400 °C) process enables to integrate the accelerometer on the sensing complementary metal-oxide semiconductor circuit by electroplating of gold; a proof mass of 1020 μm × 1020 μm in area with the thickness of 12 μm has been found to suppress the measured noise floor to 0.78 μg/Hz at 300 K, which is nearly one order of magnitude smaller than those of the conventional MEMS accelerometers made of silicon.
This paper reports a novel sensor structure and its evaluation results for an integrated complementary metal oxide semiconductor (CMOS) microelectromechanical systems (MEMS) accelerometer with a wide detection range on a chip. The proposed sensor structure has the following features: i) a layer separation technique between the proof mass and the mechanical suspensions, ii) mechanical stoppers for the proof mass to avoid destruction, and iii) a SiO 2 film underneath the proof mass to prevent stiction and electrical short. Gold was used as the MEMS structure material to reduce the proof mass size and to lower the Brownian noise to below 100 g/ ffiffiffiffiffiffi Hz p . Furthermore, the micro fabrication was carried out below 310 C for the CMOS devices to remain intact. The evaluation results indicate that the Brownian noise was 90.6 g/ ffiffiffiffiffiffi Hz p. Thus, we have confirmed that the proposed MEMS structure has the potential for use in future integrated CMOS-MEMS accelerometers.
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...
Equine herpesvirus 4 (EHV-4) is a major cause of respiratory tract disease in horses worldwide. The generation of recombinant viruses, which would lead to understanding of viral gene functions, has been hindered by the absence of suitable cell lines and small-animal models of the infection. In the present study, the genome of EHV-4 strain TH20p was cloned as a stable and infectious BAC without any deletions of the viral genes. Mini F plasmid sequences flanked by loxP sites were inserted into the intergenic region between genes 58 and 59. Coinfection of the recombinant virus with a recombinant adenovirus expressing Cre recombinase resulted in the excision of the BAC sequences. Importantly, the resulting recombinant EHV-4 replicated comparably to the wild-type virus in fetal horse kidney cells. The recombinant EHV-4 will facilitate EHV-4 research and provide the opportunity to exploit the power of BAC technology for production of recombinant viral vaccines.
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