anti ifit1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Small aberrant viral genomes induce the innate immune response to arenaviruses

doi: 10.64898/2026.03.09.710519

Figure Lengend Snippet: (a) A549 cells were infected with untreated or UV-irradiated TCRV at an MOI of 0.01 for 72 h. Whole cell lysates were analysed by western blot for TCRV NP, IFIT1, STAT1, phospho-STAT1 (Y701) expression. (b) Total RNA was extracted at 48 hpi from uninfected and MOPV, LASV or TCRV (MOI = 0.1) infected A549 cells. RNA was subsequently transfected into A549-ISRE-Nluc2AGFP cells and ISRE-induced NanoLuc luciferase activity was measured at 24 h post-transfection. The graph depicts the mean reporter activity normalised to uninfected control cellular RNA from six technical replicates. (c-f) A549-ISRE-Nluc2AGFP cells were infected with LATV or MOPV (MOI = 0.1) for 72 hpi before being treated with 1,000 iu/ml IFN-ɑ2b or 1 µg poly(I:C) RNA. ISRE-mediated NanoLuc luciferase activity was measured 24 h post-treatment. (c) The graph depicts mean relative light units normalised to the uninfected and untreated control from three independent biological replicates. Error bars represent the standard deviation of the mean. RT-qPCR analysis of (d) LATV and MOPV S RNA, (e) IFNB1 and (f) ISG15 gene expression levels normalised to the ACTB housekeeping gene. (g-i) HEK293T cells were transiently co-transfected with plasmids expressing a Firefly luciferase reporter under the control of an IFNB1 promoter, RIG-I and the indicated (g) NP or (h) Z proteins of LASV and TCRV, or (i) the NP and Z proteins of the indicated arenaviruses. The graphs depict the mean relative light units normalised to the uninhibited positive control from 6 to 10 technical replicates. Error bars represent the standard deviation of the mean. (j-k) A549 cells infected with TCRV or LASV (MOI = 0.1) for 48 hpi were analysed by total RNA-seq and viral ORF expression was determined relative to (j) the total number of viral reads or (k) to the size of ORFs and the mean viral reads per nucleotide. Statistical significance was determined by One-way ANOVA with post-hoc Turkey test (for e-f), One-way ANOVA with post-hoc Dunnett’s test (for g-i) and or by Two-way ANOVA with post-hoc Šídák’s test (for c, j-k) for multiple comparisons: **** ( p < 0.0001), *** ( p < 0.001), ** ( p < 0.01), * ( p < 0.05), ns ( p > 0.05).

Article Snippet: Target proteins were detected using primary antibodies again Actin (Invitrogen; MA5-11869), GAPDH (Sigma-Aldrich; G9545), IFIT1 (Cell Signaling Technology; 14769), STAT1 (Cell Signaling Technology; 9172), p-STAT1 [Tyr701] (Cell Signaling Technology; 9167), MX1 (Abcam; ab95926), LASV NP (GeneTex; GTX636832), and JUNV NP (BEI Resources; NR-48834).

Techniques: Infection, Irradiation, Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Control, Standard Deviation, Quantitative RT-PCR, Gene Expression, Positive Control, RNA Sequencing

Journal: bioRxiv

Article Title: Small aberrant viral genomes induce the innate immune response to arenaviruses

doi: 10.64898/2026.03.09.710519

Figure Lengend Snippet: (a) Maximum-likelihood phylogenetic tree based on the amino acid sequences of arenavirus RdRps. Arenaviruses are grouped into Old World and New World (clades A-D) viruses. Viruses used in this study are highlighted with a red asterisk (*). Old World arenaviruses are highlighted in blue colours; New World arenaviruses are highlighted in red colours throughout this study. (b-f) A549 cells were infected with the indicated arenaviruses at an MOI of 0.1 and samples were collected immediately after infection (0 hpi) or over a 72 h timecourse and analysed by RT-qPCR for the expression of (b) viral S segment RNA, (c) IFNB1 , (d) ISG15 or (e) IFIT1 gene expression or by (f) western blot analysis for Mx1, IFIT1 and GAPDH protein expression. (g) A549-ISRE-mScarlet cells were infected with the indicated arenaviruses at an MOI of 0.1 and ISRE-dependent reporter activity was measured at 4 h intervals for 120 hours post-infection (hpi). The graph depicts the mean fluorescence intensity from three independent biological replicates with error bars representing the standard deviation of the mean. (h-k) A549 cells were infected with LASV at an MOI of 0.1 or 3 and RNA or whole cell lysates were extracted at the indicated time points. A549 cells transfected with poly(I:C) RNA for 24 h were used as control. Extracted RNA was analysed by RT-qPCR for (h) LASV S segment, (i) IFNB1 or (j) ISG15 gene expression. (k) Whole cell lysates were analysed by western blot for the expression of LASV NP, IFIT1 or ϕ3-Actin. (l-n) HPMECs were infected with MOPV or LATV at an MOI of 0.1 for 48 hpi. Total RNA was analysed by RT-qPCR for (l) viral S segment expression, (m) IFNB1 or (n) ISG15 gene expression. All graphs representing RT-qPCR data depict the mean relative fold change of gene expression normalized to human GAPDH expression levels from three independent biological replications. Error bars represent the standard deviation of the mean. Statistical significance was determined by One-way ANOVA with post-hoc Tukey test for multiple comparisons: **** ( p < 0.0001), *** ( p < 0.001), ** ( p < 0.01), * ( p < 0.05), ns ( p > 0.05).

Article Snippet: Target proteins were detected using primary antibodies again Actin (Invitrogen; MA5-11869), GAPDH (Sigma-Aldrich; G9545), IFIT1 (Cell Signaling Technology; 14769), STAT1 (Cell Signaling Technology; 9172), p-STAT1 [Tyr701] (Cell Signaling Technology; 9167), MX1 (Abcam; ab95926), LASV NP (GeneTex; GTX636832), and JUNV NP (BEI Resources; NR-48834).

Techniques: Infection, Quantitative RT-PCR, Expressing, Gene Expression, Western Blot, Activity Assay, Fluorescence, Standard Deviation, Transfection, Control

Journal: bioRxiv

Article Title: Small aberrant viral genomes induce the innate immune response to arenaviruses

doi: 10.64898/2026.03.09.710519

Figure Lengend Snippet: (a-d) A549 cells were infected with the indicated arenaviruses at an MOI of 0.1. At the indicated times post infection RNA was extracted and analysed by RT-qPCR for the relative gene expression of (a) viral S segment RNA, (b) IFNB1 , or (c) ISG15 or (d) whole cell lysates were analysed by western blot analysis for viral NP, IFIT1 and or ϕ3-Actin protein expression. Poly(I:C) transfected A549 cells (24 h post-transfection) were used as control where indicated. (e-f) A549 cells were infected with (d) MOBV or (e) LASV at an MOI of 0.1. At 48 hpi, cells were fixed and analysed by immunofluorescence staining for viral NP protein and DAPI staining for DNA.

Article Snippet: Target proteins were detected using primary antibodies again Actin (Invitrogen; MA5-11869), GAPDH (Sigma-Aldrich; G9545), IFIT1 (Cell Signaling Technology; 14769), STAT1 (Cell Signaling Technology; 9172), p-STAT1 [Tyr701] (Cell Signaling Technology; 9167), MX1 (Abcam; ab95926), LASV NP (GeneTex; GTX636832), and JUNV NP (BEI Resources; NR-48834).

Techniques: Infection, Quantitative RT-PCR, Gene Expression, Western Blot, Expressing, Transfection, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Small aberrant viral genomes induce the innate immune response to arenaviruses

doi: 10.64898/2026.03.09.710519

Figure Lengend Snippet: (a) A549 cells were infected with untreated or UV-irradiated TCRV at an MOI of 0.01 for 72 h. Whole cell lysates were analysed by western blot for TCRV NP, IFIT1, STAT1, phospho-STAT1 (Y701) expression. (b) Total RNA was extracted at 48 hpi from uninfected and MOPV, LASV or TCRV (MOI = 0.1) infected A549 cells. RNA was subsequently transfected into A549-ISRE-Nluc2AGFP cells and ISRE-induced NanoLuc luciferase activity was measured at 24 h post-transfection. The graph depicts the mean reporter activity normalised to uninfected control cellular RNA from six technical replicates. (c-f) A549-ISRE-Nluc2AGFP cells were infected with LATV or MOPV (MOI = 0.1) for 72 hpi before being treated with 1,000 iu/ml IFN-ɑ2b or 1 µg poly(I:C) RNA. ISRE-mediated NanoLuc luciferase activity was measured 24 h post-treatment. (c) The graph depicts mean relative light units normalised to the uninfected and untreated control from three independent biological replicates. Error bars represent the standard deviation of the mean. RT-qPCR analysis of (d) LATV and MOPV S RNA, (e) IFNB1 and (f) ISG15 gene expression levels normalised to the ACTB housekeeping gene. (g-i) HEK293T cells were transiently co-transfected with plasmids expressing a Firefly luciferase reporter under the control of an IFNB1 promoter, RIG-I and the indicated (g) NP or (h) Z proteins of LASV and TCRV, or (i) the NP and Z proteins of the indicated arenaviruses. The graphs depict the mean relative light units normalised to the uninhibited positive control from 6 to 10 technical replicates. Error bars represent the standard deviation of the mean. (j-k) A549 cells infected with TCRV or LASV (MOI = 0.1) for 48 hpi were analysed by total RNA-seq and viral ORF expression was determined relative to (j) the total number of viral reads or (k) to the size of ORFs and the mean viral reads per nucleotide. Statistical significance was determined by One-way ANOVA with post-hoc Turkey test (for e-f), One-way ANOVA with post-hoc Dunnett’s test (for g-i) and or by Two-way ANOVA with post-hoc Šídák’s test (for c, j-k) for multiple comparisons: **** ( p < 0.0001), *** ( p < 0.001), ** ( p < 0.01), * ( p < 0.05), ns ( p > 0.05).

Article Snippet: Target proteins were detected using primary antibodies again Actin (Invitrogen; MA5-11869), GAPDH (Sigma-Aldrich; G9545), IFIT1 (Cell Signaling Technology; 14769), STAT1 (Cell Signaling Technology; 9172), p-STAT1 [Tyr701] (Cell Signaling Technology; 9167), MX1 (Abcam; ab95926), LASV NP (GeneTex; GTX636832), and JUNV NP (BEI Resources; NR-48834).

Techniques: Infection, Irradiation, Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Control, Standard Deviation, Quantitative RT-PCR, Gene Expression, Positive Control, RNA Sequencing

Journal: bioRxiv

Article Title: Small aberrant viral genomes induce the innate immune response to arenaviruses

doi: 10.64898/2026.03.09.710519

Figure Lengend Snippet: (a) Schematic of viral RNA sensing and cellular antiviral signalling pathways. (b-f) A549-ISRE-Nluc2AGFP cells with or without RIG-I or MAVS knockouts were infected with TCRV or MOPV (MOI = 0.1). At 24 hpi ISRE-induced NanoLuc luciferase activity was measured or RNA was extracted and analysed by RT-qPCR. (b) The graph depicts the mean relative light units normalised to uninfected control cells from three independent biological replicates with error bars representing the standard deviation of the mean. (c) Extracted RNA was analysed by RT-qPCR for relative MOPV S RNA, (d) TCRV S RNA, (e) IFNB1 and (f) ISG15 gene expression. (g-j) A549-Cas9 cells with or without PKR knockouts were infected with TCRV or MOPV (MOI = 0.1) for 24 hpi. Extracted RNA was analysed by RT-qPCR for relative (g) MOPV S RNA, (h) TCRV S RNA, (i) IFNB1 and (j) ISG15 gene expression. (k) A549 cells were infected with LASV or TCRV (MOI = 0.1) for 48 hpi and extracted RNA was analysed by small RNA-seq. The graphs depict the coordinates and abundance of break and rejoin points of small TCRV delVGs. (l) A549 cells were transfected with siRNAs targeting the TCRV NP mRNA transcripts, followed by infection with TCRV (MOI = 0.1) for 24 hpi. Whole cells lysates were analysed by western blot for TCRV NP, IFIT1 and GAPDH protein expression. (m) Extracted RNA was analysed by RT-qPCR for the relative expression TCRV S RNA, IFNB1 and ISG15 gene expression. (n) The small RNA fraction (<200 nt) was analysed by vgRT-PCR and denaturing 7M urea 8% PAGE. Red arrows depict TCRV-derived mini viral RNAs. The graphs related to RT-qPCR data depict the mean fold change in gene expression normalised to the ACTB housekeeping gene from three independent biological replicates, with error bars representing the standard deviation of the mean. Statistical significance was determined by Two-way ANOVA with post-hoc Dunnett’s test (for b-f) and Šídák’s test (for g-j and m) for multiple comparisons: **** ( p < 0.0001), *** ( p < 0.001), ** ( p < 0.01), * ( p < 0.05), ns ( p > 0.05).

Article Snippet: Target proteins were detected using primary antibodies again Actin (Invitrogen; MA5-11869), GAPDH (Sigma-Aldrich; G9545), IFIT1 (Cell Signaling Technology; 14769), STAT1 (Cell Signaling Technology; 9172), p-STAT1 [Tyr701] (Cell Signaling Technology; 9167), MX1 (Abcam; ab95926), LASV NP (GeneTex; GTX636832), and JUNV NP (BEI Resources; NR-48834).

Techniques: Infection, Luciferase, Activity Assay, Quantitative RT-PCR, Control, Standard Deviation, Gene Expression, RNA Sequencing, Transfection, Western Blot, Expressing, Derivative Assay