virus human lung epithelial cell a549  (ATCC)

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: NLRC5 overexpression inhibits influenza virus PR8 replication and induces RIG-I and IFN-β expression in A549 cells. A549 cells were transfected with 2 μg of vector alone, myc-NLRC5 or flag-CIITA expression vector. Twenty-four hours post-transfection, the cells were infected with PR8 virus (MOI 0, 0.01, 0.1, and 1.0) for 24 h. (A) Supernatants were tested for viral titers by plaque assay using MDCK cells. (B–F) The expression of (B) NP vRNA, (C) NP mRNA, (D) IFN-β mRNA, (E) RIG-I mRNA, and (F) IFN-α was analyzed by real-time RT-PCR, relative to β-actin. Data shown are mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. (F) Expression of myc-NLRC5 and NS1 was analyzed by immunoblotting and the immunoblot shown is from one single experiment representative of three independent experiments. β-Actin was used as a loading control. ANOVA was performed to compare vector control versus myc-NLRC5 or flag-CIITA-transfected A549 cells and p values <0.05 are indicated with an asterisk.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Over Expression, Virus, Expressing, Transfection, Plasmid Preparation, Infection, Plaque Assay, Quantitative RT-PCR, Western Blot, Control

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: Viral NS1 counteracts endogenous NLRC5, RIG-I, and IFN-β expression. A549 cells were infected with PR8 or PR8ΔNS1 (MOI 1.0) for 0, 3, 6, 12, and 24 h and the expression of (A) NP vRNA, (B) NP mRNA, (C) NLRC5 mRNA, (D) RIG-I mRNA, (E) IFN-β mRNA, and (F) IFN-α was analyzed by real-time RT-PCR, relative to β-actin. Data shown are mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. ANOVA was performed to compare PR8-infected versus PR8ΔNS1-infected A549 cells and p values <0.05 are indicated with an asterisk.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Expressing, Infection, Quantitative RT-PCR

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: NS1 complementation inhibits PR8ΔNS1-induced NLRC5. A549 cells were cotransfected with 2 μg of vector or myc-NS1 expression vector and IFN-β promoter LUC reporter using lipofectamine 2000. Twenty-four hours post-transfection, these cells were infected with PR8ΔNS1 for another 24 h. (A, B) Cells were harvested and mRNA expression of (A) NLRC5 and (B) RIG-I was determined by real-time RT-PCR, relative to β-actin. (C) IFN-β induction was assayed by LUC reporter assay. (D) NLRC5, RIG-I, and myc-NS1 expression was analyzed by immunoblotting. The immunoblot shown is from one single experiment representative of three independent experiments. β-Actin was used as a loading control. Data shown are mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. ANOVA was performed to compare vector control versus myc-NS1-transfected A549 cells and p values <0.05 are indicated with an asterisk.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Plasmid Preparation, Expressing, Transfection, Infection, Quantitative RT-PCR, Reporter Assay, Western Blot, Control

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: NS1ΔPR8 virus induces NLRC5, IFN-β, RANTES expression, and NFκB activation in a RIG-I-dependent manner. The expression of endogenous NLRC5 or RIG-I was silenced using gene-specific NLRC5 or RIG-I siRNA in A549 cells followed by infection with PR8 or PR8ΔNS1 (MOI 1.0). Cells were also cotransfected with NFκB promoter LUC reporter using lipofectamine 2000. (A–C) Cells were harvested 24 h postinfection to assess the expression of (A) NLRC5 mRNA, (B) IFN-β mRNA, and (C) RIG-I mRNA, relative to β-actin by real-time RT-PCR. (D) Cells were analyzed for endogenous RIG-I, NLRC5, and β-actin (loading control) protein expression by immunoblotting and the immunoblot shown is from one single experiment representative of three independent experiments. Cell supernatants were assayed for (E) IFN-β and (F) RANTES by ELISA. (G) NFκB activation was measured by LUC reporter assay. Data shown are mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. ANOVA was performed to compare PR8-infected versus PR8ΔNS1-infected A549 cells and p values <0.05 are indicated with an asterisk.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Virus, Expressing, Activation Assay, Infection, Quantitative RT-PCR, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Reporter Assay

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: LPS-induced IFN-β induction and NFκB activation remain unchanged in the presence or absence of NLRC5. (A–C) The expression of endogenous NLRC5 in A549 cells was silenced using gene-specific NLRC5 siRNA. (D–F) Alternatively, A549 cells were transfected with 2 μg of vector alone or myc-NLRC5 expression vector. (A–F) Cells were also cotransfected with IFN-β promoter or NFκB promoter LUC reporter using lipofectamine 2000 and treated with indicated dose of LPS. (A–F) Cells were harvested 24 h post-LPS treatment to assess (A and D) IFN-β induction and (B and E) NFκB activation by LUC reporter assay. (C and F) Cells were analyzed for myc-NLRC5, RIG-I, and β-actin (loading control) protein expression by immunoblotting and the immunoblot shown is from one single experiment representative of two independent experiments. Data are shown as mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. ANOVA was performed to compare control siRNA versus NLRC5 siRNA treated A549 cells or vector versus NLRC5-transfected A549 cells and p values <0.05 are indicated with an asterisk; ns: not significant.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Reporter Assay, Control, Western Blot

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: NLRC5 is required for robust IFN-β and RIG-I expression. A549 cells transfected with control siRNA or NLRC5 siRNA were infected with NS1-del PR8 (MOI 1.0). (A–C) Cells were harvested 0, 24, 48, 72, and 96 h postinfection and analyzed for (A) IFN-β, (B) RIG-I, and (C) NLRC5 mRNA expression, relative to β-actin by real-time RT-PCR. Data are shown as mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. ANOVA was performed to compare control siRNA versus NLRC5 siRNA treated A549 cells and p values <0.05 are indicated with an asterisk.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Expressing, Transfection, Control, Infection, Quantitative RT-PCR

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: The NLRC5 death domain and nucleotide-binding domain is critical for NLRC5-mediated antiviral function. A549 cells were transfected with vector alone or with myc-tagged wtNLRC5, NLRC5-K234A, NLRC5-ISO3, NLRC5-ΔDD, NLRC5-DD, or LRR domain of NLRC5 and subsequently infected with PR8 (MOI 1.0) for 24 h. (A) The upper panel shows the schematic representation of the NLRC5 constructs used and the lower panel shows expression of NLRC5 constructs in the cells by immunoblotting. β-Actin was used as a loading control. (B, C) Supernatants were collected and cells were harvested to determine (B) viral titers by plaque assay and (C) IFN-β mRNA expression, relative to β-actin by real-time RT-PCR. (D) Secretion of CCL5 (RANTES) in cell supernatants was measured by ELISA. Data are shown as mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. ANOVA was performed to compare control vector versus myc-NLRC5 expression vectors transfected A549 cells and p values <0.05 are indicated with an asterisk.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Binding Assay, Transfection, Plasmid Preparation, Infection, Construct, Expressing, Western Blot, Control, Plaque Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: NLRC5 stabilizes RIG-I. (A) A549 cells transfected with vector alone or myc-wtNLRC5 were infected with PR8 (MOI 1.0) for 0, 3, 6, 9, 12, 18, 24, 48, and 72 h and harvested for NLRC5, RIG-I, and NS1 expression and coimmunoprecipitation assay. β-Actin was used as a loading control. Cell lysates from 6, 12, and 24 h were immunoprecipitated with anti-myc, anti-NS1, or anti-RIG-I antibodies and immunoprecipitates were analyzed for the presence of RIG-I, NS1, and NLRC5 by immunoblotting. (B) To map the domain responsible for NLRC5 interaction with RIG-I and NS1, A549 cells were transfected for 24 h with myc-vector alone or with myc-tagged wtNLRC5, NLRC5-K234A, NLRC5-ISO3, NLRC5-ΔDD, NLRC5-DD, or NLRC5-LRR mutants and then infected with PR8 (MOI 1.0) for 3 or 9 h. Cell lysates were immunoprecipitated with anti-myc, anti-NS1, or anti-RIG-I antibodies and immunoprecipitates were analyzed for the presence of RIG-I, NS1, and NLRC5 by immunoblotting. β-Actin was used as a loading control. The input for the immunoblot was about 5% of the total cell lysate. (C) A549 cells transfected with wtNLRC5 were infected with PR8 (MOI 1.0) in the presence or absence of actinomycin D (5 μg/mL)/cyclohexamide (20 μg/mL) combination. Cell lysates were analyzed for RIG-I and NLRC5 expression at 0, 3, 6, and 9 h postinfection by immunoblotting. Data shown are from one single experiment representative of two independent experiments.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Transfection, Plasmid Preparation, Infection, Expressing, Co-Immunoprecipitation Assay, Control, Immunoprecipitation, Western Blot

Journal: Journal of Virology

Article Title: Autophagy Promotes Replication of Influenza A Virus In Vitro

doi: 10.1128/jvi.01984-18

Figure Lengend Snippet: Figure 7. IAV NP and M2 induce autophagy via AKT-mTOR pathway. (A) A549 842

Article Snippet: 141 142 Materials and Methods 143 Cells and Virus 144 Human lung epithelial cell line A549 and Madin-Darby canine kidney cells 145 (MDCKs) were obtained from China Center for Type Culture Collection (Wuhan, 146 China), and human embryonic kidney (HEK) 293T cells were purchased from ATCC 147 (Manassas, VA, USA).

Techniques:

Journal: The Journal of General Virology

Article Title: Identification and characterization of viral defective RNA genomes in influenza B virus

doi: 10.1099/jgv.0.001018

Figure Lengend Snippet: Viral replication kinetics and proliferation dynamics of FLUBV-infected A549 cells. (a) Viral titre in A549 cells infected with B/Brisbane/60/2008 virus. Supernatant samples of A549 cells infected by 1.0 m.o.i. of FLUBV were collected at 0, 6, 12 and 24 h p.i. The TCID50 of each sample was evaluated in MDCK cells. Mean TCID50 (+sd) from three independent experients in duplicate are shown. (b) A549 cell growth in the first 24 h of infection. MTS assay in 96-well plate format was performed at 0, 6, 12 and 24 h p.i. The bars represent mean cell numbers (+sd) from three independent experiments in triplicate. '*' indicates a significant difference using Student’s t-test (P<0.05).

Article Snippet: Cells and virus Human lung alveolar carcinoma epithelial cell line A549 (ATCC CCL-185) cells, MDCK cells (ATCC CCL-34), and human Calu-3 (ATCC HTB-55) were maintained in Dulbecco's minimum essential medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum (FBS, PAA Laboratories, Dartmouth, MA, USA) and 100 U ml −1 penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA).

Techniques: Infection, Virus, MTS Assay

Journal: The Journal of General Virology

Article Title: Identification and characterization of viral defective RNA genomes in influenza B virus

doi: 10.1099/jgv.0.001018

Figure Lengend Snippet: Analysis of viral gene transcription by NGS. (a) Hierarchical clustering of expression profiles. The UPGMA tree was rerooted using the RNA-seq read from the two control samples as the root. The divergence of expression profiles at 0, 6, 12 and 24 h p.i. is shown in the UPGMA dendrogram. Two independent experiments (indicated with superscripts 'a' and 'b') at each time point are shown separately. (b) PC analysis of host gene expression profile divergence. FLUBV-induced expression differences are shown using the first two components (PC1 and PC2) of PC analysis. The two independent experiments at each time point are partially (6 h p.i.) or almost completely (0, 12 and 24 h p.i.) overlapping with each other for both PC1 and PC2. Two repeats (indicated with superscripts 'a' and 'b') are shown separately. (c) Percentage of viral reads in the NGS samples. Genome-wide total gene expression was quantified using RNA-seq. Two total RNA samples at each time point were extracted from A549 cells infected with 1.0 m.o.i. of FLUBV. Percentage of viral genome reads was shown, which represents the ratio of the FLUBV genome reads to the total reads of the genomes of Homo sapiens and FLUBV. (d) Expression level changes of the eight FLUBV segments over time. The expression level was displayed using reads per kilobases per million reads(RPKM).

Article Snippet: Cells and virus Human lung alveolar carcinoma epithelial cell line A549 (ATCC CCL-185) cells, MDCK cells (ATCC CCL-34), and human Calu-3 (ATCC HTB-55) were maintained in Dulbecco's minimum essential medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum (FBS, PAA Laboratories, Dartmouth, MA, USA) and 100 U ml −1 penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA).

Techniques: Expressing, RNA Sequencing, Control, Gene Expression, Genome Wide, Infection

Journal: The Journal of General Virology

Article Title: Identification and characterization of viral defective RNA genomes in influenza B virus

doi: 10.1099/jgv.0.001018

Figure Lengend Snippet: Alternative splicing or junction sites and defective RNA genomes. (a) NS segment splicing. Diagram showing the known splicing site in the FLUBV NS segment that generates NS2 mRNA. (b) PB1 segment-derived defective RNAs. Schematic diagram shows the two newly identified defective RNAs from the PB1 segment termed PB1∆A and PB1∆B. (c) M segment-derived defective RNAs. Schematic diagram displays the two newly identified defective RNAs in the M segment termed M∆A and M∆B. The initiation, junction and termination positions (5′→3′) of an mRNA’s ORF are labelled with position numbers above. An mRNA’s two junction sequences are shown under ORFs. (a) Detection of PB1∆A, M∆A, PB1∆B, M∆B and NS2 RNAs in FLUBV-infected A549 cells. Segment-specific primers were used to amplify full-length PB1∆A, M∆A and NS2 by RT-PCR. Junction-specific primers (forward) and segmental primers (reverse) were used to amplify partial PB1∆B and M∆B transcripts. The cDNA was generated from total mRNA extracted from FLUBV-infected A549 cells. (e) Absence of PB1∆A and M∆A RNAs in HEK293T cells transfected with plasmid DNA encoding FLUBV PB1 and M segments. Segment-specific primers were used to amplify PB1∆A and M∆A by RT-PCR. NS2-specific RT-PCR was performed as a splicing control. The cDNA was generated from total mRNA extracted from transfected HEK293T cells with FLUBV PB1, M or NS segments.

Article Snippet: Cells and virus Human lung alveolar carcinoma epithelial cell line A549 (ATCC CCL-185) cells, MDCK cells (ATCC CCL-34), and human Calu-3 (ATCC HTB-55) were maintained in Dulbecco's minimum essential medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum (FBS, PAA Laboratories, Dartmouth, MA, USA) and 100 U ml −1 penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA).

Techniques: Alternative Splicing, Derivative Assay, Infection, Reverse Transcription Polymerase Chain Reaction, Generated, Transfection, Plasmid Preparation, Control

Journal: The Journal of General Virology

Article Title: Identification and characterization of viral defective RNA genomes in influenza B virus

doi: 10.1099/jgv.0.001018

Figure Lengend Snippet: Transcription profiles of defective RNAs and their relative abundances. (a) Transcription profiles of defective mRNAs. RNA-seq junction reads for the four defective RNAs (M∆A, M∆B, PB1∆A and PB1∆B) and NS2 are shown as junction reads per million total reads at indicated time points following FLUBV infection of A549 cells. (b) Ratio of M∆A vRNAs relative to full-length M vRNAs. (c) Ratio of PB1∆A vRNAs relative to full-length PB1 vRNAs. Ratios of M∆A to the full-length M segment and of PB1∆A to the full-length PB1 segment were measured respectively by droplet digital PCR (ddPCR). Both clarified supernatants and cell lysates of A549 cells infected with 1.0 m.o.i. of FLUBV were collected at 6, 12 and 24 h p.i. Total RNAs were then extracted and 200 ng RNA was used for reverse transcription with vRNA-specific primers. ddPCRs were conducted with primers and probes specific to amplify defective RNAs (the junction regions) and full-length segments. No-template controls were included in every run. The ratios of defective genomes to their corresponding full-length segments were normalized and shown as mean+sem from three separate experiments with each assaying samples in triplicate. Note that M∆A and PB1∆A were detected in the viral inoculum and copy ratios relative to their parental segments were indicated by red dashed lines in (b) and (c). Viral inoculum was generated from clarified and filtered supernatants from MDCK cells infected with 0.1 m.o.i. at 72 h p.i.

Article Snippet: Cells and virus Human lung alveolar carcinoma epithelial cell line A549 (ATCC CCL-185) cells, MDCK cells (ATCC CCL-34), and human Calu-3 (ATCC HTB-55) were maintained in Dulbecco's minimum essential medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum (FBS, PAA Laboratories, Dartmouth, MA, USA) and 100 U ml −1 penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA).

Techniques: RNA Sequencing, Infection, Digital PCR, Reverse Transcription, Generated