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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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Related in: MedlinePlus

RNAi of individual host protease genes down-regulates replication of a clinical influenza isolate.A549 cells were reverse transfected with 100 nM of the novel siRNA targeting siADAMTS7, siCPE, siDPP3, siMST1, and siPRSS12. After 48 hours, cells were infected with A/New Caledonia/20/99 at an MOI of 0.1 in the presence of 1 ug/ml TPCK-trypsin. After 48 hours of infection, cellular supernatant was tested for infectious virus production by a modified TCID50. Data is expressed as TCID50/ml. Data is representative of two independent experiments. (*p<0.05 vs. siNEG).
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pone-0037169-g002: RNAi of individual host protease genes down-regulates replication of a clinical influenza isolate.A549 cells were reverse transfected with 100 nM of the novel siRNA targeting siADAMTS7, siCPE, siDPP3, siMST1, and siPRSS12. After 48 hours, cells were infected with A/New Caledonia/20/99 at an MOI of 0.1 in the presence of 1 ug/ml TPCK-trypsin. After 48 hours of infection, cellular supernatant was tested for infectious virus production by a modified TCID50. Data is expressed as TCID50/ml. Data is representative of two independent experiments. (*p<0.05 vs. siNEG).

Mentions: Since A/WSN/33 was used in the primary and secondary screens because of its ability to grow in the absence of trypsin, a feature facilitating high throughput screening, cross-validation of ADAMTS7, CPE, DPP3, MST1, and PRSS12 genes was performed in A549 cells with A/New Caledonia/20/99. Using the same siRNAs previously used to validate the genes (Figure 1C), A549 cells treated with 100 nM of the siRNAs significantly (p<0.05) reduced A/New Caledonia/20/99 virus replication (Figure 2). The differences in viral titer were at least 1.5 logs lower compared to siNEG-treated cells for all five genes examined. These results show that ADAMTS7, CPE, DPP3, MST1, and PRSS12 contribute to influenza A virus replication, and provide promising disease intervention targets.


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 individual host protease genes down-regulates replication of a clinical influenza isolate.A549 cells were reverse transfected with 100 nM of the novel siRNA targeting siADAMTS7, siCPE, siDPP3, siMST1, and siPRSS12. After 48 hours, cells were infected with A/New Caledonia/20/99 at an MOI of 0.1 in the presence of 1 ug/ml TPCK-trypsin. After 48 hours of infection, cellular supernatant was tested for infectious virus production by a modified TCID50. Data is expressed as TCID50/ml. Data is representative of two independent experiments. (*p<0.05 vs. siNEG).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037169-g002: RNAi of individual host protease genes down-regulates replication of a clinical influenza isolate.A549 cells were reverse transfected with 100 nM of the novel siRNA targeting siADAMTS7, siCPE, siDPP3, siMST1, and siPRSS12. After 48 hours, cells were infected with A/New Caledonia/20/99 at an MOI of 0.1 in the presence of 1 ug/ml TPCK-trypsin. After 48 hours of infection, cellular supernatant was tested for infectious virus production by a modified TCID50. Data is expressed as TCID50/ml. Data is representative of two independent experiments. (*p<0.05 vs. siNEG).
Mentions: Since A/WSN/33 was used in the primary and secondary screens because of its ability to grow in the absence of trypsin, a feature facilitating high throughput screening, cross-validation of ADAMTS7, CPE, DPP3, MST1, and PRSS12 genes was performed in A549 cells with A/New Caledonia/20/99. Using the same siRNAs previously used to validate the genes (Figure 1C), A549 cells treated with 100 nM of the siRNAs significantly (p<0.05) reduced A/New Caledonia/20/99 virus replication (Figure 2). The differences in viral titer were at least 1.5 logs lower compared to siNEG-treated cells for all five genes examined. These results show that ADAMTS7, CPE, DPP3, MST1, and PRSS12 contribute to influenza A virus replication, and provide promising disease intervention targets.

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