1. Volume 27, Issue 11, Pages 3295-3304.e4 (June 2019)
Influenza Virus Exploits an Interferon-Independent lncRNA to Preserve Viral RNA Synthesis through Stabilizing Viral RNA Polymerase PB1 
Jing Wang, Yongxin Zhang, Quanjie Li, Jianyuan Zhao, Dongrong Yi, Jiwei Ding, Fei Zhao, Siqi Hu, Jinming Zhou, Tao Deng, Xiaoyu Li, Fei Guo, Chen Liang, Shan Cen 
Cell Reports 
Volume 27, Issue 11, Pages e4 (June 2019)
DOI: /j.celrep
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2. Cell Reports 2019 27, 3295-3304.e4DOI: (10.1016/j.celrep.2019.05.036)
Copyright © 2019 The Author(s) Terms and Conditions

3. Figure 1
High-Throughput esiRNA Screen for lncRNAs Involved in IAV Replication
(A) Schematic diagram of esiRNA screen. HEK293T-Gluc cells were transfected with individual esiRNAs for 48 h and then infected with IAV at an MOI of 0.5. At 24 h post-infection, Gluc activity and cell viability were determined. The screen was performed in triplicate.
(B) Plot of Z scores for all individual esiRNAs in the library. Each esiRNA was ranked by its Z score. Z score thresholds of −1.48 and 1.48 were shown as broken lines.
(C) The top 10 significantly enriched GO terms of predicted lncRNA targets in biological processes (BPs), cellular components (CCs), and molecular function (MF).
(D) Scatterplot of the top 10 most enriched KEGG pathways of predicted lncRNA targets. The y-axis represents pathway terms, and the x axis (Rich factor) represents the proportion of the targets accounted for all genes of a specific pathway term. The size of the point represents the gene count in this item. The p values are color-coded.
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4. Figure 2
IPAN Knockdown Specifically Impairs IAV Replication and Reduces Viral RNA Transcription and Replication
(A) Western blot analysis of IAV NPs in HEK293T-Gluc cells that were transfected with either esiIPAN or control esiRNA (esiEGFP), followed by infection with WSN/33 (MOI = 0.5) for 24 h. The relative levels of each protein were determined using ImageJ software (NIH; bottom).
(B–D) HEK293T-Gluc cells were transfected with various amounts of either esiIPAN or esiEGFP, as indicated, followed by infection with WSN/33 (MOI = 0.5) for 24 h. Viral infectivity was determined by measuring Gluc activity (B), and the levels of IPAN in cells were quantified by qRT-PCR. The GAPDH mRNA level was used as the internal control to normalize the level of IPAN (C). The titers of progeny virus in supernatants were determined in the 50% tissue-culture infectious dose (TCID50) assay (D).
(E) Cell viability was determined by the cell counting kit 8 (CCK8) assay in HEK293T cells transfected with either esiIPAN or esiEGFP.
(F) Viral RNA and protein levels were determined in IPAN knockout A549 cells infected with WSN/33 (MOI = 0.5) for 36 h.
(G) HEK293T-Gluc cells were transfected with either esiIPAN or esiEGFP, followed by infection with type A influenza viruses (strain WSN/33(H1N1), PR/8/34(H1N1), and Beijing/30/95(H3N2)) or type B influenza viruses (strains Beijinghaidian and Massachusetts) (MOI = 0.5) for 24 h, and viral infectivity was determined.
(H) Gluc activity was determined in IPAN knockdown HEK293T-Gluc cells that were infected with WSN/33 (MOI = 0.5) for 24 h (left) and in IPAN knockdown A549-5Ps cells that carry the IAV replicon (right).
(I) Levels of vRNA and mRNA of the NP gene were quantified by qRT-PCR in WSN/33-infected HEK293T-Gluc cells that were transfected with the indicated esiRNAs.
Graphs show the means ± SDs of three independent experiments (A–I). ∗p ≤ 0.05, ∗∗p ≤ 0.01, and NS, not significant (two-tailed Student’s t test).
See also Figure S1.
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5. Figure 3
IAV Infection Enhances IPAN Expression and Causes Translocation of IPAN from the Cytoplasm into the Nucleus
(A) Levels of IPAN in A549 cells that were infected with IAV at the indicated MOIs or heat-inactivated IAV, as quantified by qRT-PCR.
(B) Levels of IPAN in A549 cells that were infected with different influenza virus strains—HIV, EV71, or ZIKV—as quantified by qRT-PCR.
(C) Levels of IPAN, myxovirus-resistance protein A (MxA) mRNA, and ISG15 mRNA in A549 cells that were treated with IFN-α (1,000 IU/mL) for the indicated hours.
(D) Different amounts of poly(I:C) were transfected into 293T cells. Levels of IPAN and IFN-β were determined by qRT-PCR.
(E) Levels of IPAN and IFN-β in A549 cells that were infected with IAV or Sendai virus (SeV), as quantified by qRT-PCR.
(F) RNA-fluorescence in situ hybridization (FISH) and confocal imaging to show the localization of IPAN RNA (red) in IAV-infected cells. Uninfected cells (mock) or cells that were exposed to inactivated IAV were also analyzed as controls. Overexpressed IPAN was detected to show the specificity of FISH staining. Nuclei were stained with DAPI (blue). Scale bars, 5 μm.
(G) The number of IPAN foci per cell was scored, and the N:T (nucleus/total cell) ratio was calculated. Results from 50 cells of either the mock control or IAV infection are summarized in the graph. Each dot represents the N:T ratio of a single cell, and the bars denote the means ±SDs of scatterplots. ∗p ≤ 0.05 (two-tailed Student’s t test). IAV means the WSN strain, unless otherwise specified.
(A–E) Data are normalized to the control group, with the value of control arbitrarily set at 1, and are mean fold-inductions ± SDs of the RNA level from three independent experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, and NS, not significant (one-way ANOVA with Dunnett’s post-test).
See also Figure S2.
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6. Figure 4 Overexpression of IPAN Promotes IAV Replication
(A) Schematic representation of three full-length isoforms and two mutants of human IPAN.
(B) HEK293T-Gluc cells were transfected with the indicated amounts of plasmid DNA expressing different forms of IPAN, followed by infection with WSN/33 (MOI = 0.5) for 24 h. Viral infectivity was determined by measuring Gluc activity.
(C) Viral RNA levels were determined in IPAN knockout A549 cells that were transfected with plasmid DNA expressing different forms of IPAN, followed by infection with WSN/33 (MOI = 0.5) for 24 h.
(D) IAV replication kinetics of IPAN-overexpressing 293T cells were examined by qRT-PCR (MOI = 1). Levels of NP RNA in supernatants were measured at the indicated time points.
(E) Levels of mRNA, vRNA, and cRNA of the viral NP gene were quantified by qRT-PCR in IPAN-overexpressing HEK293T cells that were infected with WSN/33 (MOI = 0.5).
(F and G) 293T-Gluc cells were transfected with plasmid DNA expressing different forms of IPAN or with empty vector (EV) DNA as the control, followed by infection with WSN/33 (MOI = 0.5). Infectious titers of progeny viruses in supernatants were determined in the TCID50 assay (F), and Gluc activity in the supernatants was measured to determine viral infection (G).
Data are the means ± SDs of at least three independent experiments (B, C, and E–G) or two independent experiments (D). ∗p ≤ 0.05, ∗∗p ≤ 0.01, and NS, not significant (one-way ANOVA with Dunnett’s post-test).
See also Figure S3.
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7. Figure 5 IPAN Stabilizes IAV PB1 Protein
(A) A549-5Ps cells were transfected with esiEGFP or esiIPAN; levels of viral PA, PB1, PB2, and NP were analyzed with western blot. Data are representative of two independent experiments.
(B) The level of PB1 mRNA in A549-5Ps cells was quantified by qRT-PCR after transfection with esiEGFP or esiIPAN.
(C) Western blot to measure the level of PB1 protein in A549-5Ps cells that were transfected with esiIPAN or esiEGFP after CHX treatment for the indicated time intervals. Protein band intensity was determined using ImageJ software.
(D) Level of PB1 protein in A549-5Ps cells that were transfected with esiIPAN or esiEGFP, then treated with DMSO or MG132. Protein band intensity was determined using ImageJ software.
(E) HEK293T-Gluc cells were transfected with plasmid DNA expressing PB1 and esiIPAN or esiEGFP, followed by infection with IAV (MOI = 0.5) for 24 h. Levels of PB1 protein were determined by western blot. Gluc activity was measured to quantify viral infectivity. Data are means ± SDs of five independent experiments. ∗p ≤ 0.05 and NS, not significant (two-tailed Student’s t test).
(F) Wild-type or truncated IPAN was expressed in HEK293T-Gluc cells, followed by IAV infection. Levels of the PB1 protein levels were assessed by western blot. Protein band intensity was determined using ImageJ software. Data are means ± SDs of three independent immunoblots. ∗p ≤ 0.05 and NS, not significant (one-way ANOVA with Dunnett’s post-test).
(B–D) Graphs are means ± SDs of three independent experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, and NS, not significant (two-tailed Student’s t test).
Results of controls are arbitrarily set at 1. IAV means the WSN strain, unless otherwise specified.
See also Figure S4.
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8. Figure 6 IPAN Interacts with IAV PB1 Protein
(A) A CL-RIP experiment was performed to detect the association of IPAN RNA with the viral proteins. HEK293T cells were transfected with plasmid DNA expressing PA, PB1, PB2, or NP. Cell lysates were immunoprecipitated with indicated antibodies, followed by qRT-PCR to measure the RNA level of IPAN and GAPDH. Data are means ± SDs of two independent experiments. ∗p ≤ 0.05 and NS, not significant (two-tailed Student’s t test).
(B) A RIP experiment was performed to detect the association of IPAN RNA with viral PB1 protein. HEK293T cells were transfected with plasmid DNA expressing PB1 and different forms of IPAN. Immunoprecipitation was performed with anti-PB1 antibody. Levels of IPAN RNA in the precipitated materials were determined by qRT-PCR. Data are means ± SDs of four independent experiments. ∗p ≤ 0.05 and NS, not significant (two-tailed Student’s t test).
(C) An RNA pull-down assay was performed to detect the association of IPAN RNA with the PB1 protein. A549 cells were infected with IAV (MOI = 0.5) for 36 h. Cell lysates were incubated with biotinylated RNA probes of IPAN. The pull-down products were examined by immunoblot for the presence of IAV proteins. IAV means the WSN strain, unless otherwise specified.
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9. Figure 7 A Model to Illustrate the Role of IPAN in IAV Replication
IAV infection stimulates the expression of IPAN, which associates with viral PB1 protein to form the IPAN/PB1 complex and further protects PB1 from degradation (left). In the absence of IPAN, the host immunity may target PB1 for degradation, leading to the inhibition of viral RNA synthesis (right).
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