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MicroRNA regulation of human protease genes essential for influenza virus replication.

Meliopoulos VA, Andersen LE, Brooks P, Yan X, Bakre A, Coleman JK, Tompkins SM, Tripp RA - PLoS ONE (2012)

Bottom Line: However, the rapid emergence of drug resistance has emphasized the need for new drug targets.The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis.Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

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RNAi of DPP3 (siDPP3) inhibits influenza replication by modulation of apoptotic genes.A549 cells were reverse transfected with 50 nM of siDPP3 or siNEG. After 48 hours, cells were infected with A/WSN/33 at an MOI of 0.001. After 18 hours of infection, cellular RNA was isolated and apoptosis gene expression profiles were determined by array. Gene expression was normalized to GAPDH levels. Silencing DPP3 resulted in upregulated levels of the pro-apoptotic genes BCL2L10, TNFSF10, TNFSF25 and TNFSF8. Data is representative of three independent experiments. * p<0.05.
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pone-0037169-g004: RNAi of DPP3 (siDPP3) inhibits influenza replication by modulation of apoptotic genes.A549 cells were reverse transfected with 50 nM of siDPP3 or siNEG. After 48 hours, cells were infected with A/WSN/33 at an MOI of 0.001. After 18 hours of infection, cellular RNA was isolated and apoptosis gene expression profiles were determined by array. Gene expression was normalized to GAPDH levels. Silencing DPP3 resulted in upregulated levels of the pro-apoptotic genes BCL2L10, TNFSF10, TNFSF25 and TNFSF8. Data is representative of three independent experiments. * p<0.05.

Mentions: Dynamic pathway analysis predicted DPP3 to be involved in apoptotic pathways (Figure S3), and as a luciferase/Renilla pathway reporter system was unavailable, a human apoptosis array was performed to determine the level of mRNA expression of various pro- and anti-apoptotic host genes in relation to DPP3 expression (Figure 4). After confirming RNAi silencing (>95%) of DPP3 by PCR (data not shown), gene expression was compared in cellular RNA extracted from infected A549 cells treated with either siDPP3 or siNEG (50 nM). While most genes tested did not vary in expression levels relative to siNEG-treated cells, four pro-apoptotic genes were found to be substantially increased when DPP3 was silenced: BCL2L10, TNFSF10, TNFRSF25, and TNFSF8. Of these four genes, TNFSF10 was significantly (p<0.05) increased 6-fold, and TNFSF8 was significantly (p<0.05) increased 5-fold compared to siNEG control treated cells. TNFRSF25 was substantially increased as well. These findings show that inhibition of DPP3 expression initiates higher expression of pro-apoptotic factors, features that may negatively impact influenza virus replication and associated modulation of cellular apoptosis. Silencing of the other four target protease genes did not significantly affect levels of pro- or anti-apoptotic genes compared to cells treated with siNEG as predicted by the Ingenuity pathway analysis, although TNFSF10 was substantially increased when cells were treated with siADAMTS7 (Table S1).


MicroRNA regulation of human protease genes essential for influenza virus replication.

Meliopoulos VA, Andersen LE, Brooks P, Yan X, Bakre A, Coleman JK, Tompkins SM, Tripp RA - PLoS ONE (2012)

RNAi of DPP3 (siDPP3) inhibits influenza replication by modulation of apoptotic genes.A549 cells were reverse transfected with 50 nM of siDPP3 or siNEG. After 48 hours, cells were infected with A/WSN/33 at an MOI of 0.001. After 18 hours of infection, cellular RNA was isolated and apoptosis gene expression profiles were determined by array. Gene expression was normalized to GAPDH levels. Silencing DPP3 resulted in upregulated levels of the pro-apoptotic genes BCL2L10, TNFSF10, TNFSF25 and TNFSF8. Data is representative of three independent experiments. * p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351457&req=5

pone-0037169-g004: RNAi of DPP3 (siDPP3) inhibits influenza replication by modulation of apoptotic genes.A549 cells were reverse transfected with 50 nM of siDPP3 or siNEG. After 48 hours, cells were infected with A/WSN/33 at an MOI of 0.001. After 18 hours of infection, cellular RNA was isolated and apoptosis gene expression profiles were determined by array. Gene expression was normalized to GAPDH levels. Silencing DPP3 resulted in upregulated levels of the pro-apoptotic genes BCL2L10, TNFSF10, TNFSF25 and TNFSF8. Data is representative of three independent experiments. * p<0.05.
Mentions: Dynamic pathway analysis predicted DPP3 to be involved in apoptotic pathways (Figure S3), and as a luciferase/Renilla pathway reporter system was unavailable, a human apoptosis array was performed to determine the level of mRNA expression of various pro- and anti-apoptotic host genes in relation to DPP3 expression (Figure 4). After confirming RNAi silencing (>95%) of DPP3 by PCR (data not shown), gene expression was compared in cellular RNA extracted from infected A549 cells treated with either siDPP3 or siNEG (50 nM). While most genes tested did not vary in expression levels relative to siNEG-treated cells, four pro-apoptotic genes were found to be substantially increased when DPP3 was silenced: BCL2L10, TNFSF10, TNFRSF25, and TNFSF8. Of these four genes, TNFSF10 was significantly (p<0.05) increased 6-fold, and TNFSF8 was significantly (p<0.05) increased 5-fold compared to siNEG control treated cells. TNFRSF25 was substantially increased as well. These findings show that inhibition of DPP3 expression initiates higher expression of pro-apoptotic factors, features that may negatively impact influenza virus replication and associated modulation of cellular apoptosis. Silencing of the other four target protease genes did not significantly affect levels of pro- or anti-apoptotic genes compared to cells treated with siNEG as predicted by the Ingenuity pathway analysis, although TNFSF10 was substantially increased when cells were treated with siADAMTS7 (Table S1).

Bottom Line: However, the rapid emergence of drug resistance has emphasized the need for new drug targets.The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis.Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

Show MeSH
Related in: MedlinePlus