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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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Inhibition of Rac1 by NSC23766 leads to reduced synthesis of viral proteins.(A) A549 cells, infected with A/Puerto-Rico/8/34 rec. (moi = 5), were treated with NSC23766 (100 µM) 30 min p.i.. Cell lysates were prepared at the indicated times and subjected to Western blot analysis. The viral protein synthesis was monitored by PB1, M1, and NS1 detection and visualization of ERK2 served as a loading control. (B) A549 cells were transfected with a constitutively active CMV promoter luciferase plasmid for six hours. Subsequently, NSC23766 was added at a concentration of 100 µM. Untreated cells served as a negative control, while cycloheximide treatment (10 µg/ml) was used as a positive control. After 24 h incubation, cells were subjected to luciferase assay. Data represent means ± SD of three independent experiments including three biological samples. Statistical significance was evaluated with Student's t-test (*** p<0.001).
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pone-0088520-g004: Inhibition of Rac1 by NSC23766 leads to reduced synthesis of viral proteins.(A) A549 cells, infected with A/Puerto-Rico/8/34 rec. (moi = 5), were treated with NSC23766 (100 µM) 30 min p.i.. Cell lysates were prepared at the indicated times and subjected to Western blot analysis. The viral protein synthesis was monitored by PB1, M1, and NS1 detection and visualization of ERK2 served as a loading control. (B) A549 cells were transfected with a constitutively active CMV promoter luciferase plasmid for six hours. Subsequently, NSC23766 was added at a concentration of 100 µM. Untreated cells served as a negative control, while cycloheximide treatment (10 µg/ml) was used as a positive control. After 24 h incubation, cells were subjected to luciferase assay. Data represent means ± SD of three independent experiments including three biological samples. Statistical significance was evaluated with Student's t-test (*** p<0.001).

Mentions: To further examine the Rac1-mediated virus-supportive function during IV replication, we focused on the expression of viral proteins within the first replication cycle in the presence of NSC23766. Expression of the viral proteins PB1, M1, and NS1 was reduced upon NSC23766 treatment (Fig. 4, lanes 7 and 9) in comparison to untreated control cells (Fig. 4, lanes 6 and 8) 6 and 8 h after infection. However, to rule out a general blockage of cellular transcription or translation by NSC23766, expression of a reporter gene (luciferase) driven by a constitutive active promoter (CMV) was monitored upon NSC23766 treatment (Fig. 4B). In comparison to untreated samples, NSC23766 treatment for 24 h showed no reduction of luciferase activity, while treatment with cycloheximide, a general translation blocker, resulted in significantly lower expression of the reporter gene. Thus, it can be concluded that the inhibitor specifically reduces synthesis of IV 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)

Inhibition of Rac1 by NSC23766 leads to reduced synthesis of viral proteins.(A) A549 cells, infected with A/Puerto-Rico/8/34 rec. (moi = 5), were treated with NSC23766 (100 µM) 30 min p.i.. Cell lysates were prepared at the indicated times and subjected to Western blot analysis. The viral protein synthesis was monitored by PB1, M1, and NS1 detection and visualization of ERK2 served as a loading control. (B) A549 cells were transfected with a constitutively active CMV promoter luciferase plasmid for six hours. Subsequently, NSC23766 was added at a concentration of 100 µM. Untreated cells served as a negative control, while cycloheximide treatment (10 µg/ml) was used as a positive control. After 24 h incubation, cells were subjected to luciferase assay. Data represent means ± SD of three independent experiments including three biological samples. Statistical significance was evaluated with Student's t-test (*** p<0.001).
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pone-0088520-g004: Inhibition of Rac1 by NSC23766 leads to reduced synthesis of viral proteins.(A) A549 cells, infected with A/Puerto-Rico/8/34 rec. (moi = 5), were treated with NSC23766 (100 µM) 30 min p.i.. Cell lysates were prepared at the indicated times and subjected to Western blot analysis. The viral protein synthesis was monitored by PB1, M1, and NS1 detection and visualization of ERK2 served as a loading control. (B) A549 cells were transfected with a constitutively active CMV promoter luciferase plasmid for six hours. Subsequently, NSC23766 was added at a concentration of 100 µM. Untreated cells served as a negative control, while cycloheximide treatment (10 µg/ml) was used as a positive control. After 24 h incubation, cells were subjected to luciferase assay. Data represent means ± SD of three independent experiments including three biological samples. Statistical significance was evaluated with Student's t-test (*** p<0.001).
Mentions: To further examine the Rac1-mediated virus-supportive function during IV replication, we focused on the expression of viral proteins within the first replication cycle in the presence of NSC23766. Expression of the viral proteins PB1, M1, and NS1 was reduced upon NSC23766 treatment (Fig. 4, lanes 7 and 9) in comparison to untreated control cells (Fig. 4, lanes 6 and 8) 6 and 8 h after infection. However, to rule out a general blockage of cellular transcription or translation by NSC23766, expression of a reporter gene (luciferase) driven by a constitutive active promoter (CMV) was monitored upon NSC23766 treatment (Fig. 4B). In comparison to untreated samples, NSC23766 treatment for 24 h showed no reduction of luciferase activity, while treatment with cycloheximide, a general translation blocker, resulted in significantly lower expression of the reporter gene. Thus, it can be concluded that the inhibitor specifically reduces synthesis of IV 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