A critical role of N-myc and STAT interactor (Nmi) in foot-and-mouth disease virus (FMDV) 2C-induced apoptosis
Highlights
► FMDV 2C is one of the most highly-conserved proteins among the serotypes of FMDV. ► We found FMDV 2C interacted with a cellular protein Nmi. ► FMDV 2C induced translocation of Nmi from the cytoplasm to the speckles. ► Knockdown of Nmi inhibited FMDV 2C-induced apoptosis.
Introduction
Foot-and-mouth disease (FMD) is a highly-contagious disease with an enormous impact on trading patterns. The etiologic agent for FMD is FMD virus (FMDV), a member of the Aphthovirus genus of the Picornaviridae family that contains a single-stranded positive-sense RNA genome of 8500 nucleotides surrounded by an icosahedral capsid composed of 60 copies of each of four structural proteins, VP1-4 (Grubman and Baxt, 2004). Following FMDV infection, vesicles develop in the snout, nares, lips, tongue and soft tissues of the feet and teats of the infected animals. In the young animals, infection of the heart muscle may result in severe myocardial necrosis and death (Ku et al., 2005). Despite being one of the most studied animal viruses, the molecular mechanisms regarding FMDV induced pathogenesis are still unclear. Recent in vivo studies suggest FMDV induced apoptosis may play a role in FMDV pathogenesis. By 2 days postinfection, apoptosis was detected in most of the infected organs in swine (Ku et al., 2005). However, how FMDV induces host cells to undergo apoptosis is not known.
FMDV exhibits a high potential for genetic and antigenic variation, which has led to the classification of seven serotypes of FMDV (A, O, C, Asia1, SAT 1, SAT 2 and SAT3) (Saiz et al., 2002). Among the viral proteins encoded by the FMDV genome, 2C is one of the most highly conserved molecules, and 2C-like proteins have been identified in a number of other animal and plant RNA viruses (Gorbalenya and Koonin, 1989). For the poliovirus (PV), an important member of the Picornavirus family, the viral protein 2C shows multiple functions including membrane binding (Echeverri et al., 1998, Teterina et al., 1997), viral RNA binding (Banerjee and Dasgupta, 2001, Rodriguez and Carrasco, 1995), NTP binding (Gorbalenya and Koonin, 1989) and ATPase and GTPase activity (Pfister et al., 2000, Rodriguez and Carrasco, 1993, Sweeney et al., 2010). The 2C protein of enterovirus 71, poliovirus and coxsackievirus A16 interacts with the cellular protein reticulon 3 and in enterovirus 71 this interaction may be involved in the synthesis of viral proteins and replicative double-stranded RNA (Tang et al., 2007).
FMDV 2C protein, a 318 amino acid polypeptide, has a predicted amphipathic helix in its N-terminus (residues 17–34) (Teterina et al., 2006), a feature that has been shown for other picornaviruses to confer their ability to bind the cell membranes and is also thought to be required for both membrane rearrangement and formation of the viral replication complex (Bienz et al., 1990). Although it was found that 2C impedes trafficking of proteins between ER and Golgi and that the subcellular localization of 2C determines the trafficking site (Moffat et al., 2005, Moffat et al., 2007), the role of 2C protein in the host cell is not very clear. Using yeast two-hybrid system and immunoprecipitation approaches, we found that FMDV 2C interacted with N-myc and STAT interactor (Nmi) and induced apoptosis. We further demonstrated that FMDV 2C-induced apoptosis in BHK-21 cells was markedly inhibited by Nmi knockdown. These data suggest that Nmi may play a role in FMDV 2C-induced apoptosis.
Section snippets
Reagents
Endotoxin free plasmid preparation kits were purchased from Aidlab (Beijing, China). Anti-c-Myc (SC-40), anti-β-actin (SC-130656) and anti-GFP (SC-9996) antibodies were purchased from Santa Cruz Biotechnology (USA). Anti-FLAG M2 (F1804) antibody and propidium iodide (PI) were purchased from Sigma (USA). Anti-caspase 3 antibody was purchased from Cell Signaling Technology (USA). Anti-Nmi antibody was prepared in our lab according to the standard protocol. Tetramethyl rhodamine isothiocyanate
The FMDV 2C interacts with N-myc and STAT interactor (Nmi)
To investigate the role of FMDV 2C in host cells, we employed a yeast two-hybrid system to screen a cDNA library generated from swine spleens for the molecules that interact with FMDV 2C. We found that one of the potential 2C-interacting proteins was N-myc and STAT interactor (Nmi) (data not shown). We first confirmed the interaction by using a coimmunoprecipitation assay in an overexpression system in which both 2C and Nmi are coexpressed in HEK293 cells (data not shown). We then further
Discussion
The 2C protein is highly conserved in the Picornaviruses, and has many functions including RNA binding activity (Banerjee and Dasgupta, 2001, Rodriguez and Carrasco, 1995), NTPase (Gorbalenya and Koonin, 1989), membrane binding activity (Echeverri et al., 1998, Teterina et al., 1997) and ATPase and GTPase activity (Pfister et al., 2000, Rodriguez and Carrasco, 1993, Sweeney et al., 2010). Our data suggest that FMDV 2C has apoptosis-inducing effects on BHK-21 cells, which is regulated by a host
Acknowledgements
We thank Ms. Trisha Agrawal for proofreading the manuscript and Mr. Zhonghua Li for assistance. This work was supported by grants from the National Natural Science Foundation of China (#30725026 and 31072117) and National Basic Research Program of China (#2006CB504303).
References (34)
- et al.
Interferon-inducible Myc/STAT-interacting protein Nmi associates with IFP 35 into a high molecular mass complex and inhibits proteasome-mediated degradation of IFP 35
Journal of Biological Chemistry
(2000) - et al.
Amino-terminal region of poliovirus 2C protein is sufficient for membrane binding
Virus Research
(1998) - et al.
Amino terminal regions of poliovirus 2C protein mediate membrane binding
Virology
(1995) Bidirectional membrane traffic between the endoplasmic reticulum and golgi apparatus
Trends in Cell Biology
(1993)- et al.
VP1 of foot-and-mouth disease virus induces apoptosis via the Akt signaling pathway
Journal of Biological Chemistry
(2004) - et al.
Poliovirus protein 2C has ATPase and GTPase activities
Journal of Biological Chemistry
(1993) - et al.
Foot-and-mouth disease virus: biology and prospects for disease control
Microbes and Infection
(2002) - et al.
The high-mobility group transcription factor Sox10 interacts with the N-myc-interacting protein Nmi
Journal of Molecular Biology
(2005) - et al.
Foot-and-mouth disease virus 2C is a hexameric AAA+ protein with a coordinated ATP hydrolysis mechanism
Journal of Biological Chemistry
(2010) - et al.
Reticulon 3 binds the 2C protein of enterovirus 71 and is required for viral replication
Journal of Biological Chemistry
(2007)
Testing the modularity of the N-terminal amphipathic helix conserved in picornavirus 2C proteins and hepatitis C NS5A protein
Virology
The pH domain containing protein CKIP-1 binds to IFP35 and Nmi and is involved in cytokine signaling
Cellular Signalling
Functional association of Nmi with Stat5 and Stat1 in IL-2- and IFNgamma-mediated signaling
Cell
Interaction of picornavirus 2C polypeptide with the viral negative-strand RNA
Journal of General Virology
Nmi protein interacts with regions that differ between MycN and Myc and is localized in the cytoplasm of neuroblastoma cells in contrast to nuclear MycN
Oncogene
Isolation and characterization of Nmi, a novel partner of Myc proteins
Oncogene
Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region
Journal of Virology
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Current address: Inspection and Quarantine Technological Center, Hebei Entry-Exit Inspection and Quarantine Bureau, Shijiazhuang 050051, China.