Targeting Cell Division Cycle 25 Homolog B To Regulate Influenza Virus Replication

Influenza virus is a worldwide global health concern causing seasonal morbidity mortality and economic burden. Chemotherapeutics is available; however, rapid emergence of drug-resistant influenza virus strains has reduced its efficacy. Thus, there is a need to discover novel antiviral agents. in this study, RNA interference (RNAi) was used to screen host genes required for influenza virus replication. One pro-influenza virus host gene identified was dual-specificity phosphatase cell division cycle 25 B (CDC25B). RNAi screening of CDC25B resulted in reduced influenza A virus replication, and a CDC25B small-molecule inhibitor (NSC95397) inhibited influenza A virus replication in a dose-dependent fashion. Viral RNA synthesis was reduced by NSC95397 in favor of increased beta interferon (IFN-beta) expression, and NSC95397 was found to interfere with nuclear localization and chromatin association of NS1, an influenza virus protein. As NS1 has been shown to be chromatin associated and to suppress host transcription, it is likely that CDC25B supports NS1 nuclear function to hijack host transcription machinery in favor of viral RNA synthesis, a process that is blocked by NSC95397. Importantly, NSC95397 treatment protects mice against lethal influenza virus challenge. the findings establish CDC25B as a pro-influenza A virus host factor that may be targeted as a novel influenza A therapeutic strategy.National Institutes of Health, National Institute of Allergy and Infectious DiseasesGeorgia Research AllianceUniv Georgia, Coll Vet Med, Dept Infect Dis, Athens, GA 30602 USAUniversidade Federal de São Paulo, UNIFESP, Dept Biol Sci, São Paulo, BrazilUniversidade Federal de São Paulo, UNIFESP, Dept Biol Sci, São Paulo, BrazilNational Institutes of Health, National Institute of Allergy and Infectious Diseases: HHSN266200700006CWeb of Scienc

siRNA Genome Screening Approaches to Therapeutic Drug Repositioning

Bridging high-throughput screening (HTS) with RNA interference (RNAi) has allowed for rapid discovery of the molecular basis of many diseases, and identification of potential pathways for developing safe and effective treatments. These features have identified new host gene targets for existing drugs paving the pathway for therapeutic drug repositioning. Using RNAi to discover and help validate new drug targets has also provided a means to filter and prioritize promising therapeutics. This review summarizes these approaches across a spectrum of methods and targets in the host response to pathogens. Particular attention is given to the utility of drug repurposing utilizing the promiscuous nature of some drugs that affect multiple molecules or pathways, and how these biological pathways can be targeted to regulate disease outcome

Regulatory relationship of LIF and NEK7.

<p>Regulatory relationship of LIF and NEK7.</p

Workflow from HTS to pathway analysis of miRNA top hits.

<p>(A) A549 cells were reverse-transfected with a library of 1,200 miRNA mimics at a final concentration of 20 nM. Non-targeting miRNA mimic controls and siRNAs (siNP) to countermeasure H7N9 replication were used as negative and positive controls, respectively. 48 h after miRNA transfection, the cells were infected with H7N9 at MOI of 0.01 for 24 h. 24 h after infection, the plates were fixed and analyzed by ELISA. 10 top hits from HTS ELISA were validated in virus replication studies. Top hit has-miR-664a-3p was then investigated for pathway analysis and downstream target effects. (B) A plot of normalized Z-score values calculated based on the ELISA results showing a wide range of virus replication modulation. All miRNAs with a Z-score < -1.5 were subjected to further validation.</p

miR664i reduces the replication of other influenza A strains.

<p>(A-B) A549 cells were transfected with miR664i at 25 nM for 48 h. Cells were infected with A/Ca (H1N1) at MOI 0.1 and A/Phi at MOI 0.05 for 48 h. NTC; Non-transfected control (A) Cells were fixed with 10% formalin and stained for viral NP and counterstained with DAPI. Percentage of infected cells was measured with Cellomics ArrayScan and graphed. Data are from 3 replicate wells +/- SEM. *p<0.05. (B) Supernatants from transfected and infected A549 cells were titrated on MDCK cell. TCID₅₀ values were calculated and graphed. (3 replicate wells) ± SEM.　 *p<0.05.</p

A model for miR-664-mediated regulation of influenza A H7N9 virus infection.

<p>miR-664 is upregulated during the course of influenza A infection of A549 cells, which inhibits the expression of target genes of LIF and NEK7. When an inhibitor of miR-664 is used, such as miR664i, molecules LIF and NEK7 are expressed normally, which counteracts the replication of influenza A.</p

Validation of miRNA inhibitor top hits that reduce H7N9 replication.

<p>(A-B) A549 were transfected with miRNA inhibitors and mimics at 25 nM for 48 h and then infected with H7N9 at MOI 0.01 for 48 h. Cells were fixed with 10% formalin and stained for viral NP and counterstained with DAPI. Stained cells were visualized and analyzed using Cellomics ArrayScan high content imaging system. NTC; Non-transfected control, siNP; Positive control (A) Percentage of infected cells was graphed. The data are from 3 replicate wells +/- SEM. *p<0.05. (B) Supernatants from transfected and infected A549 cells were titrated on MDCK cells. The data are from 3 replicate wells ± SEM. *p<0.05, °p<0.05 compared to positive. (C) Representative fluorescent microscope images were taken with EVOS FL imaging system (Life Technologies). Scale bar, 400 μm. NP, green; DAPI (nuclei), blue.</p