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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 does not directly affect expression of the interferon β regulated gene mxa.(A) A549 cells were infected with A/Puerto-Rico/8/34 rec. (moi = 5) and subsequently treated with NSC23766 (100 µM) 30 min after infection. (B) Alternatively, the cells were transfected with cellular or viral RNA (0.5 µg/12-well) in presence or absence of NSC23766 (100 µM). (A–B) After six hours of incubation, total RNA was isolated for qRT-PCR and the fold increase of mxa mRNA was determined using the housekeeping gene gapdh as the internal standard. Data represent means ± SD of three independent experiments including three biological samples.
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pone-0088520-g007: NSC23766 treatment does not directly affect expression of the interferon β regulated gene mxa.(A) A549 cells were infected with A/Puerto-Rico/8/34 rec. (moi = 5) and subsequently treated with NSC23766 (100 µM) 30 min after infection. (B) Alternatively, the cells were transfected with cellular or viral RNA (0.5 µg/12-well) in presence or absence of NSC23766 (100 µM). (A–B) After six hours of incubation, total RNA was isolated for qRT-PCR and the fold increase of mxa mRNA was determined using the housekeeping gene gapdh as the internal standard. Data represent means ± SD of three independent experiments including three biological samples.

Mentions: So far, our results indicated a high antiviral activity of NSC23766 in IV-infected cells. This seemed to be contradictory to our former study, which identified Rac1 as positive regulator of interferon β expression after IV infections [12]. Thus, we investigated the effect of NSC23766 treatment on IV-induced interferon response. After infection of A549 cells and subsequent treatment with NSC23766, the changes in mRNA levels of mxa, a strictly interferon β regulated gene, were analyzed by qRT-PCR (Fig. 7A). The infection resulted in an 8-fold increase of mxa expression compared to mock-infected cells. The enhanced mxa expression was slightly reduced when Rac1 was inhibited by NSC23766 (6-fold increase compared to mock infected control). Since the NSC23766-mediated reduction in mxa expression might have been a secondary effect of the impaired viral replication we used viral RNA as a virus-derived non dynamic interferon stimulus in an additional experimental setting (Fig. 7B). A549 cells were transfected with RNA isolated from infected (viral RNA) or uninfected (cellular RNA) cells, in presence or absence of NSC23766. After six hours of incubation, mxa mRNA levels were analyzed by qRT-PCR. The transfection of viral RNA resulted in a more than 300-fold increase of mxa expression compared to cellular RNA-transfected control cells. The same induction of the interferon response was achieved when NSC23766 was added to the transfection medium. In summary, it can be concluded that the expression of interferon β is not directly affected by NSC23766-mediated Rac1 inhibition.


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 does not directly affect expression of the interferon β regulated gene mxa.(A) A549 cells were infected with A/Puerto-Rico/8/34 rec. (moi = 5) and subsequently treated with NSC23766 (100 µM) 30 min after infection. (B) Alternatively, the cells were transfected with cellular or viral RNA (0.5 µg/12-well) in presence or absence of NSC23766 (100 µM). (A–B) After six hours of incubation, total RNA was isolated for qRT-PCR and the fold increase of mxa mRNA was determined using the housekeeping gene gapdh as the internal standard. Data represent means ± SD of three independent experiments including three biological samples.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3921225&req=5

pone-0088520-g007: NSC23766 treatment does not directly affect expression of the interferon β regulated gene mxa.(A) A549 cells were infected with A/Puerto-Rico/8/34 rec. (moi = 5) and subsequently treated with NSC23766 (100 µM) 30 min after infection. (B) Alternatively, the cells were transfected with cellular or viral RNA (0.5 µg/12-well) in presence or absence of NSC23766 (100 µM). (A–B) After six hours of incubation, total RNA was isolated for qRT-PCR and the fold increase of mxa mRNA was determined using the housekeeping gene gapdh as the internal standard. Data represent means ± SD of three independent experiments including three biological samples.
Mentions: So far, our results indicated a high antiviral activity of NSC23766 in IV-infected cells. This seemed to be contradictory to our former study, which identified Rac1 as positive regulator of interferon β expression after IV infections [12]. Thus, we investigated the effect of NSC23766 treatment on IV-induced interferon response. After infection of A549 cells and subsequent treatment with NSC23766, the changes in mRNA levels of mxa, a strictly interferon β regulated gene, were analyzed by qRT-PCR (Fig. 7A). The infection resulted in an 8-fold increase of mxa expression compared to mock-infected cells. The enhanced mxa expression was slightly reduced when Rac1 was inhibited by NSC23766 (6-fold increase compared to mock infected control). Since the NSC23766-mediated reduction in mxa expression might have been a secondary effect of the impaired viral replication we used viral RNA as a virus-derived non dynamic interferon stimulus in an additional experimental setting (Fig. 7B). A549 cells were transfected with RNA isolated from infected (viral RNA) or uninfected (cellular RNA) cells, in presence or absence of NSC23766. After six hours of incubation, mxa mRNA levels were analyzed by qRT-PCR. The transfection of viral RNA resulted in a more than 300-fold increase of mxa expression compared to cellular RNA-transfected control cells. The same induction of the interferon response was achieved when NSC23766 was added to the transfection medium. In summary, it can be concluded that the expression of interferon β is not directly affected by NSC23766-mediated Rac1 inhibition.

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