Amiloride Derivatives Inhibit Coxsackievirus B3 RNA Replication

Harrison, David N.; Purcell, Damian F. J.; Anderson, David A.; Petrou, Steven

doi:10.1128/jvi.01374-07

Cited by 39 publications

(51 citation statements)

References 28 publications

(38 reference statements)

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“…EIPA had a stronger inhibitory effect than amiloride (Fig. 1B), consistent with its stronger effect on CVB3 replication in cell culture (11). S299T 3D pol was less susceptible to inhibition than WT 3D pol ( Fig.…”

Section: Resultssupporting

confidence: 72%

“…Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) inhibit CVB3 propagation in cell culture by inhibiting viral genome replication (11). Two amino acid substitutions in 3D pol (S299T and A372V) confer partial resistance of the virus to the compounds, suggesting that amiloride analogues may act as inhibitors of CVB3 3D pol (11). Here we show that amiloride and EIPA inhibit VPg uridylylation and RNA elongation by CVB3 3D pol in vitro, acting as competitive inhibitors with respect to nucleoside triphosphates (NTPs) and Mg 2ϩ .…”

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confidence: 99%

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Amiloride Is a Competitive Inhibitor of Coxsackievirus B3 RNA Polymerase

Gazina

Smidansky

Holien

et al. 2011

J Virol

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). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D pol in vitro, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D pol by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg 2؉ . Docking analysis suggested a binding site for amiloride and EIPA in 3D pol , located in close proximity to one of the Mg 2؉ ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase.The family Picornaviridae is a family of positive-sense RNA viruses which contains numerous human pathogens, causing poliomyelitis, myocarditis, meningitis, hepatitis, the common cold, and other diseases. The viral genomic RNA is ϳ7,500 nucleotides (nt) long and contains a 22-amino-acid peptide, VPg, covalently linked to the 5Ј end and a poly(A) tail at the 3Ј end. Genome replication occurs via synthesis of a complementary, negative-sense RNA strand, catalyzed by the viral RNA-dependent RNA polymerase, 3D pol , in association with a number of viral and host proteins. It is a complex process taking place in membrane-associated replication complexes in the cytoplasm of infected cells (reviewed in references 5, 7, and 23). The synthesis of both complementary and genomic RNA strands is initiated by attachment of two UMP nucleotides to a tyrosine residue of VPg, resulting in the production of VPgpUpU. VPg uridylylation requires a template. In the case of genomic strand synthesis, an internal stem-loop in the genomic RNA strand (cis-acting replication element [CRE]) is used as a template, with subsequent translocation of VPg-pUpU to the 3Ј end of the complementary strand and its elongation into a full-length genomic strand (9,15,16,26). The complementary strand synthesis does not absolutely depend on CRE-it can also be templated by the poly(A) tail of the genomic strand (9,15,16,26).VPg uridylylation and RNA elongation have been reproduced successfully in vitro by use of purified components. VPg uridylylation assays require 3D pol , VPg, CRE or poly(A), UTP, and Mg 2ϩ or Mn 2ϩ (19,20), with CRE-templated reaction stimulated by viral proteins 3CD or 3C (18, 19), whereas an elongation assay mix contains an RNA primer instead of VPg (21).Coxsackievirus B3 (CVB3) is a picornavirus responsible for 14 to 32% of human myocarditis cases (1). Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) inhibit CVB3 propagation in cell culture by inhibiting viral genome replication (11). Two amino acid substitutions in 3D pol (S299T and A372V) confer partial resistance of the virus to the compounds, suggesting that amiloride analogues may act as inhibitors of CVB3 3D pol (11). Here we show that amiloride and EIPA inhibit VPg uridylylation and RNA elongation by CVB3 3D pol in vitro, acting as competitive inhibitors with respect to...

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Section: Resultssupporting

confidence: 72%

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confidence: 99%

Amiloride Is a Competitive Inhibitor of Coxsackievirus B3 RNA Polymerase

Gazina

Smidansky

Holien

et al. 2011

J Virol

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“…EIPA was previously shown to inhibit propagation of human rhinovirus 2 and Coxsackie B3 virus in HeLa cells (59). In this study, EIPA failed to inhibit HEV71 infection.…”

Section: Discussionmentioning

confidence: 51%

The Essential Role of Clathrin-mediated Endocytosis in the Infectious Entry of Human Enterovirus 71

Hussain

Leong

et al. 2011

Journal of Biological Chemistry

126

118

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Little is currently known about the infectious entry process of human enterovirus 71 (HEV71) into host cells, which may represent potential anti-viral targeting sites. In this study a targeted small-interfering RNA (siRNA) screening platform assay was established and validated to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics, and endosomal trafficking essential for HEV71 infection. Screen evaluation was conducted via the expression of well characterized dominant-negative mutants, bioimaging studies (double-labeled immunofluorescence assays, transmission electron microscopy analysis), secondary siRNA-based dosage dependence studies, and drug inhibition assays. The infectious entry of HEV71 into rhabdomyosarcoma cells was shown to be significantly inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis (CME) that include AP2A1, ARRB1, CLTC, CLTCL1, SYNJ1, ARPC5, PAK1, ROCK1, and WASF1. The functional role of CME was verified by the observation of strong co-localization between HEV71 particles and clathrin as well as dose-dependent inhibition of HEV71 infection upon siRNA knockdown of CME-associated genes. HEV71 entry by CME was further confirmed via inhibition by dominant-negative EPS15 mutants and treatment of CME drug inhibitors, with more than 80% inhibition observed at 20 M chlorpromazine. Furthermore, HEV71 infection was shown to be sensitive to the disruption of human genes in regulating early to late endosomal trafficking as well as endosomal acidic pH. The identification of clathrin-mediated endocytosis as the entry pathway for HEV71 infection of susceptible host cells contributes to a better understanding of HEV71 pathogenesis and enables future development of anti-viral strategies against HEV71 infection.

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“…The possibility that this drug affects entry can be excluded because immunofluorescence microscopy clearly showed that virus entered the cells in its presence. For coxsackievirus B3 and for HRV2, inhibition of RNA replication and of virus release, respectively, by amiloride has been suggested (12,18). Our data, rather, point to inhibition of RNA release into the cytosol, RNA translation, or even inhibition of polyprotein processing.…”

Section: Entry Of Hrv2mentioning

confidence: 51%

“…Viral replication is not required for this process because translation of the incoming RNA produces the proteinase 2A in sufficient amounts for cleavage of this cellular substrate (13). In this way, possible effects of the drugs on later steps, such as RNA synthesis and/or virus release (12,18), can be excluded. Furthermore, there is no need to maintain the cells in the presence of the drug for extended times, thus avoiding interference by cytotoxic effects.…”

Section: Discussionmentioning

confidence: 99%

Human Rhinovirus 14 Enters Rhabdomyosarcoma Cells Expressing ICAM-1 by a Clathrin-, Caveolin-, and Flotillin-Independent Pathway

Khan

Pickl‐Herk

Gajdzik

et al. 2010

J Virol

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Intercellular adhesion molecule 1 (ICAM-1) mediates binding and entry of major group human rhinoviruses (HRVs). Whereas the entry pathway of minor group HRVs has been studied in detail and is comparatively well understood, the pathway taken by major group HRVs is largely unknown. Use of immunofluorescence microscopy, colocalization with specific endocytic markers, dominant negative mutants, and pharmacological inhibitors allowed us to demonstrate that the major group virus HRV14 enters rhabdomyosarcoma cells transfected to express human ICAM-1 in a clathrin-, caveolin-, and flotillin-independent manner. Electron microscopy revealed that many virions accumulated in long tubular structures, easily distinguishable from clathrin-coated pits and caveolae. Virus entry was strongly sensitive to the Na ؉ /H ؉ ion exchange inhibitor amiloride and moderately sensitive to cytochalasin D. Thus, cellular uptake of HRV14 occurs via a pathway exhibiting some, but not all, characteristics of macropinocytosis and is similar to that recently described for adenovirus 3 entry via ␣ v integrin/CD46 in HeLa cells.

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Amiloride Derivatives Inhibit Coxsackievirus B3 RNA Replication

Cited by 39 publications

References 28 publications

Amiloride Is a Competitive Inhibitor of Coxsackievirus B3 RNA Polymerase

Amiloride Is a Competitive Inhibitor of Coxsackievirus B3 RNA Polymerase

The Essential Role of Clathrin-mediated Endocytosis in the Infectious Entry of Human Enterovirus 71

Human Rhinovirus 14 Enters Rhabdomyosarcoma Cells Expressing ICAM-1 by a Clathrin-, Caveolin-, and Flotillin-Independent Pathway

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