Chikungunya virus requires cellular chloride channels for efficient genome replication.

Marietta Müller,Eleanor J A A Todd,Andrew Tuplin,Natalie Slivinski,Magdalena Karwatka,Henna Khalid,Jamel Mankouri,Andres Merits,Raymond Li,Gregory Gromowski

doi:10.1371/journal.pntd.0007703

Abstract

Chikungunya virus (CHIKV) is a re-emerging, pathogenic alphavirus that is transmitted to humans by Aedes spp. mosquitoes—causing fever and debilitating joint pain, with frequent long-term health implications and high morbidity. The CHIKV lifecycle is poorly understood and specific antiviral therapeutics or vaccines are lacking. In this study, we investigated the role of host-cell chloride (Cl-) channels on CHIKV replication.We demonstrate that specific pharmacological Cl- channel inhibitors significantly inhibit CHIKV replication in a dose-dependent manner, suggesting that Cl-channels are pro-viral factors in human cells. Further analysis of the effect of the inhibitors on CHIKV attachment, entry, viral protein expression and replicon replication demonstrated that Cl- channels are specifically required for efficient CHIKV genome replication. This was conserved in mosquito cells, where CHIKV replication and genome copy number was significantly reduced following Cl- channel inhibition. siRNA silencing identified chloride intracellular channels 1 and 4 (CLIC1 and CLIC4, respectively) as required for efficient CHIKV replication and protein affinity chromatography showed low levels of CLIC1 in complex with CHIKV nsP3, an essential component of the viral replication machinery. In summary, for the first time we demonstrate that efficient replication of the CHIKV genome depends on cellular Cl- channels, in both human and mosquito cells and identifies CLIC1 and CLIC4 as agonists of CHIKV replication in human cells. We observe a modest interaction, either direct or indirect, between CLIC1 and nsP3 and hypothesize that CLIC1 may play a role in the formation/maintenance of CHIKV replication complexes. These findings advance our molecular understanding of CHIKV replication and identify potential druggable targets for the treatment and prevention of CHIKV mediated disease.

Highlights

Chikungunya virus (CHIKV) is a mosquito-borne virus of the Alphavirus genus in the Togaviridae family
Using pharmacological compounds, we show that Cl- channels are required for efficient replication of the virus genome, identify two specific channels (CLIC1 and CLIC4) required for replication and demonstrate that CLIC1 may interact with viral non-structural protein 3—an essential component of the CHIKV replicase complex
Treatment with nitro-2-3-phenylpropylamino benzoic acid (NPPB) resulted in an 18-fold reduction in CHIKV progeny compared to untreated cells, while treatment with DIDS and 9-anthracene carboxylic acid (9-ACA) led to an 8-fold decrease in CHIKV titer (p 0.05) (Fig 1A)

Summary

Introduction

Chikungunya virus (CHIKV) is a mosquito-borne virus of the Alphavirus genus in the Togaviridae family. It was first isolated during an outbreak in Tanzania in 1952 [1], since which its geographic range has expanded globally to include almost 40 countries [2]. CHIKV causes chikungunya fever which is characterized by high fever, maculopapular rash, myalgia and debilitating arthralgia [4]. Due to the chronic debilitating symptoms and sequela that persist for up to 3 years [8], CHIKV has a major impact on morbidity and loss of economic productivity within at-risk populations [9]. Treatment of CHIKV-associated disease is limited to the relief of symptoms with no licensed vaccines or direct acting antivirals currently available

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