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