Journal: Microbiology Spectrum

Article Title: 6-Thioguanine Inhibits Herpes Simplex Virus 1 Infection of Eyes

doi: 10.1128/Spectrum.00646-21

Figure Lengend Snippet: Details of antibodies used in this project

Article Snippet: Anti-ICP4 (H943) antibody , Santa Cruz , SC-69809 (lot no. J1520) , 1:500 , 1:100.

Techniques: Western Blot, Immunofluorescence

Journal: Microbiology Spectrum

Article Title: A 77 Amino Acid Region in the N-Terminal Half of the HSV-1 E3 Ubiquitin Ligase ICP0 Contributes to Counteracting an Established Type 1 Interferon Response

doi: 10.1128/spectrum.00593-22

Figure Lengend Snippet: Levels of HSV-1 IE and L proteins in cells infected with ICP0 truncation mutants in cells −/+ IFN-β. HepaRG cells were untreated or pretreated with IFN-β (1000 U/mL) for 16 hours and infected at an MOI of 2.5 with KOS or each ICP0 mutant in the absence (−) or presence (+) of IFN-β. Virally-infected cells were harvested and lysed at 36 hpi, and ICP4 (IE) and VP5 (L) protein levels were determined by Western blots. β-actin levels were included as loading controls. The bar graphs with error bars (SEMs) below the Western blots represent the relative difference (−/+IFN-β) in ICP4 and VP5 protein levels (as measured by densitometry) for each virus tested, which were normalized to β-actin levels. Results were from 3 independent experiments; a representative set of Western blot images is shown above. **, P = 0.001, ***, P = 0.0001, and *** * , P ≤ 0.0001 compared to KOS (one-way ANOVA).

Article Snippet: Blots were probed for protein detection with primary antibody (diluted 1:1000 in blocking buffer) overnight at 4°C (ICP0: H11060 , Santa Cruz Biotechnology; ICP5 or VP5: 56989; ICP4: H1A021 , EastCoast; Actin: I-19, Santa Cruz Biotechnology) in blocking buffer.

Techniques: Infection, Mutagenesis, Western Blot, Virus

Journal: Microbiology Spectrum

Article Title: A 77 Amino Acid Region in the N-Terminal Half of the HSV-1 E3 Ubiquitin Ligase ICP0 Contributes to Counteracting an Established Type 1 Interferon Response

doi: 10.1128/spectrum.00593-22

Figure Lengend Snippet: Relative HSV-1 transcript levels in cells treated with IFN-β and infected with WT HSV-1 and ICP0 mutants. HepaRG cells were mock-treated or exposed to IFN-β (1000 U/mL) for 16 hours and infected at an MOI of 2.5 with KOS, n 212, n 312, or n 389 in the presence of IFN-β. Cells were harvested at 24 hpi for RNA, and (IE) ICP4, (E) DNA polymerase, and (L) VP16 RNA levels were quantified by real-time PCR. Viral cDNA levels were normalized to endogenous hTBP; the relative quantities of viral transcripts for KOS were set as the baseline (value 1). Graphs are data compiled from two independent experiments. Error bars represent SEMs. *, P = 0.032 for DNA polymerase between n 312 and n 389 (unpaired t -test).

Article Snippet: Blots were probed for protein detection with primary antibody (diluted 1:1000 in blocking buffer) overnight at 4°C (ICP0: H11060 , Santa Cruz Biotechnology; ICP5 or VP5: 56989; ICP4: H1A021 , EastCoast; Actin: I-19, Santa Cruz Biotechnology) in blocking buffer.

Techniques: Infection, Real-time Polymerase Chain Reaction

Journal: Microbiology Spectrum

Article Title: A 77 Amino Acid Region in the N-Terminal Half of the HSV-1 E3 Ubiquitin Ligase ICP0 Contributes to Counteracting an Established Type 1 Interferon Response

doi: 10.1128/spectrum.00593-22

Figure Lengend Snippet: ICP0 and viral gene expression in WT HSV-1 and ICP0 truncation mutants in the presence of IFN-β. (A) HepaRG cells were infected at an MOI of 2.5 with KOS or an ICP0 truncation mutant ( n 212, n 312, or n 389) in mock-treated or cells pre-exposed to IFN-β (1000 U/mL). Cells were harvested at 24 hpi to analyze ICP0 RNA levels by quantitative real-time PCR. Viral cDNA levels were normalized to endogenous TBP and compared to transcript levels of ICP0 in KOS. Graphs shown are data compiled from 2 sets of independent experiments. Error bars represent SEMs. (B) HepaRG cells were infected at an MOI of 2.5 with KOS, n 212, n 312, or n 389 or infected at an MOI of 40 with n 212 or n 312 in untreated or IFN-β-pretreated cultures. Cells were lysed at 36 hpi to examine ICP0, ICP4, or VP5 protein levels by Western blots. β-actin was included as a loading control. A representative set of images from 3 independent experiments is shown.

Article Snippet: Blots were probed for protein detection with primary antibody (diluted 1:1000 in blocking buffer) overnight at 4°C (ICP0: H11060 , Santa Cruz Biotechnology; ICP5 or VP5: 56989; ICP4: H1A021 , EastCoast; Actin: I-19, Santa Cruz Biotechnology) in blocking buffer.

Techniques: Gene Expression, Infection, Mutagenesis, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Microbiology Spectrum

Article Title: A 77 Amino Acid Region in the N-Terminal Half of the HSV-1 E3 Ubiquitin Ligase ICP0 Contributes to Counteracting an Established Type 1 Interferon Response

doi: 10.1128/spectrum.00593-22

Figure Lengend Snippet: IFN-β increases H3K27 trimethylation on viral lytic promoters of the ICP0-null mutant, 7134. HepaRG cells were untreated or exposed to IFN-β (1000 U/mL) for 15 hours and then infected with KOS (WT HSV-1) or 7134 (ICP0-null mutant) at an MOI of 2.5 for 24 hours. Samples were processed for ChIP assays using a H3K27 trimethylation antibody, quantifying relative H3K27me3 binding to HSV-1 LAT, ICP0, ICP4, and gC promoters by real-time PCR. Mean values for each promoter are shown ( n = 4); error bars indicate SEMs. *, P < 0.013 and ****, P < 0.0001 compared to 7134 without IFN-β treatment (two-way ANOVA with Tukey’s multiple comparison test).

Article Snippet: Blots were probed for protein detection with primary antibody (diluted 1:1000 in blocking buffer) overnight at 4°C (ICP0: H11060 , Santa Cruz Biotechnology; ICP5 or VP5: 56989; ICP4: H1A021 , EastCoast; Actin: I-19, Santa Cruz Biotechnology) in blocking buffer.

Techniques: Mutagenesis, Infection, Binding Assay, Real-time Polymerase Chain Reaction, Comparison