Journal: mBio

Article Title: HCMV promotes viral reactivation through the coordinated regulation of Notch signaling by UL8 and miR-UL36

doi: 10.1128/mbio.03377-25

Figure Lengend Snippet: HCMV UL8 interacts with components of the Notch signaling pathway. ( A ) NHDF were infected with TB40/E-GFP-UL8-BT or TB40/E-GFP at an MOI of 2. At 3 days post-infection, the cell culture medium was supplemented with biotin (50 µg/mL) for 6 h. Labeling and infection were confirmed by immunoblot on whole cell lysates using the indicated primary antibodies. ( B ) Gene Ontology (GO) enrichment analysis of proteomic data derived from three independent BioID-MS experiments identifies significantly enriched biological processes. The bar plot shows the top GO terms ranked by statistical significance (-log₁₀(FDR)). ( C ) Network diagram ( https://cytoscape.org ) illustrating the proximity-based associations of the HCMV protein UL8 (blue) with key components of the Notch signaling pathway (colored nodes: NOTCH3, PSEN2, and SNW1). Dashed lines indicate interactions identified by BioID-MS analysis, and solid lines represent protein-protein interactions derived from the STRING database ( https://string-db.org ).

Article Snippet: The following commercial antibodies were used: Notch3 (2889, Cell Signaling), RBPJ (5442, Cell Signaling), GAPDH (ab8245, Abcam), HCMV IE2 (MAB810, Sigma Aldrich), UL44 (Virusys Corporation, P1202-2), and Turbo ID (AS204440, Agrisera).

Techniques: Infection, Cell Culture, Labeling, Western Blot, Derivative Assay, Protein-Protein interactions

Journal: mBio

Article Title: HCMV promotes viral reactivation through the coordinated regulation of Notch signaling by UL8 and miR-UL36

doi: 10.1128/mbio.03377-25

Figure Lengend Snippet: UL8 associates with Notch3 and promotes its degradation. ( A ) Schematic of the UL8 C-terminal region showing the introduced mutations in the tyrosine binding motifs at Y305 and Y314, as well as in the PDZ-binding domain (DTEL). ( B ) NHDFs were transduced with adenoviral constructs expressing GFP, UL7, UL8, UL8Y305A, UL88Y314A, UL8 Y305/314A, or UL8-mutDTEL for 48 h. Using rabbit anti-Notch3 antibody, UL8 was immunoprecipitated from cell lysates, and proteins separated by SDS-PAGE. Immunoprecipitated proteins were detected by western blotting using the NanoGlo HiBiT Blotting system. A western blot of total lysate is shown with GAPDH as a loading control. The bar graph represents densitometric quantification of the total Notch three signal from the western blot shown in the lower panel, normalized to GAPDH. Relative protein expression of three independent experiments was quantified using Image J. Values are means ±standard error of the means (SEM) (error bars). Statistical significance was determined by an unpaired Student’s t-test. *, P < 0.05; **, P < 0.005 compared to Ad-GFP. ( C ) HEK293 cells were co-transfected with plasmids expressing UL8-V5 and Notch3 NICD. After 24 h, cells were left untreated or incubated with 50 µM leupeptin (Leu) and 20 nM brefeldin A1 (B-A1) for 24 h. Cells were then lysed, and collected samples were subjected to SDS-PAGE and immunoblotting using anti-V5 and anti-Notch3 antibodies. GAPDH was used as a loading control. A representative western blot, out of three, is shown. ( D ) NDHF were infected with HCMV TB40/E WT, △UL8, or UL8 Y305/314A at an MOI of 1. At 96 h post-infection, protein lysates were generated and immunoblotted for Notch3, HCMV UL44, and GAPDH as a loading control. Densitometry was performed using ImageJ software for three independent experiments and plotted in right-hand panels. Values are means ±standard error of the means (SEM) (error bars). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test *, P < 0.05. ( E ) RNA was harvested from cells infected as in ( D ) and subjected to qRT-PCR using primers for the Notch target genes HES1 and HEY1 (n = 3; *** P < 0.001, **** P < 0.0001 by two-tailed t -test). ns, P > 0.05.

Article Snippet: The following commercial antibodies were used: Notch3 (2889, Cell Signaling), RBPJ (5442, Cell Signaling), GAPDH (ab8245, Abcam), HCMV IE2 (MAB810, Sigma Aldrich), UL44 (Virusys Corporation, P1202-2), and Turbo ID (AS204440, Agrisera).

Techniques: Binding Assay, Transduction, Construct, Expressing, Immunoprecipitation, SDS Page, Western Blot, Control, Transfection, Incubation, Infection, Generated, Software, Comparison, Quantitative RT-PCR, Two Tailed Test

Journal: mBio

Article Title: HCMV promotes viral reactivation through the coordinated regulation of Notch signaling by UL8 and miR-UL36

doi: 10.1128/mbio.03377-25

Figure Lengend Snippet: miR-UL36 impairs Notch signaling through targeting Notch3 and RBPJ. ( A ) HEK293 cells were transfected with a 4× CSL-Luciferase construct as well as a plasmid expressing the intracellular domain of Notch3 and either negative control miRNA mimic, miR-US33, miR-UL36, or miR-UL112. Protein lysates were harvested 24 h post-transfection, and luciferase expression was monitored using a luminometer ( n = 3; P < 0.05 calculated using a one-sample t and Wilcoxon test). ( B and C ) NHDFs were transfected with negative control miRNA mimic, miR-UL36, or siRNA against Notch3 ( B ) or RBPJ ( C ). Protein lysates were harvested 48 h post-transfection, and immunoblots were performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. ( D and E ). NHDF were mock-infected or infected with WT HCMV or a miR-UL36 mutant virus for 48 h. Protein lysates were harvested, and immunoblotting was performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as determined by two-tailed t -test. ( F ) RNA was harvested from cells infected as in ( D and E ) and subjected to qRT-PCR using primers for the Notch target genes HES1 and HEY1. ( n = 3; * P < 0.05 by two-tailed t -test).

Article Snippet: The following commercial antibodies were used: Notch3 (2889, Cell Signaling), RBPJ (5442, Cell Signaling), GAPDH (ab8245, Abcam), HCMV IE2 (MAB810, Sigma Aldrich), UL44 (Virusys Corporation, P1202-2), and Turbo ID (AS204440, Agrisera).

Techniques: Transfection, Luciferase, Construct, Plasmid Preparation, Expressing, Negative Control, Western Blot, Software, Infection, Mutagenesis, Virus, Two Tailed Test, Quantitative RT-PCR