Journal: bioRxiv

Article Title: Calmodulin controls the tempo of HSV-1 gene-expression cascade to reshape infection heterogeneity

doi: 10.64898/2026.06.02.729706

Figure Lengend Snippet: (A) Alignment of the amino acid sequence of HSV-1 ICP4 (aa 1060–1073) with the canonical IQ motif and the IQ-like motif. (B) HeLa/puroR or HeLa/Flag-CaM cells were infected with wild-type HSV-1(F) at an MOI of 5. At 9 h post-infection, cell lysates were immunoprecipitated (IP) with an anti-Flag antibody. Input lysates and immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. (C) Schematic diagram of the structure of wild-type ICP4 (ICP4-WT) and the IQ-like motif deletion mutant (ICP4-ΔIQ), which lacks amino acids 1060–1070. (D and E) Purified Flag-tagged ICP4-WT, ICP4-ΔIQ, or EGFP were incubated with calmodulin (CaM) Sepharose beads or Protein A Sepharose (control) beads. Bound proteins were detected by immunoblotting with an anti-Flag antibody (D). (E) Quantification of the relative binding intensity (pull-down/input) from (D). (F and G) HeLa cells were infected with rICP47/vUs11/ICP4ΔIQ (rvICP4ΔIQ) or its repaired virus (rvICP4ΔIQ-repair) at an MOI of 5 and analyzed by time-lapse imaging as in . (F) Dot plots showing IE onset (TagRFP, left) and L onset (Venus, right) times (min post-infection) for individual cells. Cells in which onset was not detected are plotted above the dashed line (onset n.d.) with an imputed value (maximum observed onset + 60 min) and were included in statistical analyses. (G) Time interval between IE and L onset (L onset − IE onset) in cells where both onsets were defined. (H) HeLa cells were infected with ICP4ΔIQ or ICP4ΔIQ-repair virus at an MOI of 5 and fixed at 3.5 h post-infection. ChIP-qPCR was performed using an anti-ICP4 antibody or control IgG to quantify ICP4 occupancy at the 5′ regions of UL54 (IE), UL29 (E), and UL49 (L). (I) WT or CALM1-KO HeLa cells were infected with wild-type HSV-1(F) at an MOI of 5 and analyzed by ChIP-qPCR at 3.5 h post-infection as in (H). Data are representative of three independent experiments (B and D). Each value represents the mean ± SE of three (E, H, and I) biological replicates. In (F and G), each dot represents one cell and bars indicate medians. Adjacent bar graphs indicate the differences in median values relative to rvICP4ΔIQ. The number of analyzed cells was as follows: (F) rvICP4ΔIQ, n = 956; rvICP4ΔIQ-repair, n = 1079; (G) rvICP4ΔIQ, n = 912; rvICP4ΔIQ-repair, n = 1065. Data represent pooled measurements from two independent experiments. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple-comparison test (E, H, and I) or Mann–Whitney U-test (F and G). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Article Snippet: Commercial antibodies used in this study included mouse monoclonal antibodies to Flag (M2; Sigma), α-tubulin (DM1A; Sigma), gD (sc-21719; Santa Cruz), and ICP4 (58S; ATCC); a rabbit monoclonal antibody to calmodulin (ab45689; Abcam); and rabbit polyclonal antibodies to green fluorescent protein (GFP) (598; Medical & Biological Laboratories [MBL]) and TagRFP (AB233; Evrogen).

Techniques: Sequencing, Infection, Immunoprecipitation, Western Blot, Mutagenesis, Purification, Incubation, Control, Binding Assay, Virus, Imaging, ChIP-qPCR, Comparison, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Cytosolic nucleic acid sensing triggers type I interferon activation via the Hippo kinase LATS1

doi: 10.1016/j.jbc.2026.111204

Figure Lengend Snippet: Virus infection and cytosolic nucleic acid sensing trigger Hippo pathway activation in a LATS1-dependent manner. A and B , quantitative real-time PCR (qPCR) analysis of TEAD regulated genes Ctgf and Cyr61 in murine embryonic fibroblast (MEF) cells after infection with ( A ) vesicular stomatitis virus (VSV) (MOI 0.75; 24 h) or ( B ) herpes simplex virus-1 (HSV-1) (MOI 0.5; 8 h). C and D , qPCR of Ctgf and Cyr61 expression in MEF cells transfected with pI:C ( C ) or ISD ( D ) (2 μg/ml each; 3 h). E and F , immunoblot analysis ( left panels ) and relative quantification ( right panels ) of YAP expression in WT or Lats1 −/− MEF cells transfected with pI:C ( E ) or ISD ( F ) (2 μg/ml each; 8 h). G and H , immunoblot analysis ( left panels ) and relative quantification ( right panels ) of LATS1 (T1079) and IRF3 (S396) phosphorylation in MEF cells transfected with pI:C ( G ) or ISD ( H ) (2 μg/ml each) for the indicated times. Statistical significance was determined using Student’s t test (∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05).

Article Snippet: Primary antibodies used in this study were FLAG (Sigma Life Sciences); VSV-G, HSV-1 ICP4, HSP90, P65 (Santa Cruz Biotechnology); TBK1, p-TBK1, IRF3, p-IRF3, LATS1, p-LATS1, p-YAP, YAP/TAZ, STING, p-STING, p-P65, β-Actin (Cell Signaling Technology); β-Tubulin, GAPDH, HA (Proteintech); P65 (Santa Cruz Biotechnology).

Techniques: Virus, Infection, Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Transfection, Western Blot, Quantitative Proteomics, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Cytosolic nucleic acid sensing triggers type I interferon activation via the Hippo kinase LATS1

doi: 10.1016/j.jbc.2026.111204

Figure Lengend Snippet: LATS1 is required for IFN-I activation. A and B , qPCR analysis of Ifnb mRNA expression in WT and Lats1 −/− MEF cells infected with VSV (MOI 0.5) ( A ) or HSV-1 (MOI 0.1) for 24 h. C and D , qPCR analysis of Cxcl10 and Ccl5 mRNA in WT and Lats1 −/− MEF cells infected with VSV as in panel A ( C ) or HSV-1 as in panel B ( D ). E and F , immunoblot analysis ( left panels ) and relative quantification ( right panels ) of IRF3 (S396) phosphorylation in WT and Lats1 −/− MEF cells transfected with pI:C ( E ) or ISD ( F ) (2 μg/ml each) for the indicated times. G and H , qPCR analysis of Ifnb mRNA expression in WT and Lats1 −/− MEF cells transfected with pI:C ( G ) or ISD ( H ) (2 μg/ml each) for the indicated times. I and J , ELISA for IFN-β secretion in WT and Lats1 −/− MEF cells transfected with pI:C ( I ) or ISD ( J ) (2 μg/ml each; 6 h). K and L , qPCR analysis of Cxcl10, Ccl5, and Isg15 mRNA in WT and Lats1 −/− MEF cells transfected with pI:C ( K ) or ISD ( L ) (2 μg/ml each) for the indicated times. Statistical significance was determined using Student’s t test (∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05).

Article Snippet: Primary antibodies used in this study were FLAG (Sigma Life Sciences); VSV-G, HSV-1 ICP4, HSP90, P65 (Santa Cruz Biotechnology); TBK1, p-TBK1, IRF3, p-IRF3, LATS1, p-LATS1, p-YAP, YAP/TAZ, STING, p-STING, p-P65, β-Actin (Cell Signaling Technology); β-Tubulin, GAPDH, HA (Proteintech); P65 (Santa Cruz Biotechnology).

Techniques: Activation Assay, Expressing, Infection, Western Blot, Quantitative Proteomics, Phospho-proteomics, Transfection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Cytosolic nucleic acid sensing triggers type I interferon activation via the Hippo kinase LATS1

doi: 10.1016/j.jbc.2026.111204

Figure Lengend Snippet: Cellular antiviral host defenses are dependent on LATS1. A and B , plaque assay for viral titers from supernatants of WT and Lats1 −/− MEF cells infected with VSV (MOI 0.1) ( A ) or HSV-1 (MOI 0.4) for 16 h. C , immunoblot analysis ( left panel ) and relative quantification ( right panel ) for VSV encoded glycoprotein, G (VSV-G) in WT and Lats1 −/− MEF cells infected with VSV (MOI 0.1) for the indicated times. D , immunoblot analysis ( left panel ) and relative quantification ( right panel ) of HSV-1 encoded immediate early transcription factor, ICP4 (HSV-1 ICP4) expression in WT and Lats1 −/− MEF cells infected with HSV-1 (MOI 0.1) for the indicated times. E and F , fluorescent imaging of WT and Lats1 −/− MEF cells infected with VSV-GFP (MOI 0.1) ( E ) or HSV-1-GFP (MOI 0.1) ( F ) for 24 h. Left to right : bright field, GFP, merged channels. Scale bar = 170 μm. Statistical significance was determined using student’s t test (∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05).

Article Snippet: Primary antibodies used in this study were FLAG (Sigma Life Sciences); VSV-G, HSV-1 ICP4, HSP90, P65 (Santa Cruz Biotechnology); TBK1, p-TBK1, IRF3, p-IRF3, LATS1, p-LATS1, p-YAP, YAP/TAZ, STING, p-STING, p-P65, β-Actin (Cell Signaling Technology); β-Tubulin, GAPDH, HA (Proteintech); P65 (Santa Cruz Biotechnology).

Techniques: Plaque Assay, Infection, Western Blot, Quantitative Proteomics, Expressing, Imaging

Journal: Journal of Advanced Research

Article Title: Antimycin A inhibits alpha-herpesvirus replication by disrupting the formation of pyrimidinosomes

doi: 10.1016/j.jare.2025.05.016

Figure Lengend Snippet: Antimycin A exhibits extensive antiviral activity against alpha-herpesvirus. Antimycin A effectively inhibited HSV-1 (A-C) and HSV-2 (G-H) infections in Vero E6 cells, PRV (D-E) infection in PK-15 cells, and EHV-1 (J-K) infection in RK13 cells. Vero-E6 cells, PK-15 cells, and RK13 cells were pretreated for 12 h with increasing concentrations of Antimycin A and then infected with HSV-1 (A-C), PRV (D-E), HSV-2 (G-I), and EHV-1 (J-K) at MOIs of 0.5, 0.1, 0.5, and 0.5, respectively. At 24 hpi, cells were fixed and analyzed by fluorescence imaging. (A, D, G and J) Infection levels were quantified using a fluorescent microplate reader (black curve), while cell viability was measured using the CCK-8 Assay (orange curve). The CC50 for each compound was calculated via a four-parameter logistic nonlinear regression model in GraphPad Prism. Dotted lines indicate 50 % inhibition. Data represent the means ± SEM from n = 3 independent experiments of infectious virions, normalized to DMSO-treated wells. The IC50 values for HSV-1, PRV, HSV-2, and EHV-1 were determined by nonlinear regression analysis. (B, E, H and K) eGFP expression in infected cells, either untreated (0 μM) or treated with various concentrations (0.0015–5 μM) of Antimycin A, was visualized by fluorescence microscopy at the same time point. Representative images are shown. Bars, 300 µm. Magnification, ×10. (C, F and I) Western blot analysis was performed to quantify infection in cells infected with HSV-1, PRV, or HSV-2. For HSV-1, infection was assessed using ICP4, VP16, and gD as markers. For PRV, infection levels were quantified by immunoblotting for UL54. For HSV-2, infection was quantified by immunoblotting for VP16. β-actin was used as the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in this study included goat BHV-1 antisera, mouse anti-PRV UL47 monoclonal antibody (MAb), mouse anti-HSV-1 ICP4 monoclonal antibody (Santa Cruz, sc69809), mouse anti-HSV-1 VP16 monoclonal antibody (Santa Cruz, sc7545), mouse anti-HSV-1 gD monoclonal antibody (Santa Cruz, sc21719), and mouse anti-β-actin MAb (Proteintech, 66009–1-lg).

Techniques: Activity Assay, Infection, Fluorescence, Imaging, CCK-8 Assay, Inhibition, Expressing, Microscopy, Western Blot, Control

Journal: Journal of Advanced Research

Article Title: Antimycin A inhibits alpha-herpesvirus replication by disrupting the formation of pyrimidinosomes

doi: 10.1016/j.jare.2025.05.016

Figure Lengend Snippet: Antimycin A exhibits extensive antiviral activity against alpha-herpesvirus. Antimycin A effectively inhibited HSV-1 (A-C) and HSV-2 (G-H) infections in Vero E6 cells, PRV (D-E) infection in PK-15 cells, and EHV-1 (J-K) infection in RK13 cells. Vero-E6 cells, PK-15 cells, and RK13 cells were pretreated for 12 h with increasing concentrations of Antimycin A and then infected with HSV-1 (A-C), PRV (D-E), HSV-2 (G-I), and EHV-1 (J-K) at MOIs of 0.5, 0.1, 0.5, and 0.5, respectively. At 24 hpi, cells were fixed and analyzed by fluorescence imaging. (A, D, G and J) Infection levels were quantified using a fluorescent microplate reader (black curve), while cell viability was measured using the CCK-8 Assay (orange curve). The CC50 for each compound was calculated via a four-parameter logistic nonlinear regression model in GraphPad Prism. Dotted lines indicate 50 % inhibition. Data represent the means ± SEM from n = 3 independent experiments of infectious virions, normalized to DMSO-treated wells. The IC50 values for HSV-1, PRV, HSV-2, and EHV-1 were determined by nonlinear regression analysis. (B, E, H and K) eGFP expression in infected cells, either untreated (0 μM) or treated with various concentrations (0.0015–5 μM) of Antimycin A, was visualized by fluorescence microscopy at the same time point. Representative images are shown. Bars, 300 µm. Magnification, ×10. (C, F and I) Western blot analysis was performed to quantify infection in cells infected with HSV-1, PRV, or HSV-2. For HSV-1, infection was assessed using ICP4, VP16, and gD as markers. For PRV, infection levels were quantified by immunoblotting for UL54. For HSV-2, infection was quantified by immunoblotting for VP16. β-actin was used as the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in this study included goat BHV-1 antisera, mouse anti-PRV UL47 monoclonal antibody (MAb), mouse anti-HSV-1 ICP4 monoclonal antibody (Santa Cruz, sc69809), mouse anti-HSV-1 VP16 monoclonal antibody (Santa Cruz, sc7545), mouse anti-HSV-1 gD monoclonal antibody (Santa Cruz, sc21719), and mouse anti-β-actin MAb (Proteintech, 66009–1-lg).

Techniques: Activity Assay, Infection, Fluorescence, Imaging, CCK-8 Assay, Inhibition, Expressing, Microscopy, Western Blot, Control