Journal: Frontiers in Immunology

Article Title: US6 Gene Deletion in Herpes Simplex Virus Type 2 Enhances Dendritic Cell Function and T Cell Activation

doi: 10.3389/fimmu.2017.01523

Figure Lengend Snippet: ΔgD-2 HSV type 2 (HSV-2) is attenuated in dendritic cells (DCs). (A) DC viability, as determined by flow cytometry (gated on CD11c + , I-A b+ , Zombie + cells) at different time points after virus inoculation with an multiplicity of infection (MOI) of 1. UT, untreated. (B) Western blot analyses of structural (VP16), early (ICP5, ICP27), and late (gB) viral proteins post-inoculation of DCs. (C) Viral genome loads in inoculated DCs determined by qPCR for the UL30 gene. Data are means ± SEM of three independent experiments. Representative images are shown for western blots. Two-way analysis of variance and Tukey’s multiple comparison test were used for statistical analyses (** p < 0.01).

Article Snippet: Viral protein expression was analyzed by western blot using primary antibodies against anti-ICP5 (Santa Cruz), anti-VP16 (Santa Cruz), anti-ICP27 (Santa Cruz), and anti-β-actin (BioLegend).

Techniques: Flow Cytometry, Virus, Infection, Western Blot, Comparison

Journal: Frontiers in Immunology

Article Title: US6 Gene Deletion in Herpes Simplex Virus Type 2 Enhances Dendritic Cell Function and T Cell Activation

doi: 10.3389/fimmu.2017.01523

Figure Lengend Snippet: Dendritic cells (DCs) inoculated with ΔgD-2 or wild-type (WT) virus mainly release defective herpes simplex virus particles. (A) Four predominant viral particle phenotypes, indicated in the four panels, were observed in the extracellular space of virus-inoculated DCs (extracellular space data shown). Transmission electron microscopy. Extreme left: WT-like particles displaying an envelope, an electron-dense tegument, and an electron-dense capsid; middle left: envelope-only phenotype displaying an envelope-like structure, harboring spikes in the outer region, yet no apparent capsid or tegument; middle right: WT-like capsids, yet no envelope; extreme right: empty capsids with no envelope and no electron-dense content in capsids. Right panel: quantification of viral particle phenotypes observed. (B) Transmission electron microscopy (negative staining) of viral particles recovered from ultracentrifugated supernatant of virus-inoculated DCs. (C) Western blot analysis of the major capsid protein ICP5 in virus particles recovered from ultracentrifugated supernatants of DCs 18 h post-virus inoculation. Values indicate relative expression of ICP5. (D) Quantification of viral genome copies (determined by qPCR) in viral particles obtained from ultracentrifugated supernatants derived from virus-inoculated DCs with or without acyclovir treatment. One-way analysis of variance and Tukey’s multiple comparison test were used for statistical analyses (* p < 0.05).

Article Snippet: Viral protein expression was analyzed by western blot using primary antibodies against anti-ICP5 (Santa Cruz), anti-VP16 (Santa Cruz), anti-ICP27 (Santa Cruz), and anti-β-actin (BioLegend).

Techniques: Virus, Transmission Assay, Electron Microscopy, Negative Staining, Western Blot, Expressing, Derivative Assay, Comparison

Journal: Cell reports

Article Title: The mitochondrial protein ERAL1 suppresses RNA virus infection by facilitating RIG-I-like receptor signaling.

doi: 10.1016/j.celrep.2020.108631

Figure Lengend Snippet: Figure 5. ERAL1 translocates from mitochondrial to cytoplasm through BAX/BAK pore after virus infection (A) Immunoelectron microscopy assay in U2OS cells that were transfected with ERAL1 expression plasmid for 24 h and then treated with or without SeV for another 6 h. Mitochondria are circled in red; arrows indicate the released ERAL1. Scale bar, 100 nm. (B) U2OS cells were transfected with ERAL1 expression plasmid for 24 h and then treated with or without SeV for another 6 h. ERAL1 (green) and COX IV (red) were stained and visualized by confocal microscopy. Scale bar, 5 mm. (C) Stably expressed FLAG-ERAL1 HEK293T cells were treated with or without SeV for 6 h. Organelle separation experiment and immunoblot analysis detected the localization of ERAL1, GAPDH, and COX IV. (D) HeLa cells were treated with VSV for indicated times. Organelle separation experiment and immunoblot analysis detected the localization of ERAL1, GAPDH, and COX IV. (E) BAX-depleted HeLa cells were transfected with FLAG-ERAL1 for 24 h and then treated with VSV for indicated times. Organelle separation experiment and immunoblot analysis detected the localization of ERAL1, GAPDH, and COX IV. (F and G) WT and BAX KO HeLa cells were treated with VSV (F) or SeV (G) for 16 h. IFNb and ISG15 mRNA were quantified by qRT-PCR. (H) Control and BAX KO HeLa cells were transfected with or without 100 ng/mL 50 ppp dsRNA for 16 h. The production of IFNb mRNA was measured by qPCR. (I) RIG-I KO HEK293T cells were transfected with or without FLAG-ERAL1 and HA-RIG-I for 24 h and then treated with SeV for 6 h. Organelle separation experiment and immunoblot analysis detected the localization of ERAL1, GAPDH, and COX IV. (J) MDA5 KO HEK293T cells were transfected with FLAG-ERAL1 for 24 h and then treated with or without EMCV for 6 h. Organelle separation experiment and immunoblot analysis detected the localization of ERAL1, GAPDH, and COX IV. Statistical analysis was performed by t test for (F)–(H). Data were pooled from three experiments (F–H). Data are representative of three experiments (A–E, I, and J). *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars, SD.

Article Snippet: Rose (Yale University) N/A SeV (Cantell strain) ATCC Cat# ATCC VR-907 Encephalomyocarditis virus (EMCV) Zhengfan J. (Peking University) N/A HSV-1 (Herpes simplex virus 1, F strain) A.Iwasaki (Yale University) N/A Chemicals, peptides, and recombinant proteins Poly(I:C) HMW Invivogen Cat# tlrl-pic Poly(I:C) LMW Invivogen Cat# tlrl-picw 50ppp-dsRNA Invivogen Cat# tlrl-3prna-100 3x FLAG Peptide Sigma-Aldrich Cat# F4799 Ethidium bromide Targetmol Cat# T1220 Chloramphenicol Selleck Cat# S1677 GM-CSF Peprotech Cat# 315-03 Agarose Type I, low EEO Sigma-Aldrich Cat# A6013-10G Protease inhibitor cocktail TargetMol Cat# B14001 (Continued on next page) e1 Cell Reports 34, 108631, January 19, 2021

Techniques: Virus, Infection, Immuno-Electron Microscopy, Transfection, Expressing, Plasmid Preparation, Staining, Confocal Microscopy, Stable Transfection, Western Blot, Quantitative RT-PCR, Control

Journal: Virologica Sinica

Article Title: Nuclear translocation of Sp100A suppresses multiple DNA viruses and limits HSV-1 lytic replication in vivo

doi: 10.1016/j.virs.2026.02.003

Figure Lengend Snippet: Modulation of viral replication by Sp100A mutations on HSV-1. A HEp-2 cells were mock infected, or with the indicated viruses at an MOI of 5 for 6 h. Protein expression was evaluated by immunoblotting with anti-Flag, anti-β-actin, anti-TK, anti-ICP0, and anti-ICP8 antibodies. Note that the target proteins (Flag, TK, ICP0, and ICP8) were detected on separate blots. β-actin was used as a loading control and was run on a parallel blot using the same batch of protein lysates and identical loading amounts to confirm equal sample loading. B, C Growth kinetics of the indicated viruses in HEp-2 cells at MOIs of 0.01 ( B ) and 5 ( C ). D, E Growth kinetics of the indicated viruses in Vero cells at MOIs of 0.01 ( D ) and 5 ( E ). Data in B – E , statistical comparisons of viral titers were performed between group rHSV-1 and groups rHSV-1-A, rHSV-1-A S188A and rHSV-1-A S188D , as well as between group rHSV-1-A S188A and groups rHSV-1-A and rHSV-1-A S188D . Statistical analyses were performed using two-way ANOVA. F Plaque size comparison of the indicated viruses in HEp-2 and Vero cells. G, H Plaque size of each virus was quantified from 50 plaques using ImageJ and plotted. The data were normalized to the mean plaque size of the rHSV-1-A S188A group. Statistical analyses were performed using one-way ANOVA. Data presented as mean ± SD. ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: Antibodies and drugs used in this study included the following: rabbit polyclonal anti-Flag antibody (Catalog No. 80010, Proteintech, USA), rabbit polyclonal anti-Sp100 antibody (Catalog No. GTX131569, GeneTex, USA), mouse monoclonal anti-Flag antibody (Catalog No. AB0008; Abways, Beijing, China), mouse monoclonal anti-β-actin antibody (Catalog No. 1000166; Sino Biological, Beijing, China), rabbit monoclonal anti-GAPDH antibody (Catalog No. AB0037, Abways Technology, Beijing, China), mouse monoclonal anti-Histone H3 antibody (Catalog No. 100005-MM01, Sino Biological, Beijing, China), rabbit polyclonal anti-TSG101 antibody (Catalog No. 28283-1-AP; Proteintech; USA), mouse monoclonal anti-calnexin antibody (Catalog No. sc23954, Santa Cruz, USA), mouse monoclonal anti-ICP0 antibody (Catalog No. sc53070, Santa Cruz, USA), mouse monoclonal anti-ICP8 antibody (Catalog No. ab20194, Abcam, UK), rabbit polyclonal anti-TK antibody (custom-made, GenicBio Limited, Shanghai, China), Alexa Fluor 594-labeled goat anti-rabbit IgG (H + L) (Catalog No. 2165334, Invitrogen, USA), goat anti-mouse IgG-HRP (Catalog No. 31430, Invitrogen, USA), goat anti-rabbit IgG (H + L)-HRP (Catalog No. 32460, Invitrogen, USA), and a protease inhibitor cocktail (Catalog No. EO0492, Thermo Scientific, USA).

Techniques: Infection, Expressing, Western Blot, Control, Comparison, Virus

Journal:

Article Title: Herpes Simplex Virus Type 1 Infection Induces Activation and Recruitment of Protein Kinase C to the Nuclear Membrane and Increased Phosphorylation of Lamin B

doi: 10.1128/JVI.80.1.494-504.2006

Figure Lengend Snippet: Digital images of confocal fluorescence micrographs of mock-infected (A to D and I to L) and HSV-1 (F)-infected (E to H and M to P) HEp-2 cells (A to H) and HeLa cells (I to P). Cells were fixed in methanol at 16 h postinfection and incubated with anti-PKC, anti-lamin B, and anti-ICP4 antibody. Anti-PKC antibody was stained with FITC-conjugated secondary antibody (green channel), anti-lamin B antibody was stained with Texas Red-conjugated secondary antibody (red channel), and ICP4 was stained with Cy5-conjugated secondary antibody (blue channel).

Article Snippet: All anti-PKC and anti-lamin B antibodies were obtained from Santa Cruz Biotechnology, Inc. Anti-ICP4 antibody was obtained from the Rumbaugh-Goodwin Institute for Cancer Research.

Techniques: Fluorescence, Infection, Incubation, Staining

Journal:

Article Title: Herpes Simplex Virus Type 1 Infection Induces Activation and Recruitment of Protein Kinase C to the Nuclear Membrane and Increased Phosphorylation of Lamin B

doi: 10.1128/JVI.80.1.494-504.2006

Figure Lengend Snippet: Time course of PKC upregulation and recruitment to the nuclear rim. Digital images of confocal fluorescence micrographs were taken of HEp-2 cells infected with HSV-1 (F). At 0 h (A to C), 4 h (D to F), 8 h (G to I), 12 h (J to L), and 16 h (M to O) postinfection, cells were fixed and stained for PKC (FITC; green channel), lamin B (Texas Red; red channel), and ICP4 (Cy5; blue channel).

Article Snippet: All anti-PKC and anti-lamin B antibodies were obtained from Santa Cruz Biotechnology, Inc. Anti-ICP4 antibody was obtained from the Rumbaugh-Goodwin Institute for Cancer Research.

Techniques: Fluorescence, Infection, Staining

Journal:

Article Title: Herpes Simplex Virus Type 1 Infection Induces Activation and Recruitment of Protein Kinase C to the Nuclear Membrane and Increased Phosphorylation of Lamin B

doi: 10.1128/JVI.80.1.494-504.2006

Figure Lengend Snippet: Digital confocal images of mock-infected or HSV-1 (F)-infected HEp-2 cells immunostained with PKC-specific antibodies. At 16 h postinfection, cells were fixed with methanol and incubated with anti-lamin B antibody, anti-ICP4 antibody, and one of the following PKC-specific antibodies: anti-PKCα (A to D), anti-PKCδ (E to H), anti-PKCζ (I to L). Anti-PKC antibodies were stained with FITC-conjugated secondary antibody (green channel), anti-lamin B antibody was stained with Texas Red-conjugated secondary antibody (red channel), and ICP4 was stained with Cy5-conjugated secondary antibody (blue channel).

Article Snippet: All anti-PKC and anti-lamin B antibodies were obtained from Santa Cruz Biotechnology, Inc. Anti-ICP4 antibody was obtained from the Rumbaugh-Goodwin Institute for Cancer Research.

Techniques: Infection, Incubation, Staining

Journal:

Article Title: Herpes Simplex Virus Type 1 Infection Induces Activation and Recruitment of Protein Kinase C to the Nuclear Membrane and Increased Phosphorylation of Lamin B

doi: 10.1128/JVI.80.1.494-504.2006

Figure Lengend Snippet: Effects of UL31 and UL34 and their association on HSV-1-induced PKC recruitment. Digital confocal images show mock-infected HEp-2 cells (A to C) or HEp-2 cells infected with HSV-1 (F) (D to F), v3161 UL31-null HSV-1 mutant (G to I), vRR1072 UL34-null HSV-1 mutant (J to L), or v3480 UL34-UL31 association-defective mutant (M to O). At 16 h postinfection, cells were fixed and stained for PKC (FITC; green channel), lamin B (Texas Red; red channel), and ICP4 (Cy5; blue channel).

Article Snippet: All anti-PKC and anti-lamin B antibodies were obtained from Santa Cruz Biotechnology, Inc. Anti-ICP4 antibody was obtained from the Rumbaugh-Goodwin Institute for Cancer Research.

Techniques: Infection, Mutagenesis, Staining

Journal:

Article Title: Herpes Simplex Virus Type 1 Infection Induces Activation and Recruitment of Protein Kinase C to the Nuclear Membrane and Increased Phosphorylation of Lamin B

doi: 10.1128/JVI.80.1.494-504.2006

Figure Lengend Snippet: Digital images of confocal fluorescence micrographs of mock-infected HEp-2 cells (A to C) or HEp-2 cells infected with HSV-1 (F) (D to F), R7037 US3-null HSV-1 mutant (G to I), or vRR1204 US3-kinase-defective HSV-1 mutant (J to L). At 16 h postinfection, cells were fixed and immunostained for PKC (FITC; green channel), lamin B (Texas Red; red channel), and ICP4 (Cy5; blue channel).

Article Snippet: All anti-PKC and anti-lamin B antibodies were obtained from Santa Cruz Biotechnology, Inc. Anti-ICP4 antibody was obtained from the Rumbaugh-Goodwin Institute for Cancer Research.

Techniques: Fluorescence, Infection, Mutagenesis

Journal:

Article Title: Herpes Simplex Virus Type 1 Infection Induces Activation and Recruitment of Protein Kinase C to the Nuclear Membrane and Increased Phosphorylation of Lamin B

doi: 10.1128/JVI.80.1.494-504.2006

Figure Lengend Snippet: Digital images of confocal fluorescence micrographs of HSV-1 (F)-infected HeLa cells. At 16 h postinfection, cells were fixed and immunostained for lamin B (Texas Red; red channel), lamin A (FITC; green channel), and ICP4 (Cy5; blue channel).

Article Snippet: All anti-PKC and anti-lamin B antibodies were obtained from Santa Cruz Biotechnology, Inc. Anti-ICP4 antibody was obtained from the Rumbaugh-Goodwin Institute for Cancer Research.

Techniques: Fluorescence, Infection

Journal: Scientific Reports

Article Title: MG132 exerts anti-viral activity against HSV-1 by overcoming virus-mediated suppression of the ERK signaling pathway

doi: 10.1038/s41598-020-63438-1

Figure Lengend Snippet: Effects of MG132 on HSV-1 lytic-protein expression, adsorption and entry. ( a – c ) Suppression of HSV-1 lytic protein expression by MG132 treatment. Vero ( a ), HepG2 ( b ) and H1299 ( c ) cells were mock infected or infected with HSV-1 at a multiplicity of infection (MOI) of 1 for 30 min, and then cultured with or without various concentrations of MG132 for 0.4, 12, 15, 18, 21, 24, or 36 hours. Cell lysates were subjected to Western blotting with anti-ICP5 (L/γ gene product), anti-UL42 (E/β gene product) and anti-ICP27 (IE/α gene product) antibodies. The band intensities of viral protein were calculated using ImageJ software and normalized to those of total β-Actin. The values of viral protein/β-actin are presented at the bottom of the image, and the highest value is presented as 1.0. Original images of blotting are shown in Supplementary Fig. . ( d ) The effect of MG132 on virus attachment to cells. Vero cells were incubated with 200 PFU HSV-1 with 0.75 μM MG132 or 0.01 unit/ml heparin for 30 min at 4 °C. Heparin inhibits HSV-1 attachment to host cells and was used as a positive control. The percentage of plaque number in DMSO treated cells was defined as 100%. ( e ) The effect of MG132 on HSV-1 penetration into cells. Vero cells were incubated with 100 PFU HSV-1 for 30 min at 4 °C, and DMEM containing virus was removed. Prewarmed medium containing 0.75 μM MG132 or 10 μM ACV was added, and cells were further incubated for 30 min at 37 °C. To remove virions from cell membrane, cells were washed with citrate buffer and PBS and cultured for 48 hours. The percentage of plaque number in DMSO treated cells was defined as 100%. ( d , e ) Standard deviations were determined by analyzing the data from three independent experiments and are indicated by the error bars. * P < 0.05 and *** P < 0.001 indicate the statistical significance compared with vehicle-treated cells. N.S. indicates not statistically significant.

Article Snippet: Primary antibodies used in these experiments were Ser380-phospho-PTEN, lamin B1, cleaved caspase-3, cleaved PARP, Ser473-phospho-AKT, AKT, Tyr701-phospho-STAT1, Ser338-phospho-c-Raf, Ser217/Ser221-phospho-MEK1/2, Thr202/Tyr204-phospho-ERK1/2, Ser380-phospho-p90RSK, FGFR, and EGFR (1:1500 dilution, Cell Signaling Technology, MA, USA), Ras-GRF2 (1:1500 dilution, Abcam, OR, USA), IκBα, p65, β-catenin, ERK1, and ERK2 (1:1500 dilution, BD Biosciences, NJ, USA), β-Actin, p90RSK, ICP5, ICP8, ICP27, UL42, Ras-GRF1, and h-Ras (1:750 dilution, Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Adsorption, Infection, Cell Culture, Western Blot, Software, Virus, Incubation, Positive Control, Membrane

Journal: Rhinology journal

Article Title: Th2 biased upper airway inflammation is associated with an impaired response to viral infection with Herpes simplex virus 1

doi: 10.4193/rhino15.213

Figure Lengend Snippet: Figure 3. Inflammatory mediator release after HSV-1 infection. Data were expressed as median and interquartile ranges (IQR). * P value<0.05. HSV1

Article Snippet: HSV1 (ATCC, VR-733, strain F) was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and propagated to large quantities by infection of African green monkey kidney (Vero) cells (ATCC CCL-81; Rockville, MD, USA).

Techniques: Infection