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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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Analysis of host gene involvement in major cellular pathways.A: A549 cells were reverse cotransfected with CRE/CREB reporter plasmid or the appropriate control plasmid and 100 nM of the novel siRNA. After 24 h incubation, the transfection media was replaced with culture media. Cells were mock infected or infected with A/WSN/33 at an MOI of 0.001 the following day. After 24 h, culture supernatant was analyzed for luciferase expression. Luciferase units were normalized to Renilla expression. * p<0.05, ** p<0.01 compared to siNEG control (mock), # p<0.05, ## p<0.01 compared to siNEG control (A/WSN/33) B: A549 cells were reversed cotransfected with NF-κB reporter plasmid or the appropriate control plasmid and 100 nM of the novel siRNA. After 24 h incubation, the transfection media was replaced with culture media. Cells were mock infected or infected with A/WSN/33 at an MOI of 0.001 the following day. After 24 h, culture supernatant was analyzed for luciferase expression. Luciferase units were normalized to Renilla expression. * p<0.05, ** p<0.01 compared to siNEG control (mock), # p<0.05, ## p<0.01 compared to siNEG control (A/WSN/33). Data is representative of three independent experiments.
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pone-0037169-g003: Analysis of host gene involvement in major cellular pathways.A: A549 cells were reverse cotransfected with CRE/CREB reporter plasmid or the appropriate control plasmid and 100 nM of the novel siRNA. After 24 h incubation, the transfection media was replaced with culture media. Cells were mock infected or infected with A/WSN/33 at an MOI of 0.001 the following day. After 24 h, culture supernatant was analyzed for luciferase expression. Luciferase units were normalized to Renilla expression. * p<0.05, ** p<0.01 compared to siNEG control (mock), # p<0.05, ## p<0.01 compared to siNEG control (A/WSN/33) B: A549 cells were reversed cotransfected with NF-κB reporter plasmid or the appropriate control plasmid and 100 nM of the novel siRNA. After 24 h incubation, the transfection media was replaced with culture media. Cells were mock infected or infected with A/WSN/33 at an MOI of 0.001 the following day. After 24 h, culture supernatant was analyzed for luciferase expression. Luciferase units were normalized to Renilla expression. * p<0.05, ** p<0.01 compared to siNEG control (mock), # p<0.05, ## p<0.01 compared to siNEG control (A/WSN/33). Data is representative of three independent experiments.

Mentions: Expanding from the primary screen toward defining host cell pathways involved in influenza virus replication requires assessment of the signaling pathways, molecular networks, and biological processes that are linked to the five validated host genes. Dynamic pathway analysis identified three major pathways for the five validated genes, i.e. NF-κB pathway, CRE/CREB signaling pathway, and cellular apoptosis (Figure S3). To verify the host genes in these pathways, A549 cells were transfected with a luciferase assay reporter plasmid governed by the promoter of interest linked to a firefly luciferase gene. The level of pathway activation was determined by luciferase expression, which was normalized to a control plasmid expressing Renilla luciferase to account for transfection efficiency. Pathway analysis suggested that MST1 and PRSS12 genes participate in the CRE/CREB signaling pathway. RNAi of MST1 by siRNA targeting (siMST1) led to a significant (p<0.05) increase in CRE/CREB signaling compared to the siNEG and CRE controls in the presence or absence of influenza virus infection (Figure 3A). RNAi of PRSS12 by siPRSS12 did not affect CRE/CREB activation relative to controls. Interestingly, RNAi of DPP3 using siDPP3 significantly (p<0.05) increased CRE/CREB activation under mock-infected conditions despite DPP3 not being implicated in the CRE/CREB pathway, and silencing of ADAMTS7 significantly (p<0.05) abrogated CRE/CREB activation during infection. Pathway analysis also indicated that ADAMTS7, CPE, and MST1 genes were potentially involved in the NF-κB activation pathway (Figure S3). siADAMTS7 significantly (p<0.01) abrogated NF-κB activation regardless of infection or mock-treatment (Figure 3B). Interestingly, a similar finding was observed for DPP3, a gene not previously linked to the NF-κB signaling pathway by our analysis. siCPE did not affect NF-κB activation under mock conditions, but during infection NF-κB activation levels were significantly (p<0.05) downregulated. siMST1 also had a negative effect on NF-κB activation regardless of infection. Elevated NF-κB levels were also seen in controls receiving the NF-κB plasmid alone but this was not significant compared of siNEG controls.


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)

Analysis of host gene involvement in major cellular pathways.A: A549 cells were reverse cotransfected with CRE/CREB reporter plasmid or the appropriate control plasmid and 100 nM of the novel siRNA. After 24 h incubation, the transfection media was replaced with culture media. Cells were mock infected or infected with A/WSN/33 at an MOI of 0.001 the following day. After 24 h, culture supernatant was analyzed for luciferase expression. Luciferase units were normalized to Renilla expression. * p<0.05, ** p<0.01 compared to siNEG control (mock), # p<0.05, ## p<0.01 compared to siNEG control (A/WSN/33) B: A549 cells were reversed cotransfected with NF-κB reporter plasmid or the appropriate control plasmid and 100 nM of the novel siRNA. After 24 h incubation, the transfection media was replaced with culture media. Cells were mock infected or infected with A/WSN/33 at an MOI of 0.001 the following day. After 24 h, culture supernatant was analyzed for luciferase expression. Luciferase units were normalized to Renilla expression. * p<0.05, ** p<0.01 compared to siNEG control (mock), # p<0.05, ## p<0.01 compared to siNEG control (A/WSN/33). Data is representative of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351457&req=5

pone-0037169-g003: Analysis of host gene involvement in major cellular pathways.A: A549 cells were reverse cotransfected with CRE/CREB reporter plasmid or the appropriate control plasmid and 100 nM of the novel siRNA. After 24 h incubation, the transfection media was replaced with culture media. Cells were mock infected or infected with A/WSN/33 at an MOI of 0.001 the following day. After 24 h, culture supernatant was analyzed for luciferase expression. Luciferase units were normalized to Renilla expression. * p<0.05, ** p<0.01 compared to siNEG control (mock), # p<0.05, ## p<0.01 compared to siNEG control (A/WSN/33) B: A549 cells were reversed cotransfected with NF-κB reporter plasmid or the appropriate control plasmid and 100 nM of the novel siRNA. After 24 h incubation, the transfection media was replaced with culture media. Cells were mock infected or infected with A/WSN/33 at an MOI of 0.001 the following day. After 24 h, culture supernatant was analyzed for luciferase expression. Luciferase units were normalized to Renilla expression. * p<0.05, ** p<0.01 compared to siNEG control (mock), # p<0.05, ## p<0.01 compared to siNEG control (A/WSN/33). Data is representative of three independent experiments.
Mentions: Expanding from the primary screen toward defining host cell pathways involved in influenza virus replication requires assessment of the signaling pathways, molecular networks, and biological processes that are linked to the five validated host genes. Dynamic pathway analysis identified three major pathways for the five validated genes, i.e. NF-κB pathway, CRE/CREB signaling pathway, and cellular apoptosis (Figure S3). To verify the host genes in these pathways, A549 cells were transfected with a luciferase assay reporter plasmid governed by the promoter of interest linked to a firefly luciferase gene. The level of pathway activation was determined by luciferase expression, which was normalized to a control plasmid expressing Renilla luciferase to account for transfection efficiency. Pathway analysis suggested that MST1 and PRSS12 genes participate in the CRE/CREB signaling pathway. RNAi of MST1 by siRNA targeting (siMST1) led to a significant (p<0.05) increase in CRE/CREB signaling compared to the siNEG and CRE controls in the presence or absence of influenza virus infection (Figure 3A). RNAi of PRSS12 by siPRSS12 did not affect CRE/CREB activation relative to controls. Interestingly, RNAi of DPP3 using siDPP3 significantly (p<0.05) increased CRE/CREB activation under mock-infected conditions despite DPP3 not being implicated in the CRE/CREB pathway, and silencing of ADAMTS7 significantly (p<0.05) abrogated CRE/CREB activation during infection. Pathway analysis also indicated that ADAMTS7, CPE, and MST1 genes were potentially involved in the NF-κB activation pathway (Figure S3). siADAMTS7 significantly (p<0.01) abrogated NF-κB activation regardless of infection or mock-treatment (Figure 3B). Interestingly, a similar finding was observed for DPP3, a gene not previously linked to the NF-κB signaling pathway by our analysis. siCPE did not affect NF-κB activation under mock conditions, but during infection NF-κB activation levels were significantly (p<0.05) downregulated. siMST1 also had a negative effect on NF-κB activation regardless of infection. Elevated NF-κB levels were also seen in controls receiving the NF-κB plasmid alone but this was not significant compared of siNEG controls.

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