Limits...
PI4K-beta and MKNK1 are regulators of hepatitis C virus IRES-dependent translation.

Lupberger J, Casanova C, Fischer B, Weiss A, Fofana I, Fontaine N, Fujiwara T, Renaud M, Kopp A, Schuster C, Brino L, Baumert TF, Thoma C - Sci Rep (2015)

Bottom Line: Picornaviridae and Flaviviridae including hepatitis C virus (HCV) evade this process using internal ribosomal entry sequences (IRESs).Although HCV IRES translation is a prerequisite for HCV replication, only few host factors critical for IRES activity are known and the global regulator network remains largely unknown.We demonstrate that the HCV host cell cofactors PI4K and MKNK1 are positive regulators of HCV IRES translation representing a novel pathway with a functional relevance for the HCV life cycle and IRES-mediated translation of viral RNA.

View Article: PubMed Central - PubMed

Affiliation: Inserm U1110, Institut de Recherche sur les Maladies Virales et Hépatiques Strasbourg, France.

ABSTRACT
Cellular translation is down-regulated by host antiviral responses. Picornaviridae and Flaviviridae including hepatitis C virus (HCV) evade this process using internal ribosomal entry sequences (IRESs). Although HCV IRES translation is a prerequisite for HCV replication, only few host factors critical for IRES activity are known and the global regulator network remains largely unknown. Since signal transduction is an import regulator of viral infections and the host antiviral response we combined a functional RNAi screen targeting the human signaling network with a HCV IRES-specific reporter mRNA assay. We demonstrate that the HCV host cell cofactors PI4K and MKNK1 are positive regulators of HCV IRES translation representing a novel pathway with a functional relevance for the HCV life cycle and IRES-mediated translation of viral RNA.

No MeSH data available.


Related in: MedlinePlus

Kinase activity of MKNK1 promotes IRES-dependent translation and HCV infection.(a) A specific inhibitor of MKNK1 kinase activity preferentially impairs IRES-dependent over CAP-dependent luciferase reporter translation. Huh7.5 cells incubated for one hour with increasing doses of MKNK1 inhibitor. Treated cells were co-transfected with reporter mRNAs (IRES-firefly and cap-renilla) for four hours prior measuring of the firefly and renilla luciferase activity as described in the methods section. Data are expressed as means of the ratio of firefly/renilla luciferase activity +/− SEM. *p < 0.01 (Student’s t-test, n = 15 of three independent experiments). (b) MKNK1 inhibitor has only a minor impact on cell viability of Huh7.5 cells. After 5 h incubation with increasing concentrations of MKNK1 inhibitor the cell number was assessed by counting and the cell viability was assessed using Presto Blue. Data are expressed as means +/− SEM (n = 3 of one representative experiment). (c) MKNK1 inhibitor significantly and dose-dependently inhibits HCV infection at absent cell toxicity. Huh7.5.1 cells were pre-treated for one hour with increasing concentrations of MKNK1 inhibitor prior infection with cell culture-derived HCV (strain Luc-Jc1). Infected cells were maintained in the presence of the respective MKNK1 inhibitor concentration prior cell lysis and the measurement of the luciferase activity at day three. Data are expressed as means +/− SEM. *p < 0.01 (Student’s t-test, n = 3 of one representative experiment). All inhibitor dilutions and controls in this figure were prepared in a constant background of 1% DMSO.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4555030&req=5

f2: Kinase activity of MKNK1 promotes IRES-dependent translation and HCV infection.(a) A specific inhibitor of MKNK1 kinase activity preferentially impairs IRES-dependent over CAP-dependent luciferase reporter translation. Huh7.5 cells incubated for one hour with increasing doses of MKNK1 inhibitor. Treated cells were co-transfected with reporter mRNAs (IRES-firefly and cap-renilla) for four hours prior measuring of the firefly and renilla luciferase activity as described in the methods section. Data are expressed as means of the ratio of firefly/renilla luciferase activity +/− SEM. *p < 0.01 (Student’s t-test, n = 15 of three independent experiments). (b) MKNK1 inhibitor has only a minor impact on cell viability of Huh7.5 cells. After 5 h incubation with increasing concentrations of MKNK1 inhibitor the cell number was assessed by counting and the cell viability was assessed using Presto Blue. Data are expressed as means +/− SEM (n = 3 of one representative experiment). (c) MKNK1 inhibitor significantly and dose-dependently inhibits HCV infection at absent cell toxicity. Huh7.5.1 cells were pre-treated for one hour with increasing concentrations of MKNK1 inhibitor prior infection with cell culture-derived HCV (strain Luc-Jc1). Infected cells were maintained in the presence of the respective MKNK1 inhibitor concentration prior cell lysis and the measurement of the luciferase activity at day three. Data are expressed as means +/− SEM. *p < 0.01 (Student’s t-test, n = 3 of one representative experiment). All inhibitor dilutions and controls in this figure were prepared in a constant background of 1% DMSO.

Mentions: To analyze the impact of gene silencing on IRES- and cap-dependent translation, respectively, we co-transfected reporter mRNAs (100 ng/0.3 cm2) in gene silenced hepatoma cells 48 h post siRNA transfection as described previously2627 (Fig. 1): Renilla luciferase mRNA initiated by a m7G cap structure and firefly luciferase mRNA containing a non-physiological adenosine cap structure (‘A-cap’) and the HCV IRES element. The A-cap maintains stability of the mRNA, but is not recognized by the cap binding complex. Luciferase expression was assessed by a Mithras LB 940 (Berthold Technologies) using Dual-Luciferase Reporter Assay or Bright-Glo (Promega). Toxicity of gene silencing was assessed using MTT (Sigma) and Presto Blue (Sigma) for the tertiary screen. In the primary screen targeting 893 genes we identified 46 candidates that predominant impact HCV IRES-dependent over cap-dependent translation (Supplementary table S1). In a secondary validation screen using side-by-side transfection of firefly reporter mRNAs of cap and HCV IRES (Fig. 1) we validated 11 hits of the primary screen (Supplementary table S2) and thus confirmed that these genes predominantly affect HCV IRES- rather than cap-dependent translation. As HCV IRES translation is a key step in the viral life cycle we assessed whether the identified genes confirm as positive regulators of HCV infection. We validated the results from the two foregoing screens (performed with siRNA pools) in a tertiary screen by at least two of four individual siRNAs per target (Fig. 1) to minimize off-target effects and validated mRNA knockdown specificity of the final hits by qPCR (Supplementary figure S1). As a result we confirmed that silencing of 3 genes from the secondary screening have a reproducible and significant impact on HCV infection: phosphatidylinositol 4-kinase catalytical subunit beta (PIK4CB), MAP kinase interacting serine/threonine kinase 1 (MKNK1), and tumor protein D52-like 3 (NYD-SP25) (Supplementary table S3). We were not able to validate a specific silencing of NYD-SP25 mRNA expression and therefore cannot rule out that off-target effects being responsible for the impact in IRES-dependent translation. Using a specific inhibitor of MKNK128 we demonstrate a significant (p < 0.01, t-test) and preferential inhibition of IRES-dependent translation over cap-dependent translation of luciferase reporter genes (Fig. 2a) at absent cell toxicity (Fig. 2b). Long-term treatment with the MKNK1 inhibitor over three days significantly (p < 0.01, t-test) block HCV infection (Fig. 2c) demonstrating that the molecular mechanism of action of MKNK1 involves its kinase activity.


PI4K-beta and MKNK1 are regulators of hepatitis C virus IRES-dependent translation.

Lupberger J, Casanova C, Fischer B, Weiss A, Fofana I, Fontaine N, Fujiwara T, Renaud M, Kopp A, Schuster C, Brino L, Baumert TF, Thoma C - Sci Rep (2015)

Kinase activity of MKNK1 promotes IRES-dependent translation and HCV infection.(a) A specific inhibitor of MKNK1 kinase activity preferentially impairs IRES-dependent over CAP-dependent luciferase reporter translation. Huh7.5 cells incubated for one hour with increasing doses of MKNK1 inhibitor. Treated cells were co-transfected with reporter mRNAs (IRES-firefly and cap-renilla) for four hours prior measuring of the firefly and renilla luciferase activity as described in the methods section. Data are expressed as means of the ratio of firefly/renilla luciferase activity +/− SEM. *p < 0.01 (Student’s t-test, n = 15 of three independent experiments). (b) MKNK1 inhibitor has only a minor impact on cell viability of Huh7.5 cells. After 5 h incubation with increasing concentrations of MKNK1 inhibitor the cell number was assessed by counting and the cell viability was assessed using Presto Blue. Data are expressed as means +/− SEM (n = 3 of one representative experiment). (c) MKNK1 inhibitor significantly and dose-dependently inhibits HCV infection at absent cell toxicity. Huh7.5.1 cells were pre-treated for one hour with increasing concentrations of MKNK1 inhibitor prior infection with cell culture-derived HCV (strain Luc-Jc1). Infected cells were maintained in the presence of the respective MKNK1 inhibitor concentration prior cell lysis and the measurement of the luciferase activity at day three. Data are expressed as means +/− SEM. *p < 0.01 (Student’s t-test, n = 3 of one representative experiment). All inhibitor dilutions and controls in this figure were prepared in a constant background of 1% DMSO.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555030&req=5

f2: Kinase activity of MKNK1 promotes IRES-dependent translation and HCV infection.(a) A specific inhibitor of MKNK1 kinase activity preferentially impairs IRES-dependent over CAP-dependent luciferase reporter translation. Huh7.5 cells incubated for one hour with increasing doses of MKNK1 inhibitor. Treated cells were co-transfected with reporter mRNAs (IRES-firefly and cap-renilla) for four hours prior measuring of the firefly and renilla luciferase activity as described in the methods section. Data are expressed as means of the ratio of firefly/renilla luciferase activity +/− SEM. *p < 0.01 (Student’s t-test, n = 15 of three independent experiments). (b) MKNK1 inhibitor has only a minor impact on cell viability of Huh7.5 cells. After 5 h incubation with increasing concentrations of MKNK1 inhibitor the cell number was assessed by counting and the cell viability was assessed using Presto Blue. Data are expressed as means +/− SEM (n = 3 of one representative experiment). (c) MKNK1 inhibitor significantly and dose-dependently inhibits HCV infection at absent cell toxicity. Huh7.5.1 cells were pre-treated for one hour with increasing concentrations of MKNK1 inhibitor prior infection with cell culture-derived HCV (strain Luc-Jc1). Infected cells were maintained in the presence of the respective MKNK1 inhibitor concentration prior cell lysis and the measurement of the luciferase activity at day three. Data are expressed as means +/− SEM. *p < 0.01 (Student’s t-test, n = 3 of one representative experiment). All inhibitor dilutions and controls in this figure were prepared in a constant background of 1% DMSO.
Mentions: To analyze the impact of gene silencing on IRES- and cap-dependent translation, respectively, we co-transfected reporter mRNAs (100 ng/0.3 cm2) in gene silenced hepatoma cells 48 h post siRNA transfection as described previously2627 (Fig. 1): Renilla luciferase mRNA initiated by a m7G cap structure and firefly luciferase mRNA containing a non-physiological adenosine cap structure (‘A-cap’) and the HCV IRES element. The A-cap maintains stability of the mRNA, but is not recognized by the cap binding complex. Luciferase expression was assessed by a Mithras LB 940 (Berthold Technologies) using Dual-Luciferase Reporter Assay or Bright-Glo (Promega). Toxicity of gene silencing was assessed using MTT (Sigma) and Presto Blue (Sigma) for the tertiary screen. In the primary screen targeting 893 genes we identified 46 candidates that predominant impact HCV IRES-dependent over cap-dependent translation (Supplementary table S1). In a secondary validation screen using side-by-side transfection of firefly reporter mRNAs of cap and HCV IRES (Fig. 1) we validated 11 hits of the primary screen (Supplementary table S2) and thus confirmed that these genes predominantly affect HCV IRES- rather than cap-dependent translation. As HCV IRES translation is a key step in the viral life cycle we assessed whether the identified genes confirm as positive regulators of HCV infection. We validated the results from the two foregoing screens (performed with siRNA pools) in a tertiary screen by at least two of four individual siRNAs per target (Fig. 1) to minimize off-target effects and validated mRNA knockdown specificity of the final hits by qPCR (Supplementary figure S1). As a result we confirmed that silencing of 3 genes from the secondary screening have a reproducible and significant impact on HCV infection: phosphatidylinositol 4-kinase catalytical subunit beta (PIK4CB), MAP kinase interacting serine/threonine kinase 1 (MKNK1), and tumor protein D52-like 3 (NYD-SP25) (Supplementary table S3). We were not able to validate a specific silencing of NYD-SP25 mRNA expression and therefore cannot rule out that off-target effects being responsible for the impact in IRES-dependent translation. Using a specific inhibitor of MKNK128 we demonstrate a significant (p < 0.01, t-test) and preferential inhibition of IRES-dependent translation over cap-dependent translation of luciferase reporter genes (Fig. 2a) at absent cell toxicity (Fig. 2b). Long-term treatment with the MKNK1 inhibitor over three days significantly (p < 0.01, t-test) block HCV infection (Fig. 2c) demonstrating that the molecular mechanism of action of MKNK1 involves its kinase activity.

Bottom Line: Picornaviridae and Flaviviridae including hepatitis C virus (HCV) evade this process using internal ribosomal entry sequences (IRESs).Although HCV IRES translation is a prerequisite for HCV replication, only few host factors critical for IRES activity are known and the global regulator network remains largely unknown.We demonstrate that the HCV host cell cofactors PI4K and MKNK1 are positive regulators of HCV IRES translation representing a novel pathway with a functional relevance for the HCV life cycle and IRES-mediated translation of viral RNA.

View Article: PubMed Central - PubMed

Affiliation: Inserm U1110, Institut de Recherche sur les Maladies Virales et Hépatiques Strasbourg, France.

ABSTRACT
Cellular translation is down-regulated by host antiviral responses. Picornaviridae and Flaviviridae including hepatitis C virus (HCV) evade this process using internal ribosomal entry sequences (IRESs). Although HCV IRES translation is a prerequisite for HCV replication, only few host factors critical for IRES activity are known and the global regulator network remains largely unknown. Since signal transduction is an import regulator of viral infections and the host antiviral response we combined a functional RNAi screen targeting the human signaling network with a HCV IRES-specific reporter mRNA assay. We demonstrate that the HCV host cell cofactors PI4K and MKNK1 are positive regulators of HCV IRES translation representing a novel pathway with a functional relevance for the HCV life cycle and IRES-mediated translation of viral RNA.

No MeSH data available.


Related in: MedlinePlus