Journal: Science Advances

Article Title: Common and species-specific molecular signatures, networks, and regulators of influenza virus infection in mice, ferrets, and humans

doi: 10.1126/sciadv.abm5859

Figure Lengend Snippet: Description of datasets used in this study.

Article Snippet: HTBE cells were purchased from Lonza and passaged twice to generate a stock of cells stored in liquid nitrogen.

Techniques: Infection

Journal: Science Advances

Article Title: Common and species-specific molecular signatures, networks, and regulators of influenza virus infection in mice, ferrets, and humans

doi: 10.1126/sciadv.abm5859

Figure Lengend Snippet: Unique network modules in ( A ) human MDM cells, ( B ) HTBE cells, ( C and D ) mouse lung, and ( E ) ferret lower lung are shown. Red nodes denote up-regulation, and blue nodes denote down-regulation. Diamond-shaped nodes are key regulators. Species unique MEGENA modules were identified by enrichment for SRG (JTG) intersection signatures from all eight systems. Modules with significant enrichment in one species but not in any other were deemed unique. (A) The human (MDM) M104 unique module with 30 nodes and 78 edges is ranked number 75 and is responsible for the negative regulation of leukocyte activation. CR1L , HLA-G , MNDA , and RBM34 are key regulators in this module. Most of the genes in this pathway are up-regulated. (B) The number 67 ranked regulation of endocannabinoid signaling/endosomal vacuolar pathway with 114 nodes, 326 edges, and key regulators HDGFRP2 , PDGFC , SMPDT , TIPARP , TJAP1 , and U2AF2 is shown. (C) The number 136 ranked and predominantly down-regulated mouse module M473 relevant for sphingolipid signaling has 27 nodes and 67 edges. The sole key regulator is ANXA11 . (D) M631 is a number 49 ranked and up-regulated mouse module responsible for positive regulation of T cell cytokine production with CTSS as a single key regulator. M631 has 23 nodes and 57 edges. (E) The number 170 ranked ferret lower lung module M14 with 202 nodes and 565 edges shows minichromosome maintenance (MCM) and cell cycle functionality. Key regulators are ARHGAP21 , ARPC1B , ATP6V0C , CD3D , CD3E , CD3G , CDC42BPG , CFL1 , DPP3 , ECT2 , ENO1 , ENSMPUG00000006004 (putative MELK ), ENSMPUG00000008504 (putative TRBC2 ), IFT81 , MCM10 , MCM4 , MKI67 , TPPP3 , TRAC , TRADD , UBE2V1 , and UNC93B1 .

Article Snippet: HTBE cells were purchased from Lonza and passaged twice to generate a stock of cells stored in liquid nitrogen.

Techniques: Activation Assay

Journal: Science Advances

Article Title: Common and species-specific molecular signatures, networks, and regulators of influenza virus infection in mice, ferrets, and humans

doi: 10.1126/sciadv.abm5859

Figure Lengend Snippet: A549 cells were transfected with four individual siRNAs targeting TDRD7, the negative control scrambled or the cytotoxic siRNA Allstars. At 48 hours after transfection, ( A ) RNA was isolated and analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using primers specific for TDRD7 and TBP. ( B ) Cell viability was assessed using CellTiter-Glow. ( C ) A549 cells were transfected with indicated siRNAs. At 48 hours after transfection, cells were infected with WSN virus at a multiplicity of infection of 0.01. Supernatants were collected at 48 hours postinfection (hpi) and analyzed by plaque assay using MDCK cells. ( D ) A549, 293T, and HTBE cells were seeded overnight and then treated with increasing doses of universal IFN-β (0, 10, 100, and 1000 IU/ml) for 24 hours. RNA was then isolated and analyzed by qRT-PCR using primers specific for TDRD7 and TBP. (A to D) Data represent means ± SD of at least two independent experiments run in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 by one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. Five-week-old female BALB/c mice were administered phosphate-buffered saline (PBS), nontargeting control (NTC), or TDRD7 PPMOs (100 μg in 40 μl of PBS; the equivalent of approximately 5 mg/kg) intranasally for two consecutive days before infection. On day 0, mice were infected with A/Puerto Rico/8/34 [40 plaque-forming units (PFU)] intranasally. On days 3 and 6 after infection, five mice per condition were euthanized to harvest the lungs and determine virus titer. The graph shows the mean lung virus titer ± SD on day 3 ( E ) and day 6 ( F ) after infection. Data shown are from two independent experiments with five mice per condition. ns, not significant.

Article Snippet: HTBE cells were purchased from Lonza and passaged twice to generate a stock of cells stored in liquid nitrogen.

Techniques: Transfection, Negative Control, Isolation, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Virus, Plaque Assay, Saline, Control

Journal: Science Advances

Article Title: Common and species-specific molecular signatures, networks, and regulators of influenza virus infection in mice, ferrets, and humans

doi: 10.1126/sciadv.abm5859

Figure Lengend Snippet: The transcriptional response and induced biological processes during influenza infection in WT and TDRD7 KD HTBE cells are shown. ( A ) The multiset intersections between the TDRD7 network neighborhood (up to layer 2) and significantly responding TDRD7 enhanced (T + ) or restricted genes (T − ) by SuperExact Test are depicted. The networks of all eight systems have been used to construct the consensus network. The layer 2 network neighborhood includes all next and second next neighboring genes of TDRD7 . The heights of the bars indicate set size, and colors refer to FET P values (see scale). Green-filled circles below the bars denote corresponding intersected sets (left). The size of the network layer 2 ∩ T + intersection is 13 genes (red rectangle), whereas the network layer 2 ∩ T − intersection includes 50 members (blue rectangle). Both intersections are significant after FET (layer 2 ∩ T + : 2.28-fold enrichment (FE), P = 3.63 × 10 −3 ; layer 2 ∩ T − : 1.93-FE, P = 4.70 × 10 −6 ). The biological functions of the intersections have been assessed by functional enrichment for BioPlanet (2019) pathways. ( B ) The functional enrichment of TDRD7 -enhanced genes in the network neighborhood (layer 2 ∩ T + ) is shown involving IFN signaling. ( C ) The layer 2 ∩ T − intersection has TNF/NF-κB and IL signaling functionality. Two examples, one each of a T + and T − gene in the layer 2 neighborhood, are depicted. ( D ) Antiviral GBP6 is an IFN-inducible guanosine triphosphatase (GTPase) that requires the IFN modulation by TDRD7 for significant up-regulation at 24 hpi. ( E ) PIM1 is responsible for cell survival and may benefit the viral life cycle. ( F ) The expression of TDRD7 in WT and TDRD7 KD HTBE cells is shown. Significance between WT and TDRD7 KD expression at 24 hpi is indicated as follows: *** P < 0.001 and **** P < 0.0001, which was assessed using edgeR’s generalized linear models together with a quasi-likelihood F test.

Article Snippet: HTBE cells were purchased from Lonza and passaged twice to generate a stock of cells stored in liquid nitrogen.

Techniques: Infection, Construct, Functional Assay, Expressing

Journal: mSphere

Article Title: Nucleolar Relocalization of RBM14 by Influenza A Virus NS1 Protein

doi: 10.1128/mSphereDirect.00549-18

Figure Lengend Snippet: A targeted siRNA screen identifies IAV-required host factors. (A) Schematic representation of the assay used to screen putative host factors for their requirement in influenza virus replication. The green pathway (left) describes the A549 cell-based screen using influenza A/WSN/3 (H1N1) virus and influenza A/Pan/99 (H3N2) virus. The blue pathway (right) describes the follow-up screen performed in primary human tracheal bronchial epithelial (HTBE) cells. (B) Box-and-whisker plots showing the distribution of the raw values from the positive control and the negative control and the experimental data for the A549 cell-based screen (across both IAV subtypes), after the removal of outliers was performed using a Bayesian statistical approach. The first and third quartiles of the remaining data are indicated by the boundaries of the box, with the median indicated within the box. The whiskers represent the extreme boundaries of the data, with extreme values (values exceeding 1.5× the interquartile range beyond the median) plotted as points beyond the whiskers. The window between the positive and negative controls is indicated by a red arrow. (C) The raw data from the A549 cell-based screen were plotted against ideal probability distributions in quantile-quantile (QQ) plots (a graphical method for comparing probability distributions). If data from two distributions are similar, they lie approximately on the line y = x (red). Pearson correlation coefficients measure the degree to which the data fit the proposed probability distribution. The positive control (siRNA targeting influenza virus NP protein) follows a log-normal distribution (R2 = 0.9921), the negative control (nontargeting siRNA) follows a normal distribution (R2 = 0.9922), and the experimental data (siRNAs targeting putative host factors) follow a right-skewed Gumbel distribution (R2 = 0.9941). (D) Example data from an experimental condition are shown to illustrate the hit selection criteria. An siRNA targeting a candidate gene was required to reduce the normalized percentage of infection below 0.6 to be characterized as a hit (shown in green versus nonhits shown in red). Finally, for a gene to be scored as a hit in any screen it was required that at least two siRNAs for a gene candidate met these criteria. (E) The results from the two A549-based screens (purple and green) and the HTBE screen (red) are summarized in a Venn diagram. In the A549 cell-based screen, seven and four genes were uniquely identified in the H1N1 (purple) and H3N2 (green) variants, respectively. A total of 18 genes were identified in both A549 screens. These 18 genes (along with 8 genes from the pilot group) were additionally screened in HTBEs with influenza A/WSN/33 (H1N1) virus. Thirteen genes (brown section) were hits in all three variations of the screen. Gene candidates that were not scored as hits are shown in gray.

Article Snippet: HTBE primary cells were obtained from Lonza (donor number 7F3506; catalog no. CC-2541) and were cultured in bronchial epithelial cell growth medium (BEGM) (Lonza; catalog no. CC-3171) supplemented with a BEGM BulletKit (Lonza; catalog no. CC-3170) (complete BEGM), in collagen-coated culture ware, maintaining the cells in the undifferentiated form.

Techniques: Virus, Whisker Assay, Positive Control, Negative Control, Selection, Infection