Reticulon 3 Binds the 2C Protein of Enterovirus 71 and Is Required for Viral Replication

Tang, Wen-Fang; Yang, Shing-Ying; Wu, Bo; Jheng, Jia-Rong; Chen, Yinli; Shih, Chung-Hsuan; Lin, Kwang‐Huei; Lai, Hsin-Chi; Tang, Petrus; Horng, Jim-Tong

doi:10.1074/jbc.m611145200

Cited by 141 publications

(135 citation statements)

References 43 publications

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“…45), encapsidation (46), and morphogenesis (47). EV71 2C has been reported to interact with host protein reticulon 3, and thus modulate viral replication (9). In this study, we demonstrate a novel function of 2C protein in modulating TNF-a-mediated NF-kB activation.…”

Section: Discussionmentioning

confidence: 59%

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Enterovirus 71 2C Protein Inhibits TNF-α–Mediated Activation of NF-κB by Suppressing IκB Kinase β Phosphorylation

Zheng

Zhang

et al. 2011

The Journal of Immunology

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Enterovirus 71 (EV71), a single, positive-stranded RNA virus, has been regarded as the most important neurotropic enterovirus after the eradication of the poliovirus. EV71 infection can cause hand, foot, and mouth disease or herpangina. Cytokine storm with elevated levels of proinflammatory and inflammatory cytokines, including TNF-α, has been proposed to explain the pathogenesis of EV71-induced disease. TNF-α–mediated NF-κB signaling pathway plays a key role in inflammatory response. We hypothesized that EV71 might also moderate host inflammation by interfering with this pathway. In this study, we tested this hypothesis and identified EV71 2C protein as an antagonist of TNF-α–mediated activation of NF-κB signaling pathway. Expression of 2C protein significantly reduced TNF-α–mediated NF-κB activation in 293T cells as measured by gene reporter and gel mobility shift assays. Furthermore, overexpression of TNFR-associated factor 2-, MEK kinase 1-, IκB kinase (IKK)α-, or IKKβ-induced NF-κB activation, but not constitutively active mutant of IKKβ (IKKβ SS/EE)-induced NF-κB activation, was inhibited by 2C protein. These data together suggested that the activation of IKKβ is most likely targeted by 2C; this notion was further strengthened by immunoblot detection of IKKβ phosphorylation and IκBα phosphorylation and degradation. Coimmunoprecipitation and colocalization of 2C and IKKβ expressed in mammalian cells provided compelling evidence that 2C interacts with IKKβ. Collectively, our data indicate that EV71 2C protein inhibits IKKβ activation and thus blocks NF-κB activation.

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Section: Discussionmentioning

confidence: 59%

“…The 2C is believed to have two functions during viral RNA synthesis, as follows: as an NTPase and directing replication complexes to cell membranes (8). Recently, EV71 2C protein has been reported to interact with host protein reticulon 3, and this interaction is required for viral replication (9).…”

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confidence: 99%

Enterovirus 71 2C Protein Inhibits TNF-α–Mediated Activation of NF-κB by Suppressing IκB Kinase β Phosphorylation

Zheng

Zhang

et al. 2011

The Journal of Immunology

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“…Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of proteins (named hnRNP A1 to hnRNP U) which have RNA-binding and protein-binding motifs. Several hnRNPs, such as A1, C1/C2, E1/E2, I (PTB), and L, are involved in the translational control of cellular or viral mRNAs which contain IRESs (12,31,47,84,100). As noted previously, hnRNP A1 and hnRNP K were found to be associated with the EV71 5ЈUTR and to play essential roles in EV71 replication (51,53).…”

Section: (Iii) Itafsmentioning

confidence: 51%

“…The amino acid sequence of EV71 2C shares 65% similarity with that of poliovirus. Like the 2C protein of PV, EV71 2C plays an important role in forming the viral RNA replication complex by binding to and rearranging mammalian cytoplasmic membranes (84). Using a yeast two-hybrid system to screen a human fetal brain cDNA library, Tang et al (84) demonstrated that RTN 3 interacts with the N-terminal domain of EV71 2C.…”

Section: (Iv) Host Factors Involved In Viralmentioning

confidence: 99%

Host Factors in Enterovirus 71 Replication

Shih

Stollar

2011

J Virol

108

131

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Enterovirus 71 (EV71) infections continue to remain an important public health problem around the world, especially in the Asia-Pacific region. There is a significant mortality rate following such infections, and there is neither any proven therapy nor a vaccine for EV71. This has spurred much fundamental research into the replication of the virus. In this review, we discuss recent work identifying host cell factors which regulate the synthesis of EV71 RNA and proteins. Three of these proteins, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), far-upstream element-binding protein 2 (FBP2), and FBP1 are nuclear proteins which in EV71-infected cells are relocalized to the cytoplasm, and they influence EV71 internal ribosome entry site (IRES) activity. hnRNP A1 stimulates IRES activity but can be replaced by hnRNP A2. FBP2 is a negative regulatory factor with respect to EV71 IRES activity, whereas FBP1 has the opposite effect. Two other proteins, hnRNP K and reticulon 3, are required for the efficient synthesis of viral RNA. The cleavage stimulation factor 64K subunit (CstF-64) is a host protein that is involved in the 3 polyadenylation of cellular pre-mRNAs, and recent work suggests that in EV71-infected cells, it may be cleaved by the EV71 3C protease. Such a cleavage would impair the processing of pre-mRNA to mature mRNAs. Host cell proteins play an important role in the replication of EV71, but much work remains to be done in order to understand how they act.Enterovirus 71 (EV71) is an important neurotropic enterovirus for which there is currently no effective therapy and no vaccine. Outbreaks of infection with this virus have occurred around the world (1,2,34,43,60,72,78,98). EV71 manifests most frequently as a childhood exanthem known as hand, foot, and mouth disease (HFMD). However, acute EV71 infection can also be associated with severe neurological disease and significant mortality. Children under 5 years old are particularly susceptible to severe forms of EV71-associated neurological complications, including aseptic meningitis, brainstem and/or cerebellar encephalitis, and acute flaccid paralysis, as well as myocarditis and rapid fatal pulmonary edema with hemorrhage (9, 40, 57, 79). EV71, first described by Schmidt et al. in 1974 (74), belongs to human enterovirus species A of the genus Enterovirus, family Picornaviridae, a large diverse group of small RNA viruses, for which poliovirus is the prototype. EV71 contains a positivestrand RNA genome of approximately 7,400 nucleotides (nt). Although not all the details of EV71 replication are understood, all enteroviruses do share a similar viral genome structure and strategy of replication. The most intensively studied enterovirus is poliovirus, which can be taken as a general model for EV71 (23).Enteroviruses are nonenveloped viruses about 30 nm in diameter. They encode four structural proteins and have an icosahedral capsid of 60 identical subunits, each of which is made up of one copy of VP1, VP2, and VP3. VP4 is an internal protein. The viral RNA has...

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“…The monoclonal antibody against GAPDH was purchased from Abnova. The polyclonal antibody against 3A (raised against glutathione S-transferase [GST]-3A containing full-length EV71 3A) was described previously (23). The monoclonal antibody against EV71 3D was a gift from Shin-Ru Shih (Chang Gung University, Taoyuan, Taiwan).…”

Section: Silac-msmentioning

confidence: 99%

Host MicroRNA miR-197 Plays a Negative Regulatory Role in the Enterovirus 71 Infectious Cycle by Targeting the RAN Protein

Tang

Huang

Chien

et al. 2016

J Virol

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Enterovirus 71 (EV71), a member of Picornaviridae, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a timedependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3= untranslated region (UTR) reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication. IMPORTANCEEnterovirus 71 (EV71) is a picornavirus with a positive-sense single-stranded RNA that globally inhibits the cellular translational system, mainly by cleaving cellular eukaryotic translation initiation factor 4G (eIF4G) and poly(A)-binding protein (PABP), which inhibits the association of the ribosome with the host capped mRNA. We used a microRNA (miRNA) microarray chip to identify the host miRNA 197 (miR-197) that was downregulated by EV71. We also used quantitative mass spectrometry and a target site prediction tool to identify the miR-197 target genes. During viral infection, the expression of the target protein RAN was upregulated considerably, and there was a parallel downregulation of miR-197. The nuclear transport of viral 3D/3CD protein and of the host proteins involved in viral replication proceeded in an RAN-dependent manner. We have identified a new mechanism in picornavirus through which EV71-induced cellular miRNA downregulation can regulate host protein levels to facilitate viral replication. M icroRNAs (miRNAs) are a group of endogenous, highly conserved noncoding single-stranded RNAs (ϳ22 nucleotides [nt] in length) that are transcribed from specialized genes and undergo series processing to generate final mature miRNAs. The human genome contains more than 2,580 distinct mature miRNA genes (http://www.mirbase.org/) (1), which act as...

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Reticulon 3 Binds the 2C Protein of Enterovirus 71 and Is Required for Viral Replication

Cited by 141 publications

References 43 publications

Enterovirus 71 2C Protein Inhibits TNF-α–Mediated Activation of NF-κB by Suppressing IκB Kinase β Phosphorylation

Enterovirus 71 2C Protein Inhibits TNF-α–Mediated Activation of NF-κB by Suppressing IκB Kinase β Phosphorylation

Host Factors in Enterovirus 71 Replication

Host MicroRNA miR-197 Plays a Negative Regulatory Role in the Enterovirus 71 Infectious Cycle by Targeting the RAN Protein

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