Journal: Journal of Virology

Article Title: MicroRNA hsa-miR-324-5p Suppresses H5N1 Virus Replication by Targeting the Viral PB1 and Host CUEDC2

doi: 10.1128/JVI.01057-18

Figure Lengend Snippet: RNA virus infection downregulates the expression of miR-324-5p. (A) Schematic representation of work flow for data analysis. The volcano plot represents dysregulated miRNAs identified in lung tissue of C57BL/c mice infected with A/Vietnam/1203/2004 (H5N1) (176 PFU/mouse) at day 4 postinfection in three independent microarray data sets using the indicated screening algorithm (upper portion). Common miRNAs that were increased or decreased more than log2 FC (0.5) among the top 50 miRNAs in three independent studies were selected and are shown by the green arrows (lower portion). (B) List of candidate miRNAs targeting the H5N1 genome identified using Segal Lab and RegRNA1.0. The cutoffs used for Segal Lab and RegRNA1.0 were −20 kcal/mol and −25 kcal/mol, respectively. (C) Prediction of the miR-324-5p binding site in the PB1 gene of H5N1 and minimum free energy (MFE) of miRNA-PB1 duplex. The indicated position is relative to the first nucleotide of the PB1 gene. (D) Sequence alignment of the PB1 gene (target sequence) across different clades of H5N1 aligned using ClustalW. (E to H) Quantification of relative expression of hsa-miR-324-5p in SAECs and A549 cells with H5N1 infection (MOI, 1) at the indicated time points (E); A549 cells with NDV infection (MOI, 1) at 24 h (F); A549 cells with A/PR8/H1N1 (PR8) infection (MOI, 1) at 24 h (G); A549 cells with poly(I·C) transfection (1 μg/ml) at 24 h (H). (I) Quantification of relative expression of miR-324-5p in swine flu patients (n = 9) compared to healthy controls (n = 4). (J) Quantification of relative abundance of miR-324-5p in A549 cells in response to stimulation with IFN-β (100 IU/ml) after 12 h. (K) Quantification of relative expression of miR-223-3p in A549 cells infected with H5N1 (MOI, 1), NDV (MOI, 1), and PR8 (MOI, 1) and transfected with poly(I·C) (pIC; 1 μg/ml) at 24 h. Ctrl, control (uninfected); H5, H5N1 virus. Data are means ± SEMs from triplicate samples of a single experiment and are representative of results from three independent experiments (E to K).

Article Snippet: A549 human alveolar basal epithelial cells (Cell Repository, NCCS, India), HEK293T human embryonic kidney cells (ATCC CRL-3216), and HeLa cervical cancer cells (Cell Repository, NCCS, India) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

Techniques: Virus, Infection, Expressing, Microarray, Binding Assay, Sequencing, Transfection, Control

Journal: Journal of Virology

Article Title: MicroRNA hsa-miR-324-5p Suppresses H5N1 Virus Replication by Targeting the Viral PB1 and Host CUEDC2

doi: 10.1128/JVI.01057-18

Figure Lengend Snippet: miR-324-5p enhances ISGs and type I and type III interferons. (A) Schematic outline of transfection, infection (H5N1), RNA isolation, sequencing, and RNA-Seq analysis. Mo, mock; Inf., infected; Un-inf., uninfected. (B) Global expression analysis during m-324 overexpression in H5N1-infected A549 cells using R package. NC is plotted versus m-324 in H5N1-infected samples. Genes significantly changed (>2-fold) are colored in red and green for upregulated and downregulated, respectively. (C) Gene ontologies represented as dot plots. The circles are colored based on the significance (P value) of enrichment, while the sizes represent the number of genes that are enriched for that particular category. (D) Clustergram representing enriched KEGG pathways analysis as columns and input genes as rows; cells in the matrix (blue color) indicate if a gene is associated with the pathway. (E) Heat map analysis of upregulated genes associated with innate immune responses. (F to H) A549 cells were transfected with either m-324 or NC, followed by infection with H5N1 at an MOI of 5, and subjected to qRT-PCR analysis after 24 h to determine the relative expression of IFN-α4 and IFN-β (F), IFN-λ1, IFN-λ2, and IFN-λ3 (G), and IFIT1 and OAS1 (H) RNAs. (I) Equal numbers of A549 and MDAMB-231 cells were either left uninfected (Ctrl) or infected with H5N1 at an MOI of 1 for 24 h. IFN-β RNA levels were quantified by qRT-PCR. (J) MDAMB-231 cells were transfected with 100 nM m-324 or NC, followed by infection with H5N1 at an MOI of 1 for 24 h. Relative levels of NP RNA were quantified by qRT-PCR. Data are means ± SEMs of triplicate samples of a single experiment and are representative of results from three independent experiments (F to J). ****, P < 0.0001, ***, P < 0.001, **, P < 0.01, and *, P < 0.05, by two-tailed unpaired t test.

Article Snippet: A549 human alveolar basal epithelial cells (Cell Repository, NCCS, India), HEK293T human embryonic kidney cells (ATCC CRL-3216), and HeLa cervical cancer cells (Cell Repository, NCCS, India) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

Techniques: Transfection, Infection, Isolation, Sequencing, RNA Sequencing, Expressing, Over Expression, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of Virology

Article Title: MicroRNA hsa-miR-324-5p Suppresses H5N1 Virus Replication by Targeting the Viral PB1 and Host CUEDC2

doi: 10.1128/JVI.01057-18

Figure Lengend Snippet: miR-324-5p interacts with the PB1 gene of H5N1. (A) Quantification of copy number per picogram of small RNA of miR-324-5p in uninfected and H5N1-infected (MOI, 1) A549 cells (left) and quantification of copy number per nanogram of total RNA of miR-324-5p and PB1 in A549 cells infected with H5N1 at an MOI of 1 (right). The copy number of miR-324-5p and PB1 was calculated using miR-324-5p mimic and PB1 RNA as a standard. (B) Schematic representation of work flow for RNA immunoprecipitation (RNA-IP) assay with H5N1 virus. (C) HEK293T cells were transfected with Flag-tagged Ago2 (F-Ago2) along with m-324 (miR-324-5p mimic) or NC (negative-control mimic) and then infected with H5N1 for 24 h at an MOI of 1. Cells were subjected to RNA-IP with anti-Flag antibody-tagged beads, and the abundances of PB1, PB2, PA, NP, M1, NA, HA, and NS1 viral RNAs were quantified using qRT-PCR analysis. (D) Quantification of relative abundance of miR-324-5p in RNA-IP performed for panel B. (E) The PB1, PA, and PB2 genes were cloned into the pMIR-Report vector as shown. HEK293T cells were transfected with PB1-Luc, PA-Luc, or PB2-Luc (100 ng) along with 25 nM m-324 or NC, lysed after 24 h, and subjected to luciferase assay. CMV, cytomegalovirus. (F) The PB1 gene of A/PR8/H1N1 was cloned into the pMIR-Report vector as shown. PB1 of PR8 influenza virus was aligned with PB1 of H5N1 influenza. Mismatches between the two are indicated in red. HEK293T cells were transfected with PB1(PR8)-Luc along with 25 nM m-324 or NC, lysed after 24 h, and subjected to luciferase assay. (G) A549 cells were transfected with miR-324 or NC, followed by infection with H5N1 (left) or A/PR8/H1N1 (right) at an MOI of 1 after 24 h, and subjected to qRT-PCR analysis. (H) Quantification of the relative amounts of PB1 RNA in A549 cells that were transfected with NC or m-324, infected with H5N1 (left) or A/PR8/H1N1 (right) for 24 h at an MOI of 1, and then treated with actinomycin D (ActD; 10 μg/ml) for the indicated times. Data are means ± SEMs from triplicate samples of a single experiment and are representative of results from two independent experiments (A, C, D, and H) and three independent experiments (E to G). ****, P < 0.0001, ***, P < 0.001, **, P < 0.01, and *, P < 0.05, by two-tailed unpaired t test (E to H). ns, nonsignificant.

Article Snippet: A549 human alveolar basal epithelial cells (Cell Repository, NCCS, India), HEK293T human embryonic kidney cells (ATCC CRL-3216), and HeLa cervical cancer cells (Cell Repository, NCCS, India) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

Techniques: Infection, RNA Immunoprecipitation, Virus, Transfection, Negative Control, Quantitative RT-PCR, Clone Assay, Plasmid Preparation, Luciferase, Two Tailed Test

Journal: Journal of Virology

Article Title: MicroRNA hsa-miR-324-5p Suppresses H5N1 Virus Replication by Targeting the Viral PB1 and Host CUEDC2

doi: 10.1128/JVI.01057-18

Figure Lengend Snippet: miR-324-5p inhibits H5N1 in various cell types. (A) SAECs were transfected with m-324 or NC at 100 nM, followed by infection with H5 at an MOI of 1. After 24 h, relative levels of NP RNA were measured by qRT-PCR. (B) A549 cells were transfected with 100 nM m-324 or NC, followed by infection with H5 at an MOI of 5. NP relative RNA was quantified by qRT-PCR at 12, 24, and 48 h after infection. (C) A549 cells were transfected with 100 nM m-324 or NC, followed by infection with H5 at MOIs of 1 (left) and 5 (right). NP relative RNA was quantified by qRT-PCR after 24 h of infection. (D) HEK293T cells were transfected with miR-324-5p mimic or NC for 24 h and subjected to infection with H5 at an MOI of 1 for 24 h, and NP was detected using qRT-PCR. (E) A549 cells were transfected with increasing amounts (25, 50, and 100 nM) of m-324 or NC and subjected to infection with H5N1 at an MOI of 5. NP relative RNA was quantified by qRT-PCR after 24 h. (F) A549 cells were transfected with 100 nM m-324 or NC, followed by infection with H5N1 at MOIs of 5 (left) and 10 (right) for 24 h. Relative levels of NP RNA were quantified in cell culture supernatant. (G) HEK293T cells were transfected with plasmid encoding miR-324-5p (p324-5p) or empty vector (EV; used as a negative control) for 24 h and subjected to infection with H5 at an MOI of 1 for 12 h, and NP was detected using flow cytometer analysis with anti-NP antibody. (H and I) Effect of miR-324-5p on H5N1 replication by TCID50 (H) and plaque (I) assays. A549 cells were transfected with m-324 or NC, followed by infection with H5N1 at an MOI of 0.5. After 24 h, supernatant containing viral particles was used for infecting MDCK cells. At 96 h after H5N1 infection, the viral load was determined by TCID50 assay (H) and plaque assay (I) in a 96-well plate. (J) A549 cells were transfected with 100 nM m-324 or NC, followed by infection with A/PR8/H1N1 (PR8) at an MOI of 1. NP relative RNA was quantified by qRT-PCR after 24 h of infection. (K) HEK293T cells were transfected with m-324 or NC, followed by infection at an MOI of 5. NDV RNA was quantified after 24 h using qRT-PCR analysis. (L) HEK293T cells were transfected with either m-324 or miR-1, followed by infection with GFP-expressing NDV (NDV-GFP) at an MOI of 1, and subjected to Western blotting after 24 h using anti-GFP antibody. Data are means ± SEMs from triplicate samples of a single experiment and are representative of results from three independent experiments. (A, C, D, F, J, and K) ****, P < 0.001, and *, P < 0.01, by one-way ANOVA. (B, E, H, and I) ****, P < 0.0001, ***, P < 0.001, and **, P < 0.01, by two-tailed unpaired t test.

Article Snippet: A549 human alveolar basal epithelial cells (Cell Repository, NCCS, India), HEK293T human embryonic kidney cells (ATCC CRL-3216), and HeLa cervical cancer cells (Cell Repository, NCCS, India) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

Techniques: Transfection, Infection, Quantitative RT-PCR, Cell Culture, Plasmid Preparation, Negative Control, Flow Cytometry, TCID50 Assay, Plaque Assay, Expressing, Western Blot, Two Tailed Test

Journal: Journal of Virology

Article Title: MicroRNA hsa-miR-324-5p Suppresses H5N1 Virus Replication by Targeting the Viral PB1 and Host CUEDC2

doi: 10.1128/JVI.01057-18

Figure Lengend Snippet: miR-324-5p targets the 3′ UTR of the CUEDC2 gene. (A) Identification of genes targeted by miR-324-5p. Common genes between Targetscan (Context++ score > 0.5) and genes downregulated in m-324 RNA sequencing (log2 FC < −1) were selected and are shown with green arrows. (B) Prediction of miR-324-5p binding with the 3′ UTR of the CUEDC2 gene. NM_024040 is the RefSeq CUEDC2 transcript identifier, and “ΔG” represents energy of miR-324-5p-CUEDC2 complex. (C) Conserved miR-324-5p binding sites in the 3′ UTR of CUEDC2. (D) Ago2 interaction loci in the 3′ UTR of the CUEDC2 gene in 293S cells as visualized in Integrative Genomics Viewer (IGV). CLIP tags are represented by a heat map. Numbers represent the genomic location, and an asterisk represents the stop codon. (E) HEK293T cells were transfected with CUEDC2-Luc (CUEDC2) in the presence of m-324-5p or NC, lysed after 24 h, and subjected to luciferase assay. (F and G) A549 (F) and HeLa (G) cells were transfected with m-324 (miR-324-5p) or NC, followed by quantification of CUEDC2 RNA after 24 h. (H) Schematic of RNA-IP assay. A549 cells were transfected with Flag-tagged Ago2 (F-Ago2) in the presence of either m-324 or NC. After 48 h, cells were subjected to RNA-IP with anti-Flag antibody and CUEDC2 mRNA was quantified using qRT-PCR analysis. (I to K) Quantification of relative expression of CUEDC2 in A549 cells at 24 h after infection with H5 (MOI, 1) (I) and NDV (MOI, 1) or PR8 (MOI, 1) (J) and poly(I·C) transfection (pIC-T) (1 μg/ml) (K). (L to N) A549 cells were transfected with 1.5 μg control short hairpin RNA (shRNA) (shc001) or CUEDC2-specific shRNA (G9 or G11) for 36 h, followed by H5N1 infection (MOI, 1) for 24 h. qRT-PCR was used for quantification of CUEDC2 (L), NP RNA (M), and IFN-β expression (N). Data are means ± SEMs from triplicate samples of a single experiment and are representative of results from three independent experiments (E to N). (E to K) ****, P < 0.0001, ***, P < 0.001, **, P < 0.01, and *, P < 0.05, by two-tailed unpaired t test. (L and M) ***, P < 0.001, and **, P < 0.01, by one-way ANOVA.

Article Snippet: A549 human alveolar basal epithelial cells (Cell Repository, NCCS, India), HEK293T human embryonic kidney cells (ATCC CRL-3216), and HeLa cervical cancer cells (Cell Repository, NCCS, India) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

Techniques: RNA Sequencing, Binding Assay, Transfection, Luciferase, Quantitative RT-PCR, Expressing, Infection, Control, shRNA, Two Tailed Test