Nitric oxide inhibition of coxsackievirus replication in vitro.

Zaragoza, Carlos; Ocampo, Christopher; Saura, Marta; McMillan, Audrey; Lowenstein, Charles J.

doi:10.1172/jci119702

Cited by 85 publications

(67 citation statements)

References 76 publications

(50 reference statements)

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“…iNOS regulates the expression of NO, which has been shown to inhibit the early stage of viral replication, thereby preventing viral spread and enhancing the clearance of virus from the host (45). In vitro experiments have suggested that NO does so by inhibiting viral RNA and DNA replication and protein synthesis (46)(47)(48)(49). In this study, we demonstrate that IFN a and b induce expression of Spsb1, which helps to regulate iNOS induction and the subsequent production of NO in what appears to be a negative feedback loop.…”

Section: Discussionmentioning

confidence: 99%

TLR Regulation of SPSB1 Controls Inducible Nitric Oxide Synthase Induction

Lewis

Kolesnik

Kuang

et al. 2011

The Journal of Immunology

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The mammalian innate immune system has evolved to recognize foreign molecules derived from pathogens via the TLRs. TLR3 and TLR4 can signal via the TIR domain-containing adapter inducing IFN-β (TRIF), which results in the transcription of a small array of genes, including IFN-β. Inducible NO synthase (iNOS), which catalyzes the production of NO, is induced by a range of stimuli, including cytokines and microbes. NO is a potent source of reactive nitrogen species that play an important role in killing intracellular pathogens and forms a crucial component of host defense. We have recently identified iNOS as a target of the mammalian SPSB2 protein. The SOCS box is a peptide motif, which, in conjunction with elongins B and C, recruits cullin-5 and Rbx-2 to form an active E3 ubiquitin ligase complex. In this study, we show that SPSB1 is the only SPSB family member to be regulated by the same TLR pathways that induce iNOS expression and characterize the interaction between SPSB1 and iNOS. Through the use of SPSB1 transgenic mouse macrophages and short hairpin RNA knockdown of SPSB1, we show that SPSB1 controls both the induction of iNOS and the subsequent production of NO downstream of TLR3 and TLR4. Further, we demonstrate that regulation of iNOS by SPSB1 is dependent on the proteasome. These results suggest that SPSB1 acts through a negative-feedback loop that, together with SPSB2, controls the extent of iNOS induction and NO production.

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Section: Discussionmentioning

confidence: 99%

TLR Regulation of SPSB1 Controls Inducible Nitric Oxide Synthase Induction

Lewis

Kolesnik

Kuang

et al. 2011

The Journal of Immunology

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“…22 Conversely, NO inhibits CVB3 replication in vitro, and targeted disruption of the NOS 2 gene in mice leads to aggravated myocardial damage after infection with CVB3. 23,24 The mechanisms of the protective effect of NO in enteroviral cardiomyopathy are incompletely understood.…”

Section: See P 2162mentioning

confidence: 99%

“…32 SNAP was added 1 hour after infection as described. 23,30 Dystrophin cleavage activity was measured in HeLa cell extracts prepared in protease 2A cleavage buffer as above.…”

Section: Cell Culture and Virus Infectionmentioning

confidence: 99%

“…We elected to use HeLa cells, an established cell culture model of CVB3 infection. 23,30 Because HeLa cells do not express dystrophin, protease 2A cleavage activity on dystrophin was measured in HeLa cell extracts with the mouse dystrophin miniprotein. 11 As shown in Figure 3, lysate from uninfected HeLa cells did not contain any dystrophin cleavage activity or coxsackieviral protease 2A.…”

Section: No Inhibits Protease 2a Activity In Cvb3-infected Hela Cellsmentioning

confidence: 99%

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Nitric Oxide Inhibits Dystrophin Proteolysis by Coxsackieviral Protease 2A Through S -Nitrosylation

Badorff

Fichtlscherer²,

Rhoads³

et al. 2000

Circulation

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Background-Infection with enteroviruses like coxsackievirus B3 (CVB3) as well as genetic dystrophin deficiency can cause dilated cardiomyopathy. We recently identified cleavage and functional impairment of dystrophin by the viral protease 2A during CVB3-infection as a molecular mechanism that may contribute to the pathogenesis of enterovirusinduced cardiomyopathy. Nitric oxide (NO) is elevated in human dilated cardiomyopathy, but the relevance of this finding is unknown. In mice, NO inhibits CVB3 myocarditis. Therefore, we investigated the effects of NO on the coxsackieviral protease 2A. Methods and Results-In vitro, NO donors like PAPA-NONOate inhibited the cleavage of human and mouse dystrophin by recombinant coxsackievirus B protease 2A in a dose-dependent manner (IC 50 , 51 mol/L). In CVB3-infected HeLa cells, addition of the NO donor SNAP inhibited protease 2A catalytic activity on dystrophin. Because this inhibitory effect was reversed by the thiol-protecting agent DTT, we investigated whether NO S-nitrosylates the protease 2A. In vitro, NO nitrosylated the active-site cysteine (C110) of the coxsackieviral protease 2A, as demonstrated by site-directed mutagenesis. Within living COS-7 cells, SNAP-induced S-nitrosylation of this site was confirmed with electron spin resonance spectroscopy. Conclusions-These data demonstrate inactivation of a coxsackieviral protease 2A by NO through active-cysteine S-nitrosylation in vitro and intracellularly. Given that the enteroviral protease 2A cleaves mouse and human dystrophin, NO may be protective in human heart failure with an underlying enteroviral pathogenesis through inhibition of dystrophin proteolysis.

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“…NO produced by NOS2 has potent antiviral activity against a number of viruses (11)(12)(13)(14)(15). However, NO also contributes to inflammation and injury through formation of toxic reactive nitrogen intermediates (6,10,16).…”

mentioning

confidence: 99%

Central Role of Double-Stranded RNA-Activated Protein Kinase in Microbial Induction of Nitric Oxide Synthase

Uetani

Der

Zamanian-Daryoush

et al. 2000

The Journal of Immunology

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NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-γ, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-κB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-κB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.

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Nitric oxide inhibition of coxsackievirus replication in vitro.

Abstract: Nitric oxide is a radical molecule with antibacterial, -parasitic, and -viral properties. We investigated the mechanism of NO inhibition of Coxsackievirus B3 (

Cited by 85 publications

References 76 publications

TLR Regulation of SPSB1 Controls Inducible Nitric Oxide Synthase Induction

TLR Regulation of SPSB1 Controls Inducible Nitric Oxide Synthase Induction

Nitric Oxide Inhibits Dystrophin Proteolysis by Coxsackieviral Protease 2A Through S -Nitrosylation

Central Role of Double-Stranded RNA-Activated Protein Kinase in Microbial Induction of Nitric Oxide Synthase

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