zikv rns1  (Native Antigen Inc)

Journal: Emerging Microbes & Infections

Article Title: Erp57 facilitates ZIKV-induced DNA damage via NS2B/NS3 complex formation

doi: 10.1080/22221751.2024.2417864

Figure Lengend Snippet: ERp57 is critical for ZIKA nonstructural protein expression and function. (A) Representative immunofluorescent staining images show the localization of ZIKV NS5 (green). WT RD cells were seeded in 24-well plates with pre-coated cover glasses, then infected with ZIKA virus (MOI = 1) for the indicated time. Cells were fixed and stained with anti-NS5 (green) fluorescence antibody and DAPI (blue) and visualized under confocal microscope. (B) and (C) Both wild type RD cells and knockout cells were infected by ZIKV (MOI = 1) at the indicated time and protein level was analysed by western blot. (D) Both WT and KO RD cells were infected with ZIKV (MOI = 1) for the indicated time. Cells were fixed and stained with anti-NS1 (green) fluorescence antibody and DAPI (blue) and visualized under confocal microscope. (E) Both wild type RD cells and ERp57-KO cell line were transfected with NS1-flag plasmid or vector, then infected with ZIKV (MOI = 1) for 48 h. Protein samples were blotted to investigate the expression level of NS1 and other DDR related factors. (F) Western blot images of WT and ERp57-KO cells transfected with NS1-Flag plasmid or vector for 24 h.

Article Snippet: Anti–βactin, anti-GAPDH, anti-ERp57 and other antibodies for apoptosis factors were purchase from Santa Cruz, anti-H2AX, anti-ATM and anti-p-ATM antibodies were purchased from Cell Signaling Technology, rabbit anti-ZIKV NS1 and other nonstructural protein antibodies were purchased from GeneTex, anti-Flag tag antibodies (Sigma-Aldrich) were also used for detection at the appropriate dilutions.

Techniques: Expressing, Staining, Infection, Virus, Fluorescence, Microscopy, Knock-Out, Western Blot, Transfection, Plasmid Preparation

Journal: Journal of Virology

Article Title: Activation of ATF3 via the integrated stress response pathway regulates innate immune response to restrict Zika virus

doi: 10.1128/jvi.01055-24

Figure Lengend Snippet: ZIKV significantly induces ATF3 expression 24 hours after infection. ( A ) A549 wild-type (WT) cells were infected with ZIKV PR at a multiplicity of infection (moi) of 10 PFU/cell for 0, 12, 24, or 48 hours post-infection (hpi). Cellular (ATF4 and ATF3) and viral (NS1) proteins were assayed by Western blot with GAPDH as the loading control. Western blot is representative of at least three independent experiments. ( B–E ) Total RNA was extracted at the indicated timepoints and used as template for RT-qPCR to measure the expression of ATF4 , ZIKV, ATF3 , and CHOP mRNAs. The relative mRNA expression was determined by the 2 −ΔΔCt method using mock-infected cells as reference, and the genes were normalized to ACTB . RT-qPCR data are means ± SD of three technical replicates and determined from three independent experiments. ( F ) Viral titers in the cell culture media collected at the different infection time points were measured by plaque assay. PFU, plaque-forming units. The data represent the means ± SD of two technical replicates from three independent experiments. Statistical significance was determined by Student t -test. * P < 0.01, ** P < 0.001, *** P < 0.0005, and **** P < 0.0001; ns: not significant.

Article Snippet: The following primary antibodies were used: rabbit anti-ZIKV NS1 (GeneTex, GTX133307; 1:10,000), mouse anti-GAPDH (ProteinTech, #60004–1-lg; 1:10,000), rabbit anti-ATF3 (Abcam, #AB207434; 1:1,000), rabbit anti-ATF4 (D4B8; Cell Signaling, #11815; 1:1,000), rabbit anti-PERK (D11A8; Cell Signaling, #5683; 1:1,000), rabbit anti-eIF2ɑ (D7D3; Cell Signaling, #5324; 1:1,000), rabbit anti-p-eIF2ɑ (D9G8; Cell Signaling, #3398; 1:1,000), rabbit anti-STAT1 (D1K9Y; Cell Signaling, #14994; 1:1,000), rabbit anti-phospho-STAT1 (D4A7; Cell Signaling, #7649; 1:1,000), rabbit anti-STAT2 (D9J7L; Cell Signaling, #72604; 1:1,000), rabbit anti-phospho-STAT2 (D3P2P; Cell Signaling, #88410, 1:1,000), rabbit anti-IFIT1 (D2 × 9Z; Cell Signaling, #14769; 1:1,000), rabbit anti-MX1 (D3W7I; Cell Signaling, #37849, 1:1,000), rabbit anti-fibrillarin (Abcam, #Ab166630, 1:6,000), and mouse α-tubulin (Proteintech, #,66031–1-Ig, 1:5,000).

Techniques: Expressing, Infection, Western Blot, Control, Quantitative RT-PCR, Cell Culture, Plaque Assay

Journal: Journal of Virology

Article Title: Activation of ATF3 via the integrated stress response pathway regulates innate immune response to restrict Zika virus

doi: 10.1128/jvi.01055-24

Figure Lengend Snippet: ATF3 restricts ZIKV gene expression. The effect of ATF3 expression on ZIKV infection was examined by infecting A549 WT and ATF3 KO cells with or without ZIKV PR (moi of 1 and 10 PFU/cell) for 24 hours. ( A ) ZIKV NS1 and ATF3 proteins were analyzed by Western blot in which GAPDH was used as the loading control. The Western blot shown is a representative of three independent experiments. Total RNA from infected cells was analyzed by RT-qPCR using primers specific to ( B ) ZIKV and ( C ) ATF3 . The RNA expression was normalized to ACTB , and the relative transcript expression was calculated by the 2 −ΔΔCt method using mock-infected cells as a reference for the different cell lines. The data shown are means ± SD for three technical replicates and are from three independent experiments. ( D ) Virions released during infection in WT and ATF3 KO cells were quantified as the average viral titer (PFU/mL) using the plaque assay method. The mean PFU/mL ± SD was derived from two technical assays of three independent experiments. ( E and F ) To validate the role of ATF3 in ZIKV infection, HCT-116 WT and ATF3 KO cells were infected with ZIKV PR (moi = 10 PFU/cell) for 48 hours. ( E ) ATF3 and viral NS1 proteins were analyzed by Western blot with GAPDH as the loading control. ( F ) ZIKV RNA expression normalized to ACTB was determined by RT-qPCR (2 −ΔΔCt method). Data are from three independent experiments, and three technical replicates within each experiment are shown as mean ± SD. ( G ) Viral titers in HCT-116 cell culture media were measured by plaque assay. Statistical significance was determined by Student t -test. ** P < 0.05 and **** P < 0.0001; ns: not significant.

Article Snippet: The following primary antibodies were used: rabbit anti-ZIKV NS1 (GeneTex, GTX133307; 1:10,000), mouse anti-GAPDH (ProteinTech, #60004–1-lg; 1:10,000), rabbit anti-ATF3 (Abcam, #AB207434; 1:1,000), rabbit anti-ATF4 (D4B8; Cell Signaling, #11815; 1:1,000), rabbit anti-PERK (D11A8; Cell Signaling, #5683; 1:1,000), rabbit anti-eIF2ɑ (D7D3; Cell Signaling, #5324; 1:1,000), rabbit anti-p-eIF2ɑ (D9G8; Cell Signaling, #3398; 1:1,000), rabbit anti-STAT1 (D1K9Y; Cell Signaling, #14994; 1:1,000), rabbit anti-phospho-STAT1 (D4A7; Cell Signaling, #7649; 1:1,000), rabbit anti-STAT2 (D9J7L; Cell Signaling, #72604; 1:1,000), rabbit anti-phospho-STAT2 (D3P2P; Cell Signaling, #88410, 1:1,000), rabbit anti-IFIT1 (D2 × 9Z; Cell Signaling, #14769; 1:1,000), rabbit anti-MX1 (D3W7I; Cell Signaling, #37849, 1:1,000), rabbit anti-fibrillarin (Abcam, #Ab166630, 1:6,000), and mouse α-tubulin (Proteintech, #,66031–1-Ig, 1:5,000).

Techniques: Gene Expression, Expressing, Infection, Western Blot, Control, Quantitative RT-PCR, RNA Expression, Plaque Assay, Derivative Assay, Cell Culture

Journal: Journal of Virology

Article Title: Activation of ATF3 via the integrated stress response pathway regulates innate immune response to restrict Zika virus

doi: 10.1128/jvi.01055-24

Figure Lengend Snippet: ZIKV activates ATF3 through the ISR pathway. ( A ) Schematic of the ISR pathway. Stress conditions like virus infections, ER stress, amino acid deprivation, and oxidative stress cause stalling of cap-dependent translation by phosphorylating eIF2ɑ and inducing the translation of ATF4. ATF4 in turn activates downstream targets including ATF3 to restore cellular homeostasis. A549 WT cells were mock-infected or infected with the ZIKV PR (moi = 10 PFU/cell) in the presence or absence of ISRIB, an ISR inhibitor. Cells were harvested 24 hours post-infection, and ( B ) cellular and viral proteins analyzed by Western blot. The fold change (2 −ΔΔCt ) in ( C ) ATF4 , ( D ) ASNS , ( E ) ATF3 , and ( F ) ZIKV mRNA levels relative to ACTB mRNA was determined by RT-qPCR from three technical replicates and three biological replicates. ( G ) Viral titers in cell culture media were measured by plaque assay. Average viral titers were calculated from three independent experiments with the plaque assay being performed in duplicate. ( H ) A549 WT cells received no treatment or were incubated with DMSO (control) or ISRIB. Arbitrary luciferase units were measured as a proxy for cell viability. ( I ) A549 cells were either mock-infected or infected with ZIKV PR (moi = 10 PFU/cell) in the presence or absence of ISRIB. Cellular and nuclear fractions were prepared from cells harvested 24 hours post-infection. The resultant subcellular fractions were analyzed by Western blotting and probed with anti-NS1, ATF4, ATF3, fibrillarin, and α-tubulin antibodies. Fibrillarin and α-tubulin were used as nuclear and cytoplasmic markers, respectively. The Western blots shown are representative of three independent experiments. The quantitative data are shown as the means ± SD. The experiments were repeated three times. Statistical significance was determined by Student t -test. ** P < 0.05, *** P < 0.0005, and **** P < 0.0001; ns: not significant ( , ).

Article Snippet: The following primary antibodies were used: rabbit anti-ZIKV NS1 (GeneTex, GTX133307; 1:10,000), mouse anti-GAPDH (ProteinTech, #60004–1-lg; 1:10,000), rabbit anti-ATF3 (Abcam, #AB207434; 1:1,000), rabbit anti-ATF4 (D4B8; Cell Signaling, #11815; 1:1,000), rabbit anti-PERK (D11A8; Cell Signaling, #5683; 1:1,000), rabbit anti-eIF2ɑ (D7D3; Cell Signaling, #5324; 1:1,000), rabbit anti-p-eIF2ɑ (D9G8; Cell Signaling, #3398; 1:1,000), rabbit anti-STAT1 (D1K9Y; Cell Signaling, #14994; 1:1,000), rabbit anti-phospho-STAT1 (D4A7; Cell Signaling, #7649; 1:1,000), rabbit anti-STAT2 (D9J7L; Cell Signaling, #72604; 1:1,000), rabbit anti-phospho-STAT2 (D3P2P; Cell Signaling, #88410, 1:1,000), rabbit anti-IFIT1 (D2 × 9Z; Cell Signaling, #14769; 1:1,000), rabbit anti-MX1 (D3W7I; Cell Signaling, #37849, 1:1,000), rabbit anti-fibrillarin (Abcam, #Ab166630, 1:6,000), and mouse α-tubulin (Proteintech, #,66031–1-Ig, 1:5,000).

Techniques: Virus, Infection, Western Blot, Quantitative RT-PCR, Cell Culture, Plaque Assay, Incubation, Control, Luciferase

Journal: Journal of Virology

Article Title: Activation of ATF3 via the integrated stress response pathway regulates innate immune response to restrict Zika virus

doi: 10.1128/jvi.01055-24

Figure Lengend Snippet: ATF4 induces ATF3 expression and promotes ZIKV protein and RNA expression. A549 WT cells stably expressing either control or ATF4 targeting shRNA were treated with tunicamycin (TU) or infected with ZIKV PR (moi = 10 PFU/cell). ( A ) ATF4, ATF3, and ZIKV NS1 proteins were assayed via Western blot with GAPDH expression measured as the loading control. The blot shown is a representative of three separate experiments. ( B and C ) Fold change (2 −ΔΔCt ) in ZIKV and ATF3 RNA expression relative to ACTB mRNA was determined by RT-qPCR. The RT-qPCR results presented are the mean ± SD of three technical replicates from three separate experiments. Statistical significance was determined by Student t -test. ** P < 0.05, ns: not significant, and * non-specific band detected by the anti-NS1 antibody.

Article Snippet: The following primary antibodies were used: rabbit anti-ZIKV NS1 (GeneTex, GTX133307; 1:10,000), mouse anti-GAPDH (ProteinTech, #60004–1-lg; 1:10,000), rabbit anti-ATF3 (Abcam, #AB207434; 1:1,000), rabbit anti-ATF4 (D4B8; Cell Signaling, #11815; 1:1,000), rabbit anti-PERK (D11A8; Cell Signaling, #5683; 1:1,000), rabbit anti-eIF2ɑ (D7D3; Cell Signaling, #5324; 1:1,000), rabbit anti-p-eIF2ɑ (D9G8; Cell Signaling, #3398; 1:1,000), rabbit anti-STAT1 (D1K9Y; Cell Signaling, #14994; 1:1,000), rabbit anti-phospho-STAT1 (D4A7; Cell Signaling, #7649; 1:1,000), rabbit anti-STAT2 (D9J7L; Cell Signaling, #72604; 1:1,000), rabbit anti-phospho-STAT2 (D3P2P; Cell Signaling, #88410, 1:1,000), rabbit anti-IFIT1 (D2 × 9Z; Cell Signaling, #14769; 1:1,000), rabbit anti-MX1 (D3W7I; Cell Signaling, #37849, 1:1,000), rabbit anti-fibrillarin (Abcam, #Ab166630, 1:6,000), and mouse α-tubulin (Proteintech, #,66031–1-Ig, 1:5,000).

Techniques: Expressing, RNA Expression, Stable Transfection, Control, shRNA, Infection, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Activation of ATF3 via the integrated stress response pathway regulates innate immune response to restrict Zika virus

doi: 10.1128/jvi.01055-24

Figure Lengend Snippet: ATF3 suppresses while ATF4 promotes ZIKV RNA and protein expression. A549 WT and ATF3 KO cells expressing either control or ATF4 targeting siRNA were infected without or with ZIKV (moi = 10 PFU/cell). ( A ) ZIKV NS1, ATF4, and ATF3 proteins were analyzed by Western blot with GAPDH as the loading control. The Western blot is a representative of three independent experiments. ( B and C ) Fold change (2 −ΔΔCt ) of ATF4 and ZIKV RNA abundance relative to ACTB mRNA was determined by RT-qPCR. For each independent experiment, the RT-qPCR was performed in triplicate. N = 3. The RT-qPCR data shown are the mean ± SD. Statistical significance was determined by Student t -test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The following primary antibodies were used: rabbit anti-ZIKV NS1 (GeneTex, GTX133307; 1:10,000), mouse anti-GAPDH (ProteinTech, #60004–1-lg; 1:10,000), rabbit anti-ATF3 (Abcam, #AB207434; 1:1,000), rabbit anti-ATF4 (D4B8; Cell Signaling, #11815; 1:1,000), rabbit anti-PERK (D11A8; Cell Signaling, #5683; 1:1,000), rabbit anti-eIF2ɑ (D7D3; Cell Signaling, #5324; 1:1,000), rabbit anti-p-eIF2ɑ (D9G8; Cell Signaling, #3398; 1:1,000), rabbit anti-STAT1 (D1K9Y; Cell Signaling, #14994; 1:1,000), rabbit anti-phospho-STAT1 (D4A7; Cell Signaling, #7649; 1:1,000), rabbit anti-STAT2 (D9J7L; Cell Signaling, #72604; 1:1,000), rabbit anti-phospho-STAT2 (D3P2P; Cell Signaling, #88410, 1:1,000), rabbit anti-IFIT1 (D2 × 9Z; Cell Signaling, #14769; 1:1,000), rabbit anti-MX1 (D3W7I; Cell Signaling, #37849, 1:1,000), rabbit anti-fibrillarin (Abcam, #Ab166630, 1:6,000), and mouse α-tubulin (Proteintech, #,66031–1-Ig, 1:5,000).

Techniques: Expressing, Control, Infection, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Activation of ATF3 via the integrated stress response pathway regulates innate immune response to restrict Zika virus

doi: 10.1128/jvi.01055-24

Figure Lengend Snippet: ATF3 restricts ZIKV infection through the regulation of components within the JAK/STAT antiviral response pathway. A549 WT and ATF3 KO cells were mock or ZIKV PR infected (moi = 10 PFU/cell) and co-treated with 30 nM of the JAK1/2 inhibitor Ruxolitinib for 24 hours at 37°C. Total RNA isolated from cells was used as a template for RT-qPCR analysis. ( A ) ZIKV , ( B ) IFIT1 , and ( C ) MX1 RNA expression relative to ACTB were determined by 2 −ΔΔCt RT-qPCR method. The experiments were repeated three times, and the data shown are the mean ± SD of triplicate measurements. Statistical significance was determined by Student t -test. * P < 0.05, *** P < 0.001, and **** P < 0.0001; ns: not significant. ( D ) A representative Western blot showing the expression of ZIKV NS1, antiviral (STAT1, p-STAT1, IFIT1, and MX1) and ATF3 proteins. N = 3.

Article Snippet: The following primary antibodies were used: rabbit anti-ZIKV NS1 (GeneTex, GTX133307; 1:10,000), mouse anti-GAPDH (ProteinTech, #60004–1-lg; 1:10,000), rabbit anti-ATF3 (Abcam, #AB207434; 1:1,000), rabbit anti-ATF4 (D4B8; Cell Signaling, #11815; 1:1,000), rabbit anti-PERK (D11A8; Cell Signaling, #5683; 1:1,000), rabbit anti-eIF2ɑ (D7D3; Cell Signaling, #5324; 1:1,000), rabbit anti-p-eIF2ɑ (D9G8; Cell Signaling, #3398; 1:1,000), rabbit anti-STAT1 (D1K9Y; Cell Signaling, #14994; 1:1,000), rabbit anti-phospho-STAT1 (D4A7; Cell Signaling, #7649; 1:1,000), rabbit anti-STAT2 (D9J7L; Cell Signaling, #72604; 1:1,000), rabbit anti-phospho-STAT2 (D3P2P; Cell Signaling, #88410, 1:1,000), rabbit anti-IFIT1 (D2 × 9Z; Cell Signaling, #14769; 1:1,000), rabbit anti-MX1 (D3W7I; Cell Signaling, #37849, 1:1,000), rabbit anti-fibrillarin (Abcam, #Ab166630, 1:6,000), and mouse α-tubulin (Proteintech, #,66031–1-Ig, 1:5,000).

Techniques: Infection, Isolation, Quantitative RT-PCR, RNA Expression, Western Blot, Expressing