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The Rac1 inhibitor NSC23766 exerts anti-influenza virus properties by affecting the viral polymerase complex activity.

Dierkes R, Warnking K, Liedmann S, Seyer R, Ludwig S, Ehrhardt C - PLoS ONE (2014)

Bottom Line: The frequent emergence of new influenza viruses in the human population underlines the urgent need for antiviral therapeutics in addition to the preventative vaccination against the seasonal flu.Inhibition of Rac1 by NSC23766 resulted in impaired replication of a wide variety of influenza viruses, including a human virus strain of the pandemic from 2009 as well as highly pathogenic avian virus strains.Furthermore, we identified a crucial role of Rac1 for the activity of the viral polymerase complex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Virology (IMV), Centre of Molecular Virology (ZMBE), Westfälische Wilhelms-University, Münster, Germany.

ABSTRACT
The frequent emergence of new influenza viruses in the human population underlines the urgent need for antiviral therapeutics in addition to the preventative vaccination against the seasonal flu. To circumvent the development of resistance, recent antiviral approaches target cellular proteins needed by the virus for efficient replication. We investigated the contribution of the small GTPase Rac1 to the replication of influenza viruses. Inhibition of Rac1 by NSC23766 resulted in impaired replication of a wide variety of influenza viruses, including a human virus strain of the pandemic from 2009 as well as highly pathogenic avian virus strains. Furthermore, we identified a crucial role of Rac1 for the activity of the viral polymerase complex. The antiviral potential of NSC23766 was confirmed in mouse experiments, identifying Rac1 as a new cellular target for therapeutic treatment of influenza virus infections.

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NSC23766 treatment results in reduced viral mRNA levels caused by impaired activity of the viral polymerase complex.(A) A549 cells were infected with A/Puerto-Rico/8/34 rec. (moi = 5) and subsequently treated with NSC23766 (100 µM) 30 min post-infection. Six hours p.i., the RNA was isolated for qRT-PCR and the fold increase of ns and pb1 mRNA compared to uninfected cells was determined using the housekeeping gene gapdh as the internal standard. Treatment of cells with cycloheximide (10 µg/ml, right panel) was performed to inhibit de novo protein synthesis. (B) Viral polymerase activity was assayed in a mini-genome system using a luciferase reporter gene driven by the viral RNA promoter. A549 cells were transfected with plasmids encoding PB1, PB2, PA, and NP in addition to the luciferase reporter plasmid. Six hours post-transfection, NSC23766 was added at a final concentration of 100 µM and cells were incubated for a further 24 h. Luciferase activity was assayed in cell lysates. Data represent means ± SD of three independent experiments including three biological samples. Statistical significance was evaluated with Student's t-test (* p<0.05; ** p<0.01). (C) A549 cells were infected with A/Puerto Rico/8/34 rec. (moi = 0.01) for 30 min and were subsequently treated with NSC23766 (100 µM). After eight hours of infection nuclear extracts were prepared and subjected to Western blot analysis. The amounts of Rac1 and the viral proteins PB1, M1, and NS1 were analyzed in cytosolic and nuclear fractions. Detection of the cytosolic protein α-Tubulin and the nuclear localized protein Drosha served as control for efficient fractionation.
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pone-0088520-g005: NSC23766 treatment results in reduced viral mRNA levels caused by impaired activity of the viral polymerase complex.(A) A549 cells were infected with A/Puerto-Rico/8/34 rec. (moi = 5) and subsequently treated with NSC23766 (100 µM) 30 min post-infection. Six hours p.i., the RNA was isolated for qRT-PCR and the fold increase of ns and pb1 mRNA compared to uninfected cells was determined using the housekeeping gene gapdh as the internal standard. Treatment of cells with cycloheximide (10 µg/ml, right panel) was performed to inhibit de novo protein synthesis. (B) Viral polymerase activity was assayed in a mini-genome system using a luciferase reporter gene driven by the viral RNA promoter. A549 cells were transfected with plasmids encoding PB1, PB2, PA, and NP in addition to the luciferase reporter plasmid. Six hours post-transfection, NSC23766 was added at a final concentration of 100 µM and cells were incubated for a further 24 h. Luciferase activity was assayed in cell lysates. Data represent means ± SD of three independent experiments including three biological samples. Statistical significance was evaluated with Student's t-test (* p<0.05; ** p<0.01). (C) A549 cells were infected with A/Puerto Rico/8/34 rec. (moi = 0.01) for 30 min and were subsequently treated with NSC23766 (100 µM). After eight hours of infection nuclear extracts were prepared and subjected to Western blot analysis. The amounts of Rac1 and the viral proteins PB1, M1, and NS1 were analyzed in cytosolic and nuclear fractions. Detection of the cytosolic protein α-Tubulin and the nuclear localized protein Drosha served as control for efficient fractionation.

Mentions: After demonstrating the reducing effect of NSC23766 on viral protein synthesis, we investigated if inhibitor treatment affects viral mRNA expression. Thus, the expression levels of ns and pb1 mRNA in the presence and absence of the inhibitor were determined by qRT-PCR (Fig. 5A, left panel). Indeed, mRNA expression of the investigated genes was impaired upon NSC23766 treatment. Based on these results, we concluded that the reduced amount of viral mRNA expression leads to the observed lowering of viral proteins.


The Rac1 inhibitor NSC23766 exerts anti-influenza virus properties by affecting the viral polymerase complex activity.

Dierkes R, Warnking K, Liedmann S, Seyer R, Ludwig S, Ehrhardt C - PLoS ONE (2014)

NSC23766 treatment results in reduced viral mRNA levels caused by impaired activity of the viral polymerase complex.(A) A549 cells were infected with A/Puerto-Rico/8/34 rec. (moi = 5) and subsequently treated with NSC23766 (100 µM) 30 min post-infection. Six hours p.i., the RNA was isolated for qRT-PCR and the fold increase of ns and pb1 mRNA compared to uninfected cells was determined using the housekeeping gene gapdh as the internal standard. Treatment of cells with cycloheximide (10 µg/ml, right panel) was performed to inhibit de novo protein synthesis. (B) Viral polymerase activity was assayed in a mini-genome system using a luciferase reporter gene driven by the viral RNA promoter. A549 cells were transfected with plasmids encoding PB1, PB2, PA, and NP in addition to the luciferase reporter plasmid. Six hours post-transfection, NSC23766 was added at a final concentration of 100 µM and cells were incubated for a further 24 h. Luciferase activity was assayed in cell lysates. Data represent means ± SD of three independent experiments including three biological samples. Statistical significance was evaluated with Student's t-test (* p<0.05; ** p<0.01). (C) A549 cells were infected with A/Puerto Rico/8/34 rec. (moi = 0.01) for 30 min and were subsequently treated with NSC23766 (100 µM). After eight hours of infection nuclear extracts were prepared and subjected to Western blot analysis. The amounts of Rac1 and the viral proteins PB1, M1, and NS1 were analyzed in cytosolic and nuclear fractions. Detection of the cytosolic protein α-Tubulin and the nuclear localized protein Drosha served as control for efficient fractionation.
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pone-0088520-g005: NSC23766 treatment results in reduced viral mRNA levels caused by impaired activity of the viral polymerase complex.(A) A549 cells were infected with A/Puerto-Rico/8/34 rec. (moi = 5) and subsequently treated with NSC23766 (100 µM) 30 min post-infection. Six hours p.i., the RNA was isolated for qRT-PCR and the fold increase of ns and pb1 mRNA compared to uninfected cells was determined using the housekeeping gene gapdh as the internal standard. Treatment of cells with cycloheximide (10 µg/ml, right panel) was performed to inhibit de novo protein synthesis. (B) Viral polymerase activity was assayed in a mini-genome system using a luciferase reporter gene driven by the viral RNA promoter. A549 cells were transfected with plasmids encoding PB1, PB2, PA, and NP in addition to the luciferase reporter plasmid. Six hours post-transfection, NSC23766 was added at a final concentration of 100 µM and cells were incubated for a further 24 h. Luciferase activity was assayed in cell lysates. Data represent means ± SD of three independent experiments including three biological samples. Statistical significance was evaluated with Student's t-test (* p<0.05; ** p<0.01). (C) A549 cells were infected with A/Puerto Rico/8/34 rec. (moi = 0.01) for 30 min and were subsequently treated with NSC23766 (100 µM). After eight hours of infection nuclear extracts were prepared and subjected to Western blot analysis. The amounts of Rac1 and the viral proteins PB1, M1, and NS1 were analyzed in cytosolic and nuclear fractions. Detection of the cytosolic protein α-Tubulin and the nuclear localized protein Drosha served as control for efficient fractionation.
Mentions: After demonstrating the reducing effect of NSC23766 on viral protein synthesis, we investigated if inhibitor treatment affects viral mRNA expression. Thus, the expression levels of ns and pb1 mRNA in the presence and absence of the inhibitor were determined by qRT-PCR (Fig. 5A, left panel). Indeed, mRNA expression of the investigated genes was impaired upon NSC23766 treatment. Based on these results, we concluded that the reduced amount of viral mRNA expression leads to the observed lowering of viral proteins.

Bottom Line: The frequent emergence of new influenza viruses in the human population underlines the urgent need for antiviral therapeutics in addition to the preventative vaccination against the seasonal flu.Inhibition of Rac1 by NSC23766 resulted in impaired replication of a wide variety of influenza viruses, including a human virus strain of the pandemic from 2009 as well as highly pathogenic avian virus strains.Furthermore, we identified a crucial role of Rac1 for the activity of the viral polymerase complex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Virology (IMV), Centre of Molecular Virology (ZMBE), Westfälische Wilhelms-University, Münster, Germany.

ABSTRACT
The frequent emergence of new influenza viruses in the human population underlines the urgent need for antiviral therapeutics in addition to the preventative vaccination against the seasonal flu. To circumvent the development of resistance, recent antiviral approaches target cellular proteins needed by the virus for efficient replication. We investigated the contribution of the small GTPase Rac1 to the replication of influenza viruses. Inhibition of Rac1 by NSC23766 resulted in impaired replication of a wide variety of influenza viruses, including a human virus strain of the pandemic from 2009 as well as highly pathogenic avian virus strains. Furthermore, we identified a crucial role of Rac1 for the activity of the viral polymerase complex. The antiviral potential of NSC23766 was confirmed in mouse experiments, identifying Rac1 as a new cellular target for therapeutic treatment of influenza virus infections.

Show MeSH
Related in: MedlinePlus