Journal: PLoS Pathogens

Article Title: Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus PA-X Host Shutoff Protein

doi: 10.1371/journal.ppat.1005427

Figure Lengend Snippet: (A) Crystal violet staining of plaques generated by wild-type PR8 and the PR8-PA(fs) mutant viruses in Vero cells, showing a decrease in plaque size for PA-X-deficient mutant compared to wild-type virus plaques. (B-D) A549 cells were infected with PR8 or PR8-PA(fs) IAV at a MOI of 0.25. (B) At 12 hpi cells were fixed and the infected cells were identified by immunofluorescence staining for IAV PA protein (green) to confirm that infection rates were highly similar between the two virus inoculums. Cell nuclei were stained with Hoechst dye (blue). (C) Viral protein accumulation was determined by western blotting of the whole cell lysates collected at the indicated time points. (D) Band intensities from (C) were quantified using ImageJ software (NIH) from two independent experiments. Error bars = st. dev. (E) Total RNA was isolated from cells infected as in (A-C) and the mRNA and vRNA levels for a subset of IAV genes were measured by RT-qPCR. Data was normalized to 18S rRNA and values plotted relative to 9 hpi levels in wild-type virus infected cells. (F) The levels of the indicated viral mRNAs at 12 hpi from (E) were plotted as fold change in PR8-PA(fs) infected cells vs. PR8 wild-type infected cells. (G) Rates of RNA synthesis were determined for the indicated genes at 9 hpi using Click-iT nascent RNA labeling and plotted as fold change in PR8-PA(fs) infected cells vs. PR8 wild-type infected cells. 18S values were normalized to input RNA concentration, whereas viral mRNA values were normalized to 18S levels. Error bars = st.dev. from three independent biological replicates. *,** = p value < 0.05, 0.01 for PR8-PA(fs)- vs. PR8-infected cells.

Article Snippet: The following antibodies were used: Xrn1 (1:200, Santa Cruz-16598), IAV NS1 (1:1,000, gift from Kevin Coombs [ ], clone 8C7) M2 (1:1,000, Abcam ab5416), GFP (1:500, Santa Cruz-8334), β tubulin (1:200, Santa Cruz-9104), actin (1:4,000, HRP-conjugated, Cell Signalling #5125), IAV PA (GeneTex-125932), IAV (1:2,000, Abcam ab20841, recognizes NP, M1, and (weakly) HA proteins of PR8 strain).

Techniques: Staining, Generated, Mutagenesis, Virus, Infection, Immunofluorescence, Western Blot, Software, Isolation, Quantitative RT-PCR, Labeling, Concentration Assay

Journal: PLoS Pathogens

Article Title: Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus PA-X Host Shutoff Protein

doi: 10.1371/journal.ppat.1005427

Figure Lengend Snippet: (A) Diagram of the GFP reporter constructs used in the figure, which bear different 3’ mRNA ends: the canonical poly(A) tail added by cellular processing machinery (poly(A)), a hammerhead ribozyme (HR), the hammerhead ribozyme proceeded by a 60-nt templated poly(A) stretch (A60-HR), or the histone stem-loop structure (hisSL). The arrows indicate where the HR self-cleaves the RNA. (B-D) HEK 293Ts were transfected with indicated Pol-II driven GFP reporters and either PA-X or empty vector. Total cellular RNA was extracted and GFP mRNA levels were measured by RT-qPCR (B) or northern blotting (C). GFP expression normalized by 18S rRNA abundance is plotted (B) or reported (C, relative levels) as fold change in PA-X-expressing vs. vector-transfected cells. (D) Levels of GFP for experiments in panel B were assessed by western blot, using tubulin as a loading control. A representative image is shown. ** = p value (Student’s t -test) < 0.01 for fold change of indicated reporter vs. fold change of poly(A) GFP. (E) HEK 293Ts were transfected with indicated Pol-II driven GFP reporters and PA-X from three different IAV strains or empty vector. Total cellular RNA was extracted and GFP or actin mRNA levels were measured by RT-qPCR. GFP expression normalized by 18S rRNA abundance is plotted as fold change in PA-X-expressing vs. vector-transfected cells. Error bars = s.e.m.

Article Snippet: The following antibodies were used: Xrn1 (1:200, Santa Cruz-16598), IAV NS1 (1:1,000, gift from Kevin Coombs [ ], clone 8C7) M2 (1:1,000, Abcam ab5416), GFP (1:500, Santa Cruz-8334), β tubulin (1:200, Santa Cruz-9104), actin (1:4,000, HRP-conjugated, Cell Signalling #5125), IAV PA (GeneTex-125932), IAV (1:2,000, Abcam ab20841, recognizes NP, M1, and (weakly) HA proteins of PR8 strain).

Techniques: Construct, Transfection, Plasmid Preparation, Quantitative RT-PCR, Northern Blot, Expressing, Western Blot, Control

Journal: Cell reports

Article Title: The RNA helicase DHX16 recognizes specific viral RNA to trigger RIG-I-dependent innate antiviral immunity.

doi: 10.1016/j.celrep.2022.110434

Figure Lengend Snippet: Figure 3. DHX16 enhances activation of the IFN-I production pathway (A–D) A549s treated with siNTC or siDHX16 followed by IAV infection for 24 h (PR8 MOI = 0.1). (A) Phosphorylation of TBK1 and IRF3 (immunoblot). (B) IFN-b and IFIT2 expression (qRT-PCR). (C) IFN-b protein in supernatants (ELISA). (D) IAV viral titers (plaque assay). (E–G) A549s transfected with empty vector or DHX16 followed by IAV infection for 24 h (PR8 MOI = 0.1). (E) Phosphorylation of TBK1 and IRF3 (immunoblot). (F) IFN-b and IFITM1 expression (qRT-PCR). (G) IAV viral titers (plaque assay).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Influenza A virus (A/Puerto Rico/8/1934 (PR8) GFP-NS1) Adolfo Garcı́a-Sastre Manicassamy et al., 2010 Sendai virus (Cantell strain) Charles River Laboratories Cat #10100774 Zika virus (ZIKV/Human/Cambodia/ FSS13025/2010) Pei-Yong Shi Shan et al., 2016 SARS-CoV-2 virus (USA-WA1/2020) World Reference Center of Emerging Viruses and Arboviruses (WRCEVA) N/A SARS-CoV-2 virus (icSARS-CoV-2-mNG) Pei-Yong Shi Xie et al., 2020 Chemicals, peptides, and recombinant proteins TransIT-LT1 Mirus Bio Cat #MIR 2305 Lipofectamine 2000 Invitrogen Cat #11668019 Lipofectamine 3000 Invitrogen Cat #L3000015 Lipofectamine RNAiMAX Invitrogen Cat #13778150 Poly(I:C) HMW InvivoGen Cat #tlrl-pic-5 Poly(dA:dT) InvivoGen Cat #tlrl-patn-1 3p-hpRNA InvivoGen Cat #tlrl-hprna Biotin-labeled Poly(I:C) HMW InvivoGen Cat #tlrl-picb IFN-b 1a PBL Assay Science Cat #11410-2 N-ethylmaleimide Sigma-Aldrich Cat #E3876-25G Iodoacetamide Sigma-Aldrich Cat #I1149-25G FLAG peptide Sigma-Aldrich Cat #F3290-4MG HA peptide Thermo Scientific Cat #26184 L-Glutathione reduced Sigma-Aldrich Cat #G4251-25G anti-FLAG M2 EZview agarose beads Sigma-Aldrich Cat #F2426 anti-HA EZview agarose beads Sigma-Aldrich Cat #E6779-5X1ML anti-GST EZview agarose beads Sigma-Aldrich Cat #E6406-1ML Ni-NTA agarose beads Qiagen Cat #30210 Protein A/G agarose beads Pierce Cat #20421 High Capacity NeutrAvidin agarose beads Thermo Scientific Cat #29202 TPCK-treated trypsin Sigma-Aldrich Cat #4370285 ATP Thermo Scientific Cat # R0441 Protease inhibitor cocktail Roche Cat #11873580001 ECL chemiluminescence reagent Pierce Cat #32106 SuperSignal West Femto chemiluminescence reagent Pierce Cat #34095 BIOMOL Green reagent Enzo Life Sciences Cat #BML-AK111-0250 2-16 Ub linkage K48-poly-Ub chains Enzo Life Sciences Cat #BML-UW0670-0100 2-7 Ub linkage K48-poly-Ub chains Boston Biochem Cat #UC-230-100 2-7 Ub linkage K63-poly-Ub chains Boston Biochem Cat #UC-330-100 GST-UBD-IsoT UBPBio Cat #I1400 Critical commercial assays Trans-Blot Turbo transfer system Bio-Rad Cat #1704150 Direct-zol RNA Miniprep Kit Zymo Research Cat #R2053 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat #4368813 iTaq Universal SYBR Green Supermix Bio-Rad Cat #1725125 Dual-Luciferase reporter assay system Promega Cat #E1960 VeriKine human IFN-b ELISA kit PBL Assay Science Cat #41410-2 VeriKine mouse IFN-b ELISA kit PBL Assay Science Cat #42400-2 (Continued on next page) e2 Cell Reports 38, 110434, March 8, 2022

Techniques: Activation Assay, Infection, Phospho-proteomics, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Plaque Assay, Transfection, Plasmid Preparation

Journal: Cell reports

Article Title: The RNA helicase DHX16 recognizes specific viral RNA to trigger RIG-I-dependent innate antiviral immunity.

doi: 10.1016/j.celrep.2022.110434

Figure Lengend Snippet: Figure 3. DHX16 enhances activation of the IFN-I production pathway (A–D) A549s treated with siNTC or siDHX16 followed by IAV infection for 24 h (PR8 MOI = 0.1). (A) Phosphorylation of TBK1 and IRF3 (immunoblot). (B) IFN-b and IFIT2 expression (qRT-PCR). (C) IFN-b protein in supernatants (ELISA). (D) IAV viral titers (plaque assay). (E–G) A549s transfected with empty vector or DHX16 followed by IAV infection for 24 h (PR8 MOI = 0.1). (E) Phosphorylation of TBK1 and IRF3 (immunoblot). (F) IFN-b and IFITM1 expression (qRT-PCR). (G) IAV viral titers (plaque assay).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Influenza A virus (A/Puerto Rico/8/1934 (PR8) GFP-NS1) Adolfo Garcı́a-Sastre Manicassamy et al., 2010 Sendai virus (Cantell strain) Charles River Laboratories Cat #10100774 Zika virus (ZIKV/Human/Cambodia/ FSS13025/2010) Pei-Yong Shi Shan et al., 2016 SARS-CoV-2 virus (USA-WA1/2020) World Reference Center of Emerging Viruses and Arboviruses (WRCEVA) N/A SARS-CoV-2 virus (icSARS-CoV-2-mNG) Pei-Yong Shi Xie et al., 2020 Chemicals, peptides, and recombinant proteins TransIT-LT1 Mirus Bio Cat #MIR 2305 Lipofectamine 2000 Invitrogen Cat #11668019 Lipofectamine 3000 Invitrogen Cat #L3000015 Lipofectamine RNAiMAX Invitrogen Cat #13778150 Poly(I:C) HMW InvivoGen Cat #tlrl-pic-5 Poly(dA:dT) InvivoGen Cat #tlrl-patn-1 3p-hpRNA InvivoGen Cat #tlrl-hprna Biotin-labeled Poly(I:C) HMW InvivoGen Cat #tlrl-picb IFN-b 1a PBL Assay Science Cat #11410-2 N-ethylmaleimide Sigma-Aldrich Cat #E3876-25G Iodoacetamide Sigma-Aldrich Cat #I1149-25G FLAG peptide Sigma-Aldrich Cat #F3290-4MG HA peptide Thermo Scientific Cat #26184 L-Glutathione reduced Sigma-Aldrich Cat #G4251-25G anti-FLAG M2 EZview agarose beads Sigma-Aldrich Cat #F2426 anti-HA EZview agarose beads Sigma-Aldrich Cat #E6779-5X1ML anti-GST EZview agarose beads Sigma-Aldrich Cat #E6406-1ML Ni-NTA agarose beads Qiagen Cat #30210 Protein A/G agarose beads Pierce Cat #20421 High Capacity NeutrAvidin agarose beads Thermo Scientific Cat #29202 TPCK-treated trypsin Sigma-Aldrich Cat #4370285 ATP Thermo Scientific Cat # R0441 Protease inhibitor cocktail Roche Cat #11873580001 ECL chemiluminescence reagent Pierce Cat #32106 SuperSignal West Femto chemiluminescence reagent Pierce Cat #34095 BIOMOL Green reagent Enzo Life Sciences Cat #BML-AK111-0250 2-16 Ub linkage K48-poly-Ub chains Enzo Life Sciences Cat #BML-UW0670-0100 2-7 Ub linkage K48-poly-Ub chains Boston Biochem Cat #UC-230-100 2-7 Ub linkage K63-poly-Ub chains Boston Biochem Cat #UC-330-100 GST-UBD-IsoT UBPBio Cat #I1400 Critical commercial assays Trans-Blot Turbo transfer system Bio-Rad Cat #1704150 Direct-zol RNA Miniprep Kit Zymo Research Cat #R2053 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat #4368813 iTaq Universal SYBR Green Supermix Bio-Rad Cat #1725125 Dual-Luciferase reporter assay system Promega Cat #E1960 VeriKine human IFN-b ELISA kit PBL Assay Science Cat #41410-2 VeriKine mouse IFN-b ELISA kit PBL Assay Science Cat #42400-2 (Continued on next page) e2 Cell Reports 38, 110434, March 8, 2022

Techniques: Activation Assay, Infection, Phospho-proteomics, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Plaque Assay, Transfection, Plasmid Preparation