Journal: PLoS ONE

Article Title: Diversity in Viral Anti-PKR Mechanisms: A Remarkable Case of Evolutionary Convergence

doi: 10.1371/journal.pone.0016711

Figure Lengend Snippet: A ) First, viruses can prevent the activation of PKR by expressing proteins or RNAs that bind the kinase in an inhibitory manner, by sequestering dsRNA activator molecules or by inducing the degradation of PKR. Examples of this group are vaccinia E3 and K3, influenza NS1 and Adenovirus VAI RNAs and Rift Valley virus NS1 protein. In the second category are viruses that promote eIF2α dephosphorylation such as Herpes virus γ34.5 protein which acts as a regulatory subunit of cellular protein phosphatase 1a (PP1). Herpesvirus also express products from the first group (Us11 gene, a PKR inhibitor). In the third category, some viruses do not prevent PKR-mediated eIF2α phosphorylation, but viral mRNA can initiate translation in an eIF2-independent manner by means of a RNA structure (DLP) that stalls the scanning 40S ribosome on the initiation codon. To date, this strategy has been found only in Alphavirus. B ) Secondary structure prediction of the 5′extreme of 26S mRNAs of SINV and SFV. The stability of DLP structure (ΔG°) is expressed in kcal. mol −1 . Arrows show the initiation codon. Circled nucleotides in SINV were mutated to adenines in ΔDLP mutant virus as described previously .

Article Snippet: Primary antibodies used were: anti-phospho eIF2α (Cell Signalling, 1∶250 dilution), anti-SINV capsid (1∶500) and anti-VVp14 (1∶500).

Techniques: Activation Assay, Expressing, Virus, De-Phosphorylation Assay, Phospho-proteomics, Mutagenesis

Journal: PLoS ONE

Article Title: Diversity in Viral Anti-PKR Mechanisms: A Remarkable Case of Evolutionary Convergence

doi: 10.1371/journal.pone.0016711

Figure Lengend Snippet: A ) Susceptibility of MEFs to viruses used in this study. Cells were infected with the indicated viruses to a moi of 25 pfu/cell and 6 h later pulsed with [ 35 S]-Met for 30 min. Labeled proteins were resolved by SDS-PAGE followed of autoradiography. B ) PKR activation and eIF2α phosphorylation in wild type or PKR o/o MEFs infected with the indicated viruses at 4 and 6 hpi. Note the mobility shift of PKR band upon activation in SINV and SFV-infected cells (upper panel). eIF2α phosphorylation in wild type (+/+) and PKR knock-out cells (o/o) infected with the indicated viruses. Only Alphavirus-infected cultures showed a strong eIF2α phosphorylation. For VSV-infected cells, a slight increase in eIF2α phosphorylation was also observed.

Article Snippet: Primary antibodies used were: anti-phospho eIF2α (Cell Signalling, 1∶250 dilution), anti-SINV capsid (1∶500) and anti-VVp14 (1∶500).

Techniques: Infection, Labeling, SDS Page, Autoradiography, Activation Assay, Phospho-proteomics, Mobility Shift, Knock-Out

Journal: PLoS ONE

Article Title: Diversity in Viral Anti-PKR Mechanisms: A Remarkable Case of Evolutionary Convergence

doi: 10.1371/journal.pone.0016711

Figure Lengend Snippet: A ) IF analysis of brains from wild type and PKR knock-out mice infected with SINV at 4 dpi. Adjacent sections were incubated with anti-SINV and anti-phosphoeIF2α antibodies as described in . B ) IF analysis of spleens from mice infected with VV-Luc at 1dpi. Sections were incubated with anti-VVp14 (reactive against the envelope protein A27) and anti-phosphoeIF2α antibodies. Note that spleen cells expressing viral antigens did not react with anti- phosphoeIF2α antibodies. C ) Expression of PKR in different mouse organs from uninfected animals. Equivalent amounts of protein extracts were analyzed by immunoblot against PKR, total eIF2α and β-actin. D ) Attenuation of ΔDLP mutant virus in wild type, but not in PKR o/o mice. Animals were inoculated with 10 7 of WT and 2×10 7 of ΔDLP mutant viruses. Viral yields in mouse brains at 4 dpi were titrated by plaque assay. Results are the mean from 10 animals inoculated for each group in three independent experiments. SD from each group is also showed.

Article Snippet: Primary antibodies used were: anti-phospho eIF2α (Cell Signalling, 1∶250 dilution), anti-SINV capsid (1∶500) and anti-VVp14 (1∶500).

Techniques: Knock-Out, Infection, Incubation, Expressing, Western Blot, Mutagenesis, Virus, Plaque Assay

Journal: PLoS ONE

Article Title: Diversity in Viral Anti-PKR Mechanisms: A Remarkable Case of Evolutionary Convergence

doi: 10.1371/journal.pone.0016711

Figure Lengend Snippet: A ) Previous infection with some viruses prevented eIF2α phosphorylation after SINV virus superinfection. The protocol followed for mixed infections is outlined. Wild type MEFs were infected with the indicated viruses at a moi of 2 pfu/cell. Three hours later, cells were superinfected with SINV at moi of 25 pfu/cell and 5 h later lysed in sample buffer for immunoblot analysis against SINV capsid (SINV C, upper panel) and anti-phosphoeIF2α (bottom panel). B ) Expression of VV E3L gene rescued translation of ΔDLP SINV mutant. MEF-E3L cells were induced for the expression of VV E3L by tetracycline withdrawal and infected with SINV-WT or SINV-ΔDLP mutant. Five hours later, cells were labeled with [ 35 S]-Met+Cys for 30 min and analyzed by SDS-PAGE followed of autoradiography and by immunoblot against anti-E3, anti-PKR and anti-phospho eIF2α. Parallel cultures were infected at moi of 5 pfu/cell and viral yields were determined by plaque assay 2 days later. Data are the mean ±SD from three and two independent experiments in PKR +/+ and PKR o/o cells, respectively.

Article Snippet: Primary antibodies used were: anti-phospho eIF2α (Cell Signalling, 1∶250 dilution), anti-SINV capsid (1∶500) and anti-VVp14 (1∶500).

Techniques: Infection, Phospho-proteomics, Virus, Western Blot, Expressing, Mutagenesis, Labeling, SDS Page, Autoradiography, Plaque Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The NF-κB/leukemia inhibitory factor/STAT3 signaling pathway in antibody-mediated suppression of Sindbis virus replication in neurons

doi: 10.1073/pnas.2016691117

Figure Lengend Snippet: Anti-E2 antibody induces rapid canonical NF-κB activation and late, prolonged STAT3 activation in SINV-infected dAP-7 cells. (A) RPPA results indicating changes in protein expression or posttranslational modification in whole cell lysates from differentiated AP-7 cells infected with SINV and treated with media (mock), anti-E2 antibody, or anti-E1 antibody at 4 h after infection (small arrows). Fold-change values are relative to mock-infected lysates at each time point. (B) Immunoblot confirmation of sustained phosphorylated STAT3 Y705 expression and IκBα degradation in whole cell lysates from differentiated AP-7 cell lysates treated with media or anti-E2 antibody. The blot was stripped and probed with antibody against total STAT3 and actin as loading controls. (C) Densitometric analysis of levels of phosphorylated STAT3 normalized to total STAT3. Data in A and C are presented as mean ± SD from three independent experiments ***P < 0.001, ****P < 0.0001, #P < 0.05, ####P < 0.0001.

Article Snippet: The following primary and secondary antibodies were used: polyclonal anti-SINV (1:1,000) ( 10 ), monoclonal anti-phospho-STAT3 Y705 (1:1,000, Cell Signaling Technology #9131), monoclonal anti-STAT3 (1:1,000, Cell Signaling Technology #12640), monoclonal anti-β-actin (1:10,000, #MAB1501, EMD Millipore), horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling Technology), and sheep anti-mouse IgG (GE Healthcare).

Techniques: Activation Assay, Infection, Expressing, Modification, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The NF-κB/leukemia inhibitory factor/STAT3 signaling pathway in antibody-mediated suppression of Sindbis virus replication in neurons

doi: 10.1073/pnas.2016691117

Figure Lengend Snippet: Treatment of SINV-infected dAP-7 cells with recombinant LIF and IL-6 does not affect viral replication. Differentiated AP-7 cells were infected with SINV and treated with 10 μg/mL recombinant rat LIF or IL-6 4 h after infection. (A) Immunoblot analysis of phosphorylated STAT3 Y705, total STAT3, SINV structural proteins, and β-actin levels. (B) Densitometric analysis of phosphorylated STAT3 normalized to STAT3 and SINV pE2 normalized to β-actin. (C) Cell viability determined by trypan blue exclusion and expressed as a percentage of day 0 cells. (D) Intracellular SINV RNA levels quantified by qRT-PCR and expressed as log10 copies per GAPDH RNA. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001.

Article Snippet: The following primary and secondary antibodies were used: polyclonal anti-SINV (1:1,000) ( 10 ), monoclonal anti-phospho-STAT3 Y705 (1:1,000, Cell Signaling Technology #9131), monoclonal anti-STAT3 (1:1,000, Cell Signaling Technology #12640), monoclonal anti-β-actin (1:10,000, #MAB1501, EMD Millipore), horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling Technology), and sheep anti-mouse IgG (GE Healthcare).

Techniques: Infection, Recombinant, Western Blot, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The NF-κB/leukemia inhibitory factor/STAT3 signaling pathway in antibody-mediated suppression of Sindbis virus replication in neurons

doi: 10.1073/pnas.2016691117

Figure Lengend Snippet: Combined anti-E2 antibody and exogenous LIF treatment results in greater suppression of SINV replication. Differentiated AP-7 cells were infected with SINV and treated with media (mock), LIF (10 μg/mL), anti-E2 antibody (5 μg/mL), or LIF and anti-E2 antibody at 4 h after infection. (A) Immunoblot analysis for phosphorylated STAT3 Y705, SINV structural proteins, and total STAT3. (B) Densitometric analysis of phosphorylated STAT3 normalized to total STAT3 and SINV pE2 normalized to β-actin. (C) Intracellular SINV RNA levels quantified by qRT-PCR and normalized to copy numbers of GAPDH RNA. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: The following primary and secondary antibodies were used: polyclonal anti-SINV (1:1,000) ( 10 ), monoclonal anti-phospho-STAT3 Y705 (1:1,000, Cell Signaling Technology #9131), monoclonal anti-STAT3 (1:1,000, Cell Signaling Technology #12640), monoclonal anti-β-actin (1:10,000, #MAB1501, EMD Millipore), horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling Technology), and sheep anti-mouse IgG (GE Healthcare).

Techniques: Infection, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Sindbis Virus Infection Causes Cell Death by nsP2-Induced Transcriptional Shutoff or by nsP3-Dependent Translational Shutoff

doi: 10.1128/JVI.01388-18

Figure Lengend Snippet: Defined mutations in nsP2 and nsP3 make SINV capable of noncytopathic replication in vertebrate cells without a profound effect on viral replication rates. (A) Schematic presentation of the designed recombinant viral genomes, infectivity of the in vitro-synthesized RNAs, and viral titers at 24 h postelectroporation. (B) NIH 3T3 cells were infected with the indicated variants at an MOI of 50 PFU/cell. At 18 h PI, media were harvested, and viral titers and concentrations of IFN-β were assessed as described in Materials and Methods. Data are shown as means ± SD of results from 3 biological repeats. (C) NIH 3T3 cells were infected with the indicated SINV variants at an MOI of 20 PFU/cell. Cell lysates were prepared at 8 h PI and analyzed using RPB1-, nsP2-, STAT1-, pSTAT1-, and tubulin-specific Abs. (D) NIH 3T3 cells and their Mavs KO derivatives were infected with the indicated variants at an MOI of 20 PFU/cell. Media were replaced every 24 h, and viral titers were determined by plaque assay on BHK-21 cells.

Article Snippet: The following primary antibodies were used: anti-tubulin (rat monoclonal antibody [MAb]; University of Alabama at Birmingham [UAB] core facility), rabbit polyclonal antibodies against SINV nsP3 (custom-made), SINV nsP2-specific mouse MAb (custom-made), anti-STAT1 MAb (rabbit MAb; Epitomics), anti-pSTAT1 MAb (mouse MAb) (pY701; BD Transduction Laboratories), and anti-RPB1 (8wG16 [Covance], 4H8 [Active Motif], or F12 [Santa Cruz Biotechnology]).

Techniques: Recombinant, Infection, In Vitro, Synthesized, Plaque Assay