Journal: mBio

Article Title: Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression

doi: 10.1128/mBio.02829-21

Figure Lengend Snippet: IFIT3 inhibits adenovirus (Ad) replication and early gene expression. (A) Effects of 401 individual (interferon-stimulated genes [ISGs]) on Ad5-enhanced green fluorescent protein (EGFP) spread in A549 cells. ISGs inhibiting more than two standard deviations from the mean in two independent screens are indicated with red dots. Log 10 spread represents the ratio of infected cells at 60 hpi/24 hpi. (B) The top bar graph shows the percent of A549 cells transduced by Lentivirus-ISG vectors as measured by RFP expression. The bottom bar graph shows confirmation assays of selected ISGs on Ad5 spread in A549 cells in follow up experiments. (C) Wild-type Ad5 (HAdV-C5) replication was analyzed in HDF cells compared to cells transduced with the empty Lentivirus vector or vector expressing IFIT3. Cells were infected with HAdV-C5 at 200 P/cell, harvested at 5 and 48 hpi, and viral DNA replication quantified by quantitative PCR. After normalizing the viral DNA copy numbers to glyceraldehyde-3- phosphate dehydrogenase (GAPDH), the fold-increase in viral copy numbers was calculated by normalizing the amount of DNA present at 48 h to the amount present at 5 h. The data are plotted as mean ± SD, n = 3. ( * , P ≤ 0.05; ** * , P ≤ 0.001). (D) HDF, HDF-Empty, and HDF-IFIT3 cells were infected with HAdV-C5 at 200 P/cell. Ad early mRNA levels were quantified by quantitative real time PCR with RNA samples isolated 48 hpi. The results were normalized to GAPDH mRNA levels and fold change in HDF-IFIT3 cells was compared to HDF and HDF-Empty cell lines plotted as mean ± SD, n = 3. ( * , P ≤ 0.05; * * , P ≤ 0.01; ** * , P ≤ 0.001). (E) HDF, HDF-Empty, and HDF-IFIT3 cells were infected with HAdV-C5 at 200 P/cell. Total cell extracts were harvested at 48 hpi and Ad early (E1A) and late (Hexon) proteins and IFIT3 were analyzed by Western blotting using specific antisera. α-Tubulin is shown as a loading control for the samples. The positions of relevant molecular weight markers are indicated in the left.

Article Snippet: After blocking in 10% goat serum, coverslips were incubated with IFIT3 antibody (GeneTex, 112442, 1:300) and monoclonal antibodies against Ad DNA binding protein (A1-6 and B6-8 , 1:100 each) for 24 h at 4°C.

Techniques: Gene Expression, Infection, Expressing, Transduction, Plasmid Preparation, Real-time Polymerase Chain Reaction, Isolation, Western Blot, Control, Molecular Weight

Journal: mBio

Article Title: Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression

doi: 10.1128/mBio.02829-21

Figure Lengend Snippet: IFIT3 knockout in HDF cells does not affect interferon (IFN)-mediated repression of Ad replication and immediate early gene expression. (A) HDF cells were depleted of IFIT3 using a CRISPR-Cas9 strategy. Cells were either treated with IFNα (1000 U/ml) for 24 h or left untreated to confirm IFIT3 knockout. IFIT3 protein was analyzed by Western blotting. (B) HDF and HDF-IFIT3 knockout cells were treated with IFNα for 24 h or left untreated, and then infected with HAdV-C5 at 200 P/cell. Cells were harvested at 5 and 48 hpi and viral DNA replication was quantified by qPCR. Viral DNA copy numbers were first normalized to glyceraldehyde-3- phosphate dehydrogenase (GAPDH), then the fold-increase in viral copy numbers was calculated by normalizing the amount of DNA present at 48 h to the amount present at 5 h, finally viral copy numbers minus IFN treatment were set at 1 and viral copy numbers plus IFNs were calculated relative to this level. The values are plotted as mean ± SD, n = 3. (* * , P ≤ 0.01; ** * , P ≤ 0.001). (C) Ad5 E1A and IFIT3 proteins were analyzed by Western blotting.

Article Snippet: After blocking in 10% goat serum, coverslips were incubated with IFIT3 antibody (GeneTex, 112442, 1:300) and monoclonal antibodies against Ad DNA binding protein (A1-6 and B6-8 , 1:100 each) for 24 h at 4°C.

Techniques: Knock-Out, Gene Expression, CRISPR, Western Blot, Infection

Journal: mBio

Article Title: Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression

doi: 10.1128/mBio.02829-21

Figure Lengend Snippet: IFIT3 does not block nuclear accumulation of Ad genomes. HDF and HDF-IFIT3 cells were infected with HAdV-C5 at 1000 P/cell and immunostained for Ad core Protein VII (FITC) and a nuclear protein, Lamin B (TRITC) at 6 hpi. Merged images are shown on the right.

Article Snippet: After blocking in 10% goat serum, coverslips were incubated with IFIT3 antibody (GeneTex, 112442, 1:300) and monoclonal antibodies against Ad DNA binding protein (A1-6 and B6-8 , 1:100 each) for 24 h at 4°C.

Techniques: Blocking Assay, Infection

Journal: mBio

Article Title: Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression

doi: 10.1128/mBio.02829-21

Figure Lengend Snippet: Induction of interferon (IFN) signaling by IFIT3. (A, B) HDF, HDF-Empty and HDF-IFIT3 cell extracts were analyzed by Western blotting using antibodies against (A) IFIT1, IFIT2 and IFIT3, (B) total STAT1 and phosho-STAT1 (Y701). The bands near the bottom of the gel in (A), IFIT2 and IFIT3, are nonspecific, cross-reactive proteins. (C) RNA was isolated from HDF, HDF-Empty and HDF-IFIT3 cells and IFNβ and specific interferon-stimulated genes (ISGs) (IFIT1, ISG15, OAS3, and MX1) mRNA levels were quantified by RT-qPCR. The results were normalized to glyceraldehyde-3- phosphate dehydrogenase (GAPDH) mRNA levels and fold change in HDF-IFIT3 cells was compared to HDF and HDF-Empty cell lines, and plotted as mean ± SD, n = 3. (D) HDF, HDF-Empty and HDF-IFIT3 cell extracts were analyzed by Western blotting for total TBK1, phospho-TBK1 (S172), total IRF3, and phopsho-IRF3 (S396). Human THP1 monocytes were left untreated or treated with IFN-stimulatory DNA (ISD) as a positive control for activation of TBK1 signaling lanes 1 and 2. (E) HDF, HDF-IFIT3, and HDF-IFIT3+JAK1 knockout cells were infected with HAdV-C5 at 200 P/cell and viral DNA replication was quantified by qPCR at 48 hpi, as described for Fig. 1C . The data are plotted as mean ± sd, n = 3. (* * , P ≤ 0.01). (F) HDF, HDF-IFIT3, and HDF-IFIT3+JAK1 knockout cells were infected with HAdV-C5 at 200 P/cell and protein expression analyzed by Western blotting at 48 hpi using antibodies against total STAT1, phopsho-STAT1, JAK1, IFIT3, IFIT1, and Ad5 E1A.

Article Snippet: After blocking in 10% goat serum, coverslips were incubated with IFIT3 antibody (GeneTex, 112442, 1:300) and monoclonal antibodies against Ad DNA binding protein (A1-6 and B6-8 , 1:100 each) for 24 h at 4°C.

Techniques: Western Blot, Isolation, Quantitative RT-PCR, Positive Control, Activation Assay, Knock-Out, Infection, Expressing

Journal: mBio

Article Title: Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression

doi: 10.1128/mBio.02829-21

Figure Lengend Snippet: TBK1 and STING knockout blocks IFIT3 induction of interferon (IFN) signaling and the inhibition of adenovirus (Ad) replication. (A) HDF, HDF-Empty and HDF-IFIT3 cells were left untreated or treated with the TBK1 inhibitor BX795 (2 μM) for 24 h. IFN-β mRNA levels were quantified by quantitative real time PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. The data are plotted as mean ± SD, n = 3. (** * , P ≤ 0.001). (B) HDF, HDF-IFIT3 (IFIT3), and HDF-IFIT3+TBK1 knockout cells (CR-TBK1) were infected with HAdV-C5 at 200 P/cell and viral DNA replication was quantified by qPCR at 48 hpi as described for Fig. 1C . The data are plotted as mean ± SD, n = 3. (* * , P ≤ 0.01; ** * , P ≤ 0.001). (C) HDF, HDF-IFIT3 (IFIT3), and HDF-IFIT3+TBK1 knockout cells (CR-TBK1) were infected with HAdV-C5 at 200 P/cell and protein expression analyzed by Western blotting at 48 hpi using antibodies against total STAT1, phopsho-STAT1, TBK1, IFIT1, IFIT3, and Ad5 E1A. (D) HDF, HDF-IFIT3 (IFIT3), and HDF-IFIT3+STING knockout cells (CR-STING) were infected with HAdV-C5 at 200 P/cell and viral DNA replication was quantified by qPCR at 48 hpi as described for Fig. 1C . The data are plotted as mean ± SD, n = 3. (** * , P ≤ 0.001). (E) HDF, HDF-IFIT3 (IFIT3), and HDF-IFIT3+STING knockout cells (CR-STING) were infected with HAdV-C5 at 200 P/cell and protein expression was analyzed by Western blotting at 48 hpi using antibodies against total STAT1, phospho-STAT1, IFIT1, IFIT3, STING, and Ad5 E1A. (F) HDF, HDF-IFIT3 (IFIT3), and HDF-IFIT3+cyclic GMP-AMP synthase (cGAS) knockout cells (CR-cGAS) were infected with HAdV-C5 at 200 P/cell and viral DNA replication was quantified by qPCR at 48 hpi as described for Fig. 1C . The data are plotted as mean ± SD, n = 3. (* * , P ≤ 0.01). (G) HDF, HDF-IFIT3 (IFIT3), and HDF-IFIT3+cGAS knockout cells (IFIT3) were infected with HAdV-C5 at 200 P/cell and protein expression analyzed by Western blotting at 48 hpi using antibodies against total STAT1, phospho-STAT1, IFIT1, IFIT3, cGAS, and Ad5 E1A.

Article Snippet: After blocking in 10% goat serum, coverslips were incubated with IFIT3 antibody (GeneTex, 112442, 1:300) and monoclonal antibodies against Ad DNA binding protein (A1-6 and B6-8 , 1:100 each) for 24 h at 4°C.

Techniques: Knock-Out, Inhibition, Real-time Polymerase Chain Reaction, Infection, Expressing, Western Blot

Journal: mBio

Article Title: Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression

doi: 10.1128/mBio.02829-21

Figure Lengend Snippet: Mitochondrial antiviral signaling (MAVS) knockout blocks the effect of IFIT3 on adenovirus (Ad) replication and early gene expression. (A) HDF, HDF-IFIT3 (IFIT3), and HDF-IFIT3+MAVS (CR-MAVS) knockout cells (CR-MAVS) were infected with HAdV-C5 at 200 P/cell and viral DNA replication was quantified by qPCR at 48 hpi as described for Fig. 1C . The data are plotted as mean ± sd, n = 3. (* * , P ≤ 0.01). (B) HDF, HDF-IFIT3 (IFIT3), and HDF-IFIT3+MAVS knockout cells (CR-MAVS) were infected with HAdV-C5 at 200 P/cell and cell lysates were analyzed by Western blotting at 48 hpi using antibodies against STAT1, phospho-STAT1, MAVS, IFIT1, IFIT3, and Ad5 E1A.

Article Snippet: After blocking in 10% goat serum, coverslips were incubated with IFIT3 antibody (GeneTex, 112442, 1:300) and monoclonal antibodies against Ad DNA binding protein (A1-6 and B6-8 , 1:100 each) for 24 h at 4°C.

Techniques: Knock-Out, Gene Expression, Infection, Western Blot

Journal: mBio

Article Title: Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression

doi: 10.1128/mBio.02829-21

Figure Lengend Snippet: IFIT1 and IFIT2 expression do not inhibit adenovirus (Ad) replication and early gene expression. (A, B) Cell extracts from HDF cells and HDF cells expressing IFIT1, IFIT2, or IFIT3 were analyzed by Western blotting using antibodies against IFIT1, IFIT2 and IFIT3. HDF cells were treated with interferon (IFN)α for 24 h as a positive control. (C) HDF cells and HDF cells expressing IFIT1, IFIT2, or IFIT3 were infected with HAdV-C5 at 200 P/cell and viral DNA replication was quantified by qPCR at 48 hpi as described for Fig. 1C . The data are plotted as mean ± SD, n = 3. ( * , P ≤ 0.05; ** * , P ≤ 0.001; ns = not statistically significant). (D) HDF cells and HDF cells expressing IFIT1, IFIT2, or IFIT3 were infected with HAdV-C5 at 200 P/cell and cell lysates were analyzed by Western blotting at 48 hpi using antibodies against Ad5 E1A.

Article Snippet: After blocking in 10% goat serum, coverslips were incubated with IFIT3 antibody (GeneTex, 112442, 1:300) and monoclonal antibodies against Ad DNA binding protein (A1-6 and B6-8 , 1:100 each) for 24 h at 4°C.

Techniques: Expressing, Gene Expression, Western Blot, Positive Control, Infection

Journal: mBio

Article Title: Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression

doi: 10.1128/mBio.02829-21

Figure Lengend Snippet: IFIT1 is required for IFIT3 inhibition of Ad replication and early gene expression. (A) HDF-IFIT3 cells were depleted of IFIT1 using CRISPR-Cas9. HDF, HDF-IFIT3 (IFIT3), and HDF-IFIT3+IFIT1 knockout cells (CR-IFIT1) were infected with HAdV-C5 at 200 P/cell and viral DNA replication was quantified using qPCR at 48 hpi as described for Fig. 1C . The data are plotted as mean ± SD, n = 3. ( * , P ≤ 0.05; * * , P ≤ 0.01). (B) Cell HDF, HDF-IFIT3 (IFIT3), and HDF-IFIT3+IFIT1 knockout cells (CR-IFIT1) were infected with HAdV-C5 at 200 P/cell and cell lysates were analyzed by Western blotting at 48 hpi using antibodies against total STAT1, phospho-STAT1, IFIT1, IFIT3, and Ad5 E1A. (C) HDF-IFIT3 cells were depleted of IFIT2 using CRISPR-Cas9. HDF, HDF-IFIT3 (IFIT3), and HDF-IFIT3+IFIT2 knockout cells (CR-IFIT2) were infected with HAdV-C5 at 200 P/cell and viral DNA replication was quantified using quantitative PCR at 48 hpi as described for Fig. 1C . The data are plotted as mean ± SD, n = 3. ( * , P ≤ 0.05; ** * , P ≤ 0.001). (D) HDF, HDF-IFIT3 (IFIT3), and HDF-IFIT3+IFIT2 knockout cells (CR-IFIT2) were infected with HAdV-C5 at 200 P/cell and cell lysates were analyzed by Western blotting at 48 hpi using antibodies against IFIT2, IFIT3, and Ad5 E1A. The asterisk in the IFIT2 blot indicates IFIT3 which cross-reacts with the IFIT2 antibody.

Article Snippet: After blocking in 10% goat serum, coverslips were incubated with IFIT3 antibody (GeneTex, 112442, 1:300) and monoclonal antibodies against Ad DNA binding protein (A1-6 and B6-8 , 1:100 each) for 24 h at 4°C.

Techniques: Inhibition, Gene Expression, CRISPR, Knock-Out, Infection, Western Blot, Real-time Polymerase Chain Reaction

Journal: mBio

Article Title: Human IFIT3 Protein Induces Interferon Signaling and Inhibits Adenovirus Immediate Early Gene Expression

doi: 10.1128/mBio.02829-21

Figure Lengend Snippet: Schematic diagram of IFIT3 as an adaptor protein in innate immunity. Expression of IFIT3 in HDF cells leads to increased expression of IFIT1 and IFIT2, which forms a complex that activates the STING and mitochondrial antiviral signaling (MAVS) pathways. This leads to phosphorylation of TBK1 which, in turn, phosphorylates and activates IRF3. Phosphorylated IRF3 dimerizes and translocates to the nucleus where it activates interferon (IFN)β gene expression. IFNβ is secreted, binds to the IFNAR, and activates canonical IFN signaling and the induction of interferon-stimulated genes (ISG) expression. One or more ISGs block Ad E1A immediate early gene expression and viral DNA replication. A:G represents cGAMP, an activator of STING at the Golgi; MAVS association with mitochondria is shown.

Article Snippet: After blocking in 10% goat serum, coverslips were incubated with IFIT3 antibody (GeneTex, 112442, 1:300) and monoclonal antibodies against Ad DNA binding protein (A1-6 and B6-8 , 1:100 each) for 24 h at 4°C.

Techniques: Expressing, Phospho-proteomics, Gene Expression, Blocking Assay