Journal: Frontiers in Microbiology

Article Title: Duck Hepatitis A Virus Type 1 Induces eIF2α Phosphorylation-Dependent Cellular Translation Shutoff via PERK/GCN2

doi: 10.3389/fmicb.2021.624540

Figure Lengend Snippet: PERK and GCN2 are involved in eIF2α phosphorylation during DHAV-1 infection. (A) Screen the kinases that affect eIF2α phosphorylation. DEFs were infected with DHAV-1 at MOI of 1. After 22 h of infection, different concentrations of kinase inhibitors were added to DEFs for 2 h. Then, DEFs were harvested for immunoblot analysis with the indicated antibodies. (B) PERK inhibitor GSK2606414 inhibits eIF2α phosphorylation induced by DHAV-1. (C) GCN2 inhibitor GCN2-IN-1 inhibits eIF2α phosphorylation induced by DHAV-1. (D) PKR inhibitor C16 cannot inhibit eIF2α phosphorylation induced by DHAV-1. (E) Transfection of poly(I:C) activate PKR kinase. (F) DHAV-1 and poly(I:C) stimulate PKR transcription. Differences between two groups were analyzed using Student’s t -test and considered as significant at * p < 0.05, ** p < 0.01, and *** p < 0.001. The bands marked by asterisk (*) are non-specific proteins.

Article Snippet: C16 (PKR Inhibitor) and GSK2606414 (PERK inhibitor) were purchased from APExBIO, and GCN2-IN-1 (GCN2 inhibitor) was purchased from MCE.

Techniques: Phospho-proteomics, Infection, Western Blot, Transfection

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: In vivo, kinase PKR regulates the pro‐inflammatory and Aif1 gene expression response to ZIKV infection. CC071 mice (5–6 weeks‐old) were treated with DMSO or the inhibitor of PKR (IPKR) 1 h before and 3 days after IC inoculation of either NaCl (NI) or 10 5 FFU of ZIKV. Mice were necropsied 6 days after infection with one hemisphere used for immunofluorescence and the other one used for RNA extraction and gene expression analysis. (a–d) Levels of RNAs purified from whole brain extracts were determined by RT‐qPCR with respect to Hrpt1 used as reference gene. Symbols represent individual mice with n = 10 (NI + iPKR), n = 12 (ZIKV + DMSO) and n = 11 (ZIKV + iPKR). Dot plots show means with one dot for each brain and significance assessed by one‐way ANOVA Tukey's multiple comparison test. p ‐value <0.0001 (****), <0.001 (***), <0.01 (**), <0.05 (*) and ns = not significant; p ‐values near significance are indicated. (e) Correlation analysis of the expression levels of the Il1b , C4 , and Aif1 genes (as determined in NI + iPKR and ZIKV + DMSO conditions) with either Eif2ak2 expression or ZIKV RNA expression. Symbols represent individual mice. (f) Brain sections of ZIKV‐infected mice treated either with DMSO (ZIKV + DMSO) or PKR inhibitor C16 (ZIKV + iPKR) were labeled with anti‐NeuN (specific of neurons), anti‐IBA1 (specific of microglia) and anti‐NS2B (specific of ZIKV) antibodies and with DNA labeled with DAPI. Merge images of maximum intensity projection of confocal sections (z projection) are shown. Scale bars = 10 μm.

Article Snippet: The inhibitor of PKR (iPKR) C16 (Merck, 527450) at a 50 μM final concentration (unless indicated otherwise) or the corresponding volume of DMSO was added directly in the culture medium for 1 h before treatment or infection as described by Pham et al. ( ).

Techniques: In Vivo, Expressing, Infection, Immunofluorescence, RNA Extraction, Purification, Quantitative RT-PCR, Comparison, RNA Expression, Labeling

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: PKR regulates the morphological transformation of microglia induced by ZIKV infection. Immunohistochemical analysis of 3 NI_iPKR, 4 ZIKV_DMSO and 7 ZIKV_iPKR mice among those analyzed in Figure . (a) Representative images of brain sections stained with an anti‐IBA1 immunoperoxidase antibody. Images obtained with a slide scanner microscope were analyzed using the QuPath software in order to assess the cell body area and the number of microglial cells in the region of the whole hippocampus (yellow selection) and the CA (green selection). (b) Enlarged representative images of microglial cells with the cell body region selected by QuPath indicated in yellow. (c) Cell body areas of independent microglial cells as determined using QuPath. Symbols represent individual microglial cells. In the case of the whole hippocampus, n = 735 (NI_IPKR), n = 1763 (ZIKV_DMSO) and n = 1988 (ZIKV_iPKR) cells. In the case of the CA region, n = 188 (NI_iPKR), n = 477 (ZIKV_DMSO) and n = 437 (ZIKV_iPKR) cells. The values were converted to log 10 . Dot plots show medians with significance assessed by the non‐parametric Mann–Whitney test with p ‐value <0.05 (*) and ns = not significant; p ‐values for ZIKV‐DMSO versus ZIKV_iPKR conditions are indicated. (d) Correlation analysis of the average cell body area of microglial cells present in each CA region with either Eif2ak2 mRNA or ZIKV RNA expression measured in the corresponding brains. Symbols represent average values per brain. (e) Symbols represent individual mice with n = 3 (NI_iPKR), n = 4 (ZIKV‐DMSO), and n = 7 (ZIKV_iPKR). Dot plots show means with significance assessed by one‐way ANOVA Tukey's multiple comparison test with ns = not significant.

Article Snippet: The inhibitor of PKR (iPKR) C16 (Merck, 527450) at a 50 μM final concentration (unless indicated otherwise) or the corresponding volume of DMSO was added directly in the culture medium for 1 h before treatment or infection as described by Pham et al. ( ).

Techniques: Transformation Assay, Infection, Immunohistochemical staining, Staining, Microscopy, Software, Selection, MANN-WHITNEY, RNA Expression, Comparison

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: Kinase PKR is a main regulator of the phosphorylation of STAT1 and of IRF1 expression in microglial cells. (a–h, j) Primary cultured microglial cells (PCMCs) were incubated with DMSO or the inhibitor of PKR (C16) for 1 h before being (a, e) either non‐ (NI) or NDV‐infected and (c, g, j) either non‐treated (NT) or treated with conditioned media from non‐ (CM_NI) or ZIKV‐infected (CM_ZIKV) primary cultured neurons. Total protein extracts were collected 5 h after NDV infection and 6 h after treatment with CMs and submitted to a Western blot analysis. (b, d, f, h) Dot plots show means with one dot for each independent experiment represented in different colors. Significance was assessed by one‐way ANOVA Tukey's multiple comparison test. p ‐value <0.0001 (****), <0.05 (*), and ns = not significant. (i–k) Primary cultured neurons (PCNs) and microglial cells (PCMCs) were (i, j) either non‐ (NI) or NDV‐infected for 5 h or (k) treated with recombinant IFNB for 6 h. (i, j) Protein extracts or (k) RNAs were collected and submitted to Western blot or qPCR analysis respectively. (l) The level of expression of the Irf1 and Irf7 genes in total RNAs purified from whole brain extracts of mice described in Figure was analyzed by RT‐qPCR with respect to Hrpt1 used as reference gene. Symbols represent individual mice with n = 10 (NI + iPKR), n = 12 (ZIKV + DMSO), and n = 11 (ZIKV + iPKR). Dot plots show means with significance assessed by one‐way ANOVA Tukey's multiple comparison test. p ‐value <0.0001 (****) and ns = not significant.

Article Snippet: The inhibitor of PKR (iPKR) C16 (Merck, 527450) at a 50 μM final concentration (unless indicated otherwise) or the corresponding volume of DMSO was added directly in the culture medium for 1 h before treatment or infection as described by Pham et al. ( ).

Techniques: Expressing, Cell Culture, Incubation, Infection, Western Blot, Comparison, Recombinant, Purification, Quantitative RT-PCR

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: Kinase PKR is a major regulator of non‐infected microglia's inflammatory and phagocytic response to ZIKV‐infected neurons. Primary cultured microglial cells (PCMCs) were incubated with DMSO or the inhibitor of PKR (C16) for 1 h before being either non‐treated (NT), treated with conditioned media from non‐ (CM_NI) or ZIKV‐infected (CM_ZIKV) primary cultured neurons or ZIKV alone (NT_ZIKV) for 6 h. In Figure are shown the qPCR results obtained from three independent primary cultures of microglia treated with conditioned media collected from three independent primary cultures of neurons. RNAs were collected and gene expression analysis was carried out by RT‐qPCR with respect to Rplp0 used as reference gene for genes associated with (a) the pro‐inflammatory response, (b) IFN‐I response with the level of Ifnb1 expression present in the three ZIKV‐infected PCNs from which the CMs were collected (PCN_ZIKV 64 h) also shown, (c) DAM response. Dot plots show means with one dot for each independent experiment represented in different colors. Significance was assessed by ratio‐paired t ‐test. (d) After 15 min incubation with C3_SRBCs, phagocytosis index was determined as in Figure . Dot plots show means with one dot for each independent experiment. Significance was assessed by two‐way ANOVA Tukey's multiple comparison test. p ‐value <0.01 (**), <0.05 (*), and ns = not significant; p ‐values near significance are indicated.

Article Snippet: The inhibitor of PKR (iPKR) C16 (Merck, 527450) at a 50 μM final concentration (unless indicated otherwise) or the corresponding volume of DMSO was added directly in the culture medium for 1 h before treatment or infection as described by Pham et al. ( ).

Techniques: Infection, Cell Culture, Incubation, Expressing, Quantitative RT-PCR, Comparison

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: Endogenous IFNs‐I increase the pro‐inflammatory and phagocytic capacity of newcastle disease virus (NDV)‐infected microglia. (a–d) Primary cultures of microglia (PCMCs) were either non‐infected (NI) or infected with NDV in the presence or absence of anti‐IFNAR antibody. (a–c) Gene expression analysis using RNAs collected 5 h post‐infection were carried out by RT‐qPCR using Rplp0 as reference gene. Dot plots show means with one dot for each independent culture represented in different colors. Significance was assessed by ratio paired t test. p ‐value <0.0001 (****), <0.001 (***), <0.01 (**), <0.05 (*), and ns = not significant; p ‐values near significance are indicated. (d) After 15 min incubation with C3_SRBCs, phagocytosis index was determined as in Figure . Dot plots show means with one dot for each independent experiment. Significance was assessed by two‐way ANOVA Tukey's multiple comparison test. p ‐value <0.01 (**), <0.05 (*), and ns = not significant; p ‐values near significance are indicated. (e–g) PCMCs (e, f) or PCNs (g) were incubated with DMSO or the inhibitor of PKR (IPKR) 1 h before NDV infection. Gene expression analysis using RNAs from PCMCs and PCNs, collected 5 and 8 h post‐infection respectively, were carried out by RT‐qPCR using Rplp0 as reference gene. Dot plots show means with one dot for each independent culture represented in different colors. Significance was assessed by ratio paired t test. p ‐value <0.001 (***), <0.01 (**), <0.05 (*) and ns = not significant; p‐values near significance are indicated.

Article Snippet: The inhibitor of PKR (iPKR) C16 (Merck, 527450) at a 50 μM final concentration (unless indicated otherwise) or the corresponding volume of DMSO was added directly in the culture medium for 1 h before treatment or infection as described by Pham et al. ( ).

Techniques: Virus, Infection, Expressing, Quantitative RT-PCR, Incubation, Comparison

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: In vivo, kinase PKR regulates the pro‐inflammatory and Aif1 gene expression response to ZIKV infection. CC071 mice (5–6 weeks‐old) were treated with DMSO or the inhibitor of PKR (IPKR) 1 h before and 3 days after IC inoculation of either NaCl (NI) or 10 5 FFU of ZIKV. Mice were necropsied 6 days after infection with one hemisphere used for immunofluorescence and the other one used for RNA extraction and gene expression analysis. (a–d) Levels of RNAs purified from whole brain extracts were determined by RT‐qPCR with respect to Hrpt1 used as reference gene. Symbols represent individual mice with n = 10 (NI + iPKR), n = 12 (ZIKV + DMSO) and n = 11 (ZIKV + iPKR). Dot plots show means with one dot for each brain and significance assessed by one‐way ANOVA Tukey's multiple comparison test. p ‐value <0.0001 (****), <0.001 (***), <0.01 (**), <0.05 (*) and ns = not significant; p ‐values near significance are indicated. (e) Correlation analysis of the expression levels of the Il1b , C4 , and Aif1 genes (as determined in NI + iPKR and ZIKV + DMSO conditions) with either Eif2ak2 expression or ZIKV RNA expression. Symbols represent individual mice. (f) Brain sections of ZIKV‐infected mice treated either with DMSO (ZIKV + DMSO) or PKR inhibitor C16 (ZIKV + iPKR) were labeled with anti‐NeuN (specific of neurons), anti‐IBA1 (specific of microglia) and anti‐NS2B (specific of ZIKV) antibodies and with DNA labeled with DAPI. Merge images of maximum intensity projection of confocal sections (z projection) are shown. Scale bars = 10 μm.

Article Snippet: When indicated, 20 μg of the PKR inhibitor C16 (Merck, 527450) were administered 45 min before and 3 days after infection.

Techniques: In Vivo, Expressing, Infection, Immunofluorescence, RNA Extraction, Purification, Quantitative RT-PCR, Comparison, RNA Expression, Labeling

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: Kinase PKR is a main regulator of the phosphorylation of STAT1 and of IRF1 expression in microglial cells. (a–h, j) Primary cultured microglial cells (PCMCs) were incubated with DMSO or the inhibitor of PKR (C16) for 1 h before being (a, e) either non‐ (NI) or NDV‐infected and (c, g, j) either non‐treated (NT) or treated with conditioned media from non‐ (CM_NI) or ZIKV‐infected (CM_ZIKV) primary cultured neurons. Total protein extracts were collected 5 h after NDV infection and 6 h after treatment with CMs and submitted to a Western blot analysis. (b, d, f, h) Dot plots show means with one dot for each independent experiment represented in different colors. Significance was assessed by one‐way ANOVA Tukey's multiple comparison test. p ‐value <0.0001 (****), <0.05 (*), and ns = not significant. (i–k) Primary cultured neurons (PCNs) and microglial cells (PCMCs) were (i, j) either non‐ (NI) or NDV‐infected for 5 h or (k) treated with recombinant IFNB for 6 h. (i, j) Protein extracts or (k) RNAs were collected and submitted to Western blot or qPCR analysis respectively. (l) The level of expression of the Irf1 and Irf7 genes in total RNAs purified from whole brain extracts of mice described in Figure was analyzed by RT‐qPCR with respect to Hrpt1 used as reference gene. Symbols represent individual mice with n = 10 (NI + iPKR), n = 12 (ZIKV + DMSO), and n = 11 (ZIKV + iPKR). Dot plots show means with significance assessed by one‐way ANOVA Tukey's multiple comparison test. p ‐value <0.0001 (****) and ns = not significant.

Article Snippet: When indicated, 20 μg of the PKR inhibitor C16 (Merck, 527450) were administered 45 min before and 3 days after infection.

Techniques: Expressing, Cell Culture, Incubation, Infection, Western Blot, Comparison, Recombinant, Purification, Quantitative RT-PCR

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: Kinase PKR is a major regulator of non‐infected microglia's inflammatory and phagocytic response to ZIKV‐infected neurons. Primary cultured microglial cells (PCMCs) were incubated with DMSO or the inhibitor of PKR (C16) for 1 h before being either non‐treated (NT), treated with conditioned media from non‐ (CM_NI) or ZIKV‐infected (CM_ZIKV) primary cultured neurons or ZIKV alone (NT_ZIKV) for 6 h. In Figure are shown the qPCR results obtained from three independent primary cultures of microglia treated with conditioned media collected from three independent primary cultures of neurons. RNAs were collected and gene expression analysis was carried out by RT‐qPCR with respect to Rplp0 used as reference gene for genes associated with (a) the pro‐inflammatory response, (b) IFN‐I response with the level of Ifnb1 expression present in the three ZIKV‐infected PCNs from which the CMs were collected (PCN_ZIKV 64 h) also shown, (c) DAM response. Dot plots show means with one dot for each independent experiment represented in different colors. Significance was assessed by ratio‐paired t ‐test. (d) After 15 min incubation with C3_SRBCs, phagocytosis index was determined as in Figure . Dot plots show means with one dot for each independent experiment. Significance was assessed by two‐way ANOVA Tukey's multiple comparison test. p ‐value <0.01 (**), <0.05 (*), and ns = not significant; p ‐values near significance are indicated.

Article Snippet: When indicated, 20 μg of the PKR inhibitor C16 (Merck, 527450) were administered 45 min before and 3 days after infection.

Techniques: Infection, Cell Culture, Incubation, Expressing, Quantitative RT-PCR, Comparison