Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein

Utt, Age; Quirin, Tania; Saul, Sirle; Hellström, Kirsi; Ahola, Tero; Merits, Andres

doi:10.1371/journal.pone.0151616

Cited by 70 publications

(153 citation statements)

References 70 publications

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“…In order to investigate this, we employed a trans-replication system, in which RNA replication and nonstructural protein expression are uncoupled by use of two separate plasmids, one encoding the viral replicase (nonstructural polyprotein) and one encoding reporter RNA as a template for the viral replicase, expressing Renilla luciferase. We used both the previously characterized SFV (23) and a recently developed CHIKV trans-replication system (28). In both of these systems, berberine had no effect on luciferase signals resulting from the replication of the independently provided template RNA by the viral replication machinery, which was in contrast to the two drugs abamectin (ABM) and ivermectin (IVM), which significantly reduced these signals ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…), cell culture supernatant was discarded and total RNA was purified using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Viral genomic and subgenomic RNAs were detected using a digoxigenin (DIG)-labeled RNA detection probe complementary to the 3=untrans-lated region (UTR) of the CHIKV genome as described earlier (28).…”

Section: Methodsmentioning

confidence: 99%

“…The SFV trans-replication system polyprotein P123Z4 and template Tshort (template containing sequence encoding Rluc at the 5= end) and Stluc (template containing sequence encoding Rluc under the subgenomic promoter) plasmids have been described elsewhere (23). CHIKV trans-replication system polyprotein P1234 and the T7-Rluc-Tom template (template containing sequence encoding Rluc at the 5= end and the sequence encoding the red fluorescent protein Tomato under the subgenomic promoter) constructs have recently been described (28). Briefly, BSR T7/5 cells expressing bacteriophage T7 RNA polymerase seeded onto 96-well plates were transfected with the respective nonstructural polyprotein expression plasmids and plasmids encoding corresponding templates using LTX Lipofectamine according to the manufacturer's instructions and as previously described (23).…”

Section: Methodsmentioning

confidence: 99%

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The Antiviral Alkaloid Berberine Reduces Chikungunya Virus-Induced Mitogen-Activated Protein Kinase Signaling

Varghese

Thaa

Amrun

et al. 2016

J Virol

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120

103

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Chikungunya virus (CHIKV) has infected millions of people in the tropical and subtropical regions since its reemergence in the last decade. We recently identified the nontoxic plant alkaloid berberine as an antiviral substance against CHIKV in a highthroughput screen. Here, we show that berberine is effective in multiple cell types against a variety of CHIKV strains, also at a high multiplicity of infection, consolidating the potential of berberine as an antiviral drug. We excluded any effect of this compound on virus entry or on the activity of the viral replicase. A human phosphokinase array revealed that CHIKV infection specifically activated the major mitogen-activated protein kinase (MAPK) signaling pathways extracellular signal-related kinase (ERK), p38 and c-Jun NH 2 -terminal kinase (JNK). Upon treatment with berberine, this virus-induced MAPK activation was markedly reduced. Subsequent analyses with specific inhibitors of these kinases indicated that the ERK and JNK signaling cascades are important for the generation of progeny virions. In contrast to specific MAPK inhibitors, berberine lowered virus-induced activation of all major MAPK pathways and resulted in a stronger reduction in viral titers. Further, we assessed the in vivo efficacy of berberine in a mouse model and measured a significant reduction of CHIKV-induced inflammatory disease. In summary, we demonstrate the efficacy of berberine as a drug against CHIKV and highlight the importance of the MAPK signaling pathways in the alphavirus infectious cycle. IMPORTANCEChikungunya virus (CHIKV) is a mosquito-borne virus that causes severe and persistent muscle and joint pain and has recently spread to the Americas. No licensed drug exists to counter this virus. In this study, we report that the alkaloid berberine is antiviral against different CHIKV strains and in multiple human cell lines. We demonstrate that berberine collectively reduced the virus-induced activation of cellular mitogen-activated protein kinase signaling. The relevance of these signaling cascades in the viral life cycle was emphasized by specific inhibitors of these kinase pathways, which decreased the production of progeny virions. Berberine significantly reduced CHIKV-induced inflammatory disease in a mouse model, demonstrating efficacy of the drug in vivo. Overall, this work makes a strong case for pursuing berberine as a potential anti-CHIKV therapeutic compound and for exploring the MAPK signaling pathways as antiviral targets against alphavirus infections. C hikungunya virus (CHIKV) is the causative agent of chikungunya fever, a disease spread by Aedes mosquitoes and characterized by a sudden onset of febrile illness, nausea, headache, and most importantly, severe and persistent musculoskeletal pain (1). It reemerged in tropical Asia and Africa 1 decade ago and since 2013 has infected more than 1.5 million people in the Americas (2). So far, no licensed vaccine or antiviral treatment exists to counter this disease. Over the years, many research groups have focused on...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The Antiviral Alkaloid Berberine Reduces Chikungunya Virus-Induced Mitogen-Activated Protein Kinase Signaling

Varghese

Thaa

Amrun

et al. 2016

J Virol

Self Cite

120

103

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“…Negative-strand synthesis is linked to formation of viral replication compartments termed spherules, which are small, vesicular structures that form at the plasma membrane (PM) and serve as the site of vRNA replication (Figure 2). The nsPs are thought to localize at the neck of the spherules, which house dsRNA intermediates, protecting them from degradation and recognition by cellular pattern-recognition receptors (64,(80)(81)(82)(83)(84). As infection proceeds, spherules are internalized to form large cytopathic vacuoles (CPV-I), which contain markers from endosomal and lysosomal membranes (Figure 2 and refs.…”

Section: Ecology and Epidemiologymentioning

confidence: 99%

Chikungunya virus: epidemiology, replication, disease mechanisms, and prospective intervention strategies

Silva¹,

Dermody²

2017

Journal of Clinical Investigation

325

338

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“…A combination of cell-based and cell-free assays was successfully used to identify inhibitors targeting nsP1 of CHIKV (22). In addition, the activities of ns proteins can indirectly be analyzed using recently developed CHIKV trans-replication system or an assay based on the use of extracts derived from infected cells (23,24).…”

mentioning

confidence: 99%

Design and Validation of Novel Chikungunya Virus Protease Inhibitors

Das

Puusepp

Varghese

et al. 2016

Antimicrob Agents Chemother

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dChikungunya virus (CHIKV; genus Alphavirus) is the causative agent of chikungunya fever. CHIKV replication can be inhibited by some broad-spectrum antiviral compounds; in contrast, there is very little information about compounds specifically inhibiting the enzymatic activities of CHIKV replication proteins. These proteins are translated in the form of a nonstructural (ns) P1234 polyprotein precursor from the CHIKV positive-strand RNA genome. Active forms of replicase enzymes are generated using the autoproteolytic activity of nsP2. The available three-dimensional (3D) structure of nsP2 protease has made it a target for in silico drug design; however, there is thus far little evidence that the designed compounds indeed inhibit the protease activity of nsP2 and/or suppress CHIKV replication. In this study, a set of 12 compounds, predicted to interact with the active center of nsP2 protease, was designed using target-based modeling. The majority of these compounds were shown to inhibit the ability of nsP2 to process recombinant protein and synthetic peptide substrates. Furthermore, all compounds found to be active in these cell-free assays also suppressed CHIKV replication in cell culture, the 50% effective concentration (EC 50 ) of the most potent inhibitor being ϳ1.5 M. Analysis of stereoisomers of one compound revealed that inhibition of both the nsP2 protease activity and CHIKV replication depended on the conformation of the inhibitor. Combining the data obtained from different assays also indicates that some of the analyzed compounds may suppress CHIKV replication using more than one mechanism. C hikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) is the causative agent of chikungunya fever, a disease that has affected millions in the last decade. It is characterized by high fever, arthralgia, myalgia, headache, and rash. The disease is usually self-limiting; however long-lasting chronic symptoms are observed in nearly 50% of CHIKV-infected patients (1). As there is no approved vaccine or specific licensed antiviral compounds (2), the treatment of CHIKV infection is largely based on relief of symptoms.CHIKV infection can be inhibited by targeting host factors essential for virus infection. Targeting of several metabolic pathways has revealed anti-CHIKV effects of several licensed drugs (3). Compounds most likely targeting virus entry or host cell-specific components required for virus infection have been described (4-8). Several nucleosides or nucleotides, acting as pseudosubstrates for CHIKV RNA-dependent RNA polymerase and/or using another, more general mechanism of action, have shown anti-CHIKV activity (9). In addition, anti-CHIKV effects have been described for several groups of novel synthetic compounds (10-12).Computer-aided design based on molecular docking and molecular dynamics simulations and pharmacophore approach allows identifying potential in silico hits as active inhibitors for different CHIKV replicase proteins. This approach, however, requires the three-dimensional (3D) structure...

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Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein

Cited by 70 publications

References 70 publications

The Antiviral Alkaloid Berberine Reduces Chikungunya Virus-Induced Mitogen-Activated Protein Kinase Signaling

The Antiviral Alkaloid Berberine Reduces Chikungunya Virus-Induced Mitogen-Activated Protein Kinase Signaling

Chikungunya virus: epidemiology, replication, disease mechanisms, and prospective intervention strategies

Design and Validation of Novel Chikungunya Virus Protease Inhibitors

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