Journal: mBio

Article Title: Herpes Simplex Virus 1 Manipulates Host Cell Antiviral and Proviral DNA Damage Responses

doi: 10.1128/mBio.03552-20

Figure Lengend Snippet: Reagents used for immunoblotting

Article Snippet: Anti-ICP0 , East Coast Bio , H1A027 , 1:2,000.

Techniques:

Journal: Cell Insight

Article Title: IRF1 amplifies HSV-1-triggered antiviral innate immunity in a feed-forward manner

doi: 10.1016/j.cellin.2025.100255

Figure Lengend Snippet: IRF1 restricts HSV-1 replication . (A) HT1080 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were infected with HSV-1 (MOI = 0.01). Viral titers in the supernatants were quantified at 48 h post-infection. (B–E) THP-1 cells transduced with control sgRNA (Ctrl) or sgRNA targeting IRF1 were mock-infected or infected with HSV-1 (MOI = 1). The expression of ICP0, ICP8, and UL19 was quantified by RT-qPCR (B–D). The mock-infected samples were labeled as N.D. (not detected), and the signals from wild-type (WT) cells infected with HSV-1 were normalized to 1. WCLs were analyzed by immunoblotting at 8 h post-infection (E). (F) HEK293T cells were transfected with an IFN-β promoter reporter plasmid mixture with increasing amounts of IRF1 expression plasmids (0, 0.1, 0.2, or 0.5 μg). Luciferase activities were measured at 24 h post-transfection. (G–H) HT1080 cells stably expressing vector control, IRF1-WT, or IRF1-R82A were infected with HSV-1-GFP (MOI = 0.05), and GFP expression was imaged at 24 h post-infection (G). Scale bars,100 μm. Viral titers in the supernatants were quantified at 24 h post-infection (H).

Article Snippet: The following antibodies and reagents were used for immunoblotting and immunoprecipitation: Mouse anti-FLAG monoclonal antibody (1:10,000, Dia-An Biotechnology, catalog no. 2064); Mouse anti-HA monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2063); Mouse anti-β-actin monoclonal antibody (1:5000, Dia-An Biotechnology, catalog no. 2060); Mouse anti-GAPDH monoclonal antibody (1:1000, Santa Cruz, sc-47724); Histone H3 antibody (1:1000, Santa Cruz, sc-517576); Rabbit anti-MITA/STING polyclonal antibody (1:5000, Proteintech, catalog no. 19851-1-AP); Rabbit anti-IRF3 polyclonal antibody (1:1000, Proteintech, catalog no. 11312-1-AP); Rabbit anti-TBK1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 3504); Rabbit anti-phospho-IRF3 (S386) monoclonal antibody (1:1000, Abcam, AB76493); Rabbit anti-phospho-TBK1 (S172) monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 5483); Rabbit anti-IRF1 monoclonal antibody (1:1000, Cell Signaling Technology, catalog no. 8478); Rabbit IgG (Proteintech, catalog no. 20010049); Mouse anti-ICP0 monoclonal antibody (1:1000, Santa Cruz, sc-53070); Mouse anti-ICP8 monoclonal antibody (1:1000, Santa Cruz, sc-53329); Mouse anti-ICP27 monoclonal antibody (1:1000, Santa Cruz, sc-69806); Mouse anti-ICP5 monoclonal antibody (1:1000, Santa Cruz, sc-56989); IRDye 800CW Goat anti-Rabbit and Goat anti-Mouse secondary antibodies (1:10,000, LI-COR); Anti-FLAG beads (Dia-An Biotechnology); Protein A/G agarose (GE healthcare).

Techniques: Transduction, Control, Infection, Expressing, Quantitative RT-PCR, Labeling, Western Blot, Transfection, Plasmid Preparation, Luciferase, Stable Transfection

Journal: Cell & Bioscience

Article Title: RNA-binding protein PTBP1 mediates HSV-1 attachment and infection through regulation of heparan sulfate 3-O-sulfotransferase gene expression

doi: 10.1186/s13578-025-01495-7

Figure Lengend Snippet: Depletion of PTBP1 expression reduces HSV-1 binding and infection due to loss of 3OST3. A Depletion of PTBP1 expression on HSV-1 cell binding. Left panel: schematic of the viral binding assay. Middle panel: qPCR analysis of viral genomic DNA in the parental (WT) and PTBP1-ko HeLa cells. Right panel: immunoblotting of HSV-1 gB protein. GAPDH was used as a loading control. B and C PTBP1 ko on HSV-1 infection in HeLa cells. The cells were infected with HSV-1 for 24 h. HSV-1 ICP0 and ICP27 production (B) and HSV-1 titers in culture medium (C) were detected. D and E . PTBP1 ko on HSV-1 infection in 293 T cells. The cells were infected with HSV-1 for 24 h. HSV-1 ICP0 and ICP27 production (D) and HSV-1 titers in culture medium (E) were detected. F Re-expression of PTBP1 on HSV-1 infection. PTBP1-ko 293 T cells were transfected for ectopic expression of PTBP1 for 48 h. The cells were then infected with HSV-1 for 24 h. The expression of PTBP1 and HSV-1 ICP proteins were examined by immunoblotting. Vec, empty vector. G HS3ST3A1 ko using CRISPR/Cas9 by targeting exon 1 of HS3ST3A1 . H Expression of 3OST family genes in 293 T cells determined by RT-qPCR using GAPDH as an internal control for normalization. The gene expression was presented relative to HS3ST3A1. I and J Genetic ablation of HS3ST3A1 on HSV-1 infection in 293 T cells. HSV-1 infection was determined by immunoblotting (I) and by plaque forming assay (J). K Re-expression of HS3ST3A1 restores cell susceptibility to HSV-1 infection. HS3ST3A1-ko 293 T cells were transfected with pCMV3-HS3ST3A1 for 48 h and then infected with HSV-1 infection for 24 h. The HS3ST3A1 and viral gene ICP27 expression were examined by immunoblotting. L and M Expression of HS3ST3A1 in PTBP1-ko cells restores cell susceptibility to HSV-1 infection. HSV-1 infection was determined by immunoblotting (L) and by RT-qPCR (M). N Overexpression of PTBP1 in HS3ST3A1-ko cells on HSV-1 infection. HS3ST3A1-ko 293 T cells were transfected with a PTBP1 expression plasmid or empty vector, followed by HSV-1 infection. PTBP1 and viral protein ICP0 expression was assessed by immunoblotting. The experiments were performed 3 times independently. Data are shown as mean ± SD. ns, no significance, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: The antibodies against PTBP1 (Abcam, ab133734), HS3ST3A1 (SinoBiological, 209,208-T08), GAPDH (Abcam, ab9484), HS3ST3A1 (Santa Cruz, sc390024, mouse specific), the HSV-1 ICP0 (Santa Cruz, sc53070), ICP27 (Santa Cruz, sc69806), and gB (Santa Cruz, sc56987) were obtained from commercial sources.

Techniques: Expressing, Binding Assay, Infection, Western Blot, Control, Transfection, Plasmid Preparation, CRISPR, Quantitative RT-PCR, Gene Expression, Over Expression

Journal: Cell & Bioscience

Article Title: RNA-binding protein PTBP1 mediates HSV-1 attachment and infection through regulation of heparan sulfate 3-O-sulfotransferase gene expression

doi: 10.1186/s13578-025-01495-7

Figure Lengend Snippet: Requirement of PTBP1 for HS3ST3A1/B1 pre-mRNA processing. A Profiling of PTBP1 binding to HS3ST3A1 and HS3ST3B1 pre-mRNA. The publicly available data ( GSE57278 ) was retrieved and used to obtain RNA binding profiles of HS3ST3A1 and HS3ST3B1 pre-mRNA association with PTBP1 in 293 T cells. B Validation of PTBP1 and HS3ST3A1/B1 pre-mRNA interactions by RNA-immunoprecipitation assay. RIP was performed using 293 T cells with an anti-PTBP1 antibody or IgG as a control. Two sets of primers (L and R) targeting the corresponding intronic region of HS3ST3A1 or B1 were used to detect immunoprecipitation enrichment by qPCR. C RT-PCR validation of HS3ST3A1/B1 pre-mRNA processing in parental HeLa (wt HeLa) and PTBP1-ko cells. Diagram showing the pre-mRNA of HS3ST3A1 and HS3ST3B1 and primers used for the detection of pre-mRNA and mRNA by RT-PCR. Total RNA was first treated with gDNA wiper to remove contamination from gDNA, followed by reverse transcription. The expression of corresponding genes was detected by PCR amplification (+ RT) using non-transcriptase treated samples (-RT) as controls. Primer combinations spanning the 2 exons (F1/R2, F2/R2, and F2/R3) were included to demonstrate lack of gene splicing events in the PTBP1-ko cells. The experiment was performed 3 times independently. GAPDH was used as a loading control. D Diagram showing the corresponding sequences of WT PTBP1 and the C23S mutant. E Detection of PTBP1 dimer formation by immunoblotting. PTBP1-ko 293 T cells were transfected with WT or PTBP1 C23S mutant for ectopic re-expression of PTBP1. The cell lysates were separated by SDS-PAGE under reducing condition or non-denaturing native condition. GAPDH was used as a loading control. The experiment was performed 3 times. F Re-expression of WT PTBP1, but not C23S mutant, reinstates proper HS3ST3A1/B1 expression detected by RT-PCR. G and H Ectopic expression of WT but not PTBP1 C23S mutant rescues infectivity to HSV-1 infection. PTBP1-ko 293 T cells were transfected with WT or PTBP1 C23S mutant for 48 h. PTBP1 expression and HSV-1 ICP27 production in the parental 293 T (WT) and ko cells were detected by immunoblotting using GAPDH as a loading control (G). Similarly, viral gene expression was confirmed by RT-qPCR (H). The experiments were performed 3 times independently with representative gels shown. Data are presented as mean ± SD. ns, no significance, * p ≤ 0.05, *** p ≤ 0.001

Article Snippet: The antibodies against PTBP1 (Abcam, ab133734), HS3ST3A1 (SinoBiological, 209,208-T08), GAPDH (Abcam, ab9484), HS3ST3A1 (Santa Cruz, sc390024, mouse specific), the HSV-1 ICP0 (Santa Cruz, sc53070), ICP27 (Santa Cruz, sc69806), and gB (Santa Cruz, sc56987) were obtained from commercial sources.

Techniques: Binding Assay, RNA Binding Assay, Biomarker Discovery, RNA Immunoprecipitation, Control, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Expressing, Amplification, Mutagenesis, Western Blot, Transfection, SDS Page, Infection, Gene Expression, Quantitative RT-PCR