Journal: Virologica Sinica

Article Title: Ubiquitously expressed transcript isoform 2 (UXT-V2) restricts HSV-2 replication by targeting glycoprotein B for degradation through ubiquitin-proteasome pathway

doi: 10.1016/j.virs.2025.08.004

Figure Lengend Snippet: UXT-V2 inhibits HSV-2 replication. A–C HeLa cells were transfected with different doses of empty vector or plasmids expressing His-UXT-V2 (0.2, 0.5 or 1 μg), followed by infection with HSV-2 at an MOI of 0.2 PFU/cell. At 12, 18, and 24 hpi, the cells were collected for qRT-PCR ( A ), Western blotting ( B ), and plaque assay ( C ). D UXT-V2 in sgControl and sgUXT-V2 HeLa cells was assessed by immunoblot analysis. E sgControl and sgUXT-V2 HeLa cells were cultured for 24 h. Cell proliferation was assessed using the CCK-8 cell counting assay. F–H WT and UXT-V2-KO HeLa cells were infected with HSV-2 at an MOI of 0.2 PFU/cell. At 12, 18, and 24 hpi, the cells were collected for qRT-PCR ( F ), Western blotting ( G ), and plaque assay ( H ). For graphs, data shown are mean ± SD of three independent experiments with each condition performed in triplicate (∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001). For images, one representative experiment out of three is shown.

Article Snippet: Human embryonic kidney 293T (HEK293T) cell line, baby hamster kidney cell line BHK-21, African green monkey kidney cell line Vero and Vero E6, and human cervical epithelial cell line HeLa were purchased from the American Type Culture Collection.

Techniques: Transfection, Plasmid Preparation, Expressing, Infection, Quantitative RT-PCR, Western Blot, Plaque Assay, Cell Culture, CCK-8 Assay, Cell Counting

Journal: Virologica Sinica

Article Title: Ubiquitously expressed transcript isoform 2 (UXT-V2) restricts HSV-2 replication by targeting glycoprotein B for degradation through ubiquitin-proteasome pathway

doi: 10.1016/j.virs.2025.08.004

Figure Lengend Snippet: UXT-V2 inhibits HSV-2 replication via NF-κB independent pathway. A ARPE-19 cells were transfected with an empty vector or different doses of plasmids expressing His-UXT-V1 (0.5 or 1 μg) or His-UXT-V2 (0.5 or 1 μg), followed by infection with HSV-2 at an MOI of 0.2 PFU/cell at 6 hpt for 18 h. The virus yields were measured by plaque assay. B HeLa cells were transfected with 0.3 μg PRD(III-I) 4 -Luc or NF-κB–Luc reporter plasmid along with 1.5 μg empty vector or 1.5 μg plasmid expressing His-UXT-V1 or His-UXT-V2, followed by infection with HSV-2 at an MOI of 0.2 PFU/cell at 6 hpt for 18 h. The luciferase activity was measured to determine the activation fold. HEK293T cells were transfected with 0.3 μg NF-κB-Luc reporter plasmid along with an empty vector or 1 μg plasmid expressing His-UXT-V2 for 24 h, followed by treatment with or without 10 ng/mL TNF-α for 6 h. Luciferase activity was measured to determine the activation fold. C HEK293T cells were transfected with 0.3 μg NF-κB-Luc reporter plasmid along with an empty vector or 1 μg plasmid expressing His-UXT-V2 for 24 h, followed by treatment with or without 10 ng/mL TNF-α for 6 h. Luciferase activity was measured to determine the activation fold. D BHK-21 cells were transfected with an empty vector or different doses of plasmids expressing His-UXT-V1 (0.5 or 2 μg) or His-UXT-V2 (0.5 or 2 μg), followed by infection with JEV at an MOI of 0.5 PFU/cell at 6 hpt. The supernatants and cells were harvested, and the virus yields were measured by plaque assay at 24 hpi. E BHK-21 cells were transfected with an empty vector or 1.5 μg plasmid expressing His-UXT-V2, followed by infection with JEV at an MOI of 0.5 PFU/cell at 6 hpt. The supernatants and cells were harvested, and the virus yields were measured by plaque assay at 8, 16, 24 and 36 hpi. F Vero E6 cells were transfected with an empty vector or different doses of plasmids expressing His-UXT-V1 (0.5 or 2 μg) or His-UXT-V2 (0.5 or 2 μg), followed by infection with ZIKV at an MOI of 0.5 PFU/cell at 6 hpt. The supernatants and cells were harvested and the virus yields were measured by plaque assay at 24 hpi. G Vero E6 cells were transfected with an empty vector or 1.5 μg plasmid expressing His-UXT-V2, followed by infection with ZIKV at an MOI of 0.5 PFU/cell at 6 hpt. The supernatants and cells were harvested, and the virus yields were measured by plaque assay at 8, 16, 24 and 36 hpi. H Vero E6 cells transfected with the control or the two UXT shRNAs, respectively, were infected with HSV-2 at an MOI of 0.2 PFU/cell for 24 h. The virus yields were measured by plaque assay. For graphs, data shown are mean ± SD of three independent experiments with each condition performed in triplicate (∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001; ns, not significant). For images, one representative experiment out of three is shown.

Article Snippet: Human embryonic kidney 293T (HEK293T) cell line, baby hamster kidney cell line BHK-21, African green monkey kidney cell line Vero and Vero E6, and human cervical epithelial cell line HeLa were purchased from the American Type Culture Collection.

Techniques: Transfection, Plasmid Preparation, Expressing, Infection, Virus, Plaque Assay, Luciferase, Activity Assay, Activation Assay, Control

Journal: Virologica Sinica

Article Title: Ubiquitously expressed transcript isoform 2 (UXT-V2) restricts HSV-2 replication by targeting glycoprotein B for degradation through ubiquitin-proteasome pathway

doi: 10.1016/j.virs.2025.08.004

Figure Lengend Snippet: UXT-V2 downregulates the expression of HSV-2 gB. A HSV-2 proteins fished out by LC-MS/MS analysis. Figure was created using ggplot2. B HEK293T cells were co-transfected with 2 μg empty vector or 2 μg His-UXT-V2 together with 2 μg plasmid expressing Flag-gB. At 24 hpi, cells were harvested and lysed, and the extracts were subjected to IP using the anti-Flag MAb or control IgG. C HeLa cells were infected with or without HSV-2 at an MOI of 0.2 PFU/cell. At 24 hpi, cells were harvested and lysed, and the extracts were subjected to IP using the anti-gB MAb. D HEK293T cells were co-transfected with 1.5 μg empty vector or 1.5 μg plasmid expressing His-UXT-V2 together with 0.5 μg plasmid expressing various Flag-tagged HSV-2 proteins for 24 h. The expression of Flag-tagged HSV-2 proteins were measured by Western blotting. E HEK293T cells were co-transfected with 1.5 μg empty vector or 1.5 μg plasmid expressing His-UXT-V2 together with 0.5 μg plasmid expressing Flag-gB for 12 or 18 h. Cells were harvested and the expression of Flag-gB was detected by Western blotting. F HEK293T cells were co-transfected with 1.5 μg empty vector or 1.5 μg plasmid expressing His-UXT-V1 or His-UXT-V2 together with 0.5 μg plasmid expressing Flag-gB for 12 or 18 h. Cells were harvested and the expression of Flag-gB was measured by Western blotting. G ARPE-19 cells were transfected with 60 nM NC or #4 (UXT-V2 siRNA), followed by transfection with an empty vector or different doses of plasmids expressing Flag-gB (0.5 or 2 μg) for 24 h. Cells were harvested and the expression of Flag-gB was detected by Western blotting. H HeLa cells were transfected with an empty vector or 2 μg plasmid expressing His-UXT-V2, followed by infection with HSV-2 at an MOI of 5 PFU/cell. At 6 or 8 hpi, cells were harvested, and the expression level of HSV-2 gB or gD was analyzed by Western blotting. I WT and UXT-V2-KO HeLa cells were infected with HSV-2 at an MOI of 5 PFU/cell. At 6 or 8 hpi, cells were harvested and the expression level of HSV-2 gB or gD was measured by Western blotting. J HeLa cells were co-transfected with an empty vector or different doses of plasmids expressing His-UXT-V2 (0.5 or 2 μg) together with 0.5 μg plasmid expressing Flag-gB, followed by infection with HSV-2 at an MOI of 0.2 PFU/cell at 6 hpt for 18 h. Virus yields were measured by plaque assay. The graph shows compensating efficiency of gB on HSV-2 yields. For graphs, data shown are mean ± SD of three independent experiments with each condition performed in triplicate (∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001; ns, not significant). For images, one representative experiment out of three is shown.

Article Snippet: Human embryonic kidney 293T (HEK293T) cell line, baby hamster kidney cell line BHK-21, African green monkey kidney cell line Vero and Vero E6, and human cervical epithelial cell line HeLa were purchased from the American Type Culture Collection.

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Transfection, Plasmid Preparation, Control, Infection, Western Blot, Virus, Plaque Assay

Journal: Virologica Sinica

Article Title: Ubiquitously expressed transcript isoform 2 (UXT-V2) restricts HSV-2 replication by targeting glycoprotein B for degradation through ubiquitin-proteasome pathway

doi: 10.1016/j.virs.2025.08.004

Figure Lengend Snippet: UXT-V2 promotes K48-linked polyubiquitination of HSV-2 gB. A HEK293T cells were co-transfected with 0.5 μg empty vector or different doses of plasmids expressing His-UXT-V2 (0.2, 0.5 or 1.5 μg) together with 0.5 μg plasmid expressing HA-Ub. At 24 hpt, the expression of His-UXT-V2 and ubiquitinated cellular proteins was detected by Western blotting. B HEK293T cells were transfected with 0.5 μg empty vector or different doses of plasmids expressing His-UXT-V2 (0.2, 0.5 or 1.5 μg). At 24 hpt, the expression of His-UXT-V2 and ubiquitinated cellular proteins was detected by Western blotting. C HEK293T cells were co-transfected with 5 μg empty vector or 5 μg plasmid expressing His-UXT-V2 together with 4 μg plasmid expressing Flag-gB for 24 h. Cells were immunoprecipitated with the anti-Flag MAb. D HEK293T cells were co-transfected with 2 μg empty vector or 2 μg plasmid expressing His-UXT-V2 together with 2 μg plasmid expressing Flag-gB and 2 μg plasmid expressing HA-Ub, HA-K48 or HA-K63 for 24 h. Cells were immunoprecipitated with the indicated antibodies. E HeLa cells were co-transfected with 2 μg empty vector or 2 μg plasmid expressing His-UXT-V2 together with 2 μg plasmid expressing HA-Ub, HA-K48 or HA-K63, followed by infection with HSV-2 at an MOI of 2 PFU/cell at 6 hpt for 18 h. Cells were immunoprecipitated with the indicated antibodies. For images, one representative experiment out of three is shown.

Article Snippet: Human embryonic kidney 293T (HEK293T) cell line, baby hamster kidney cell line BHK-21, African green monkey kidney cell line Vero and Vero E6, and human cervical epithelial cell line HeLa were purchased from the American Type Culture Collection.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Infection

Journal: Virologica Sinica

Article Title: Ubiquitously expressed transcript isoform 2 (UXT-V2) restricts HSV-2 replication by targeting glycoprotein B for degradation through ubiquitin-proteasome pathway

doi: 10.1016/j.virs.2025.08.004

Figure Lengend Snippet: UXT-V2 facilitates TRIM21-dependent proteasomal degradation of gB. A E3 ubiquitin ligases fished out by LC-MS/MS analysis. Figure was created using ggplot2. B HEK293T cells were co-transfected either with 2 μg empty vector or 2 μg HA-TRIM21 expression plasmid, along with 2 μg plasmid expressing Flag-gB. At 24 hpt, cells were harvested and lysed, followed by IP using the anti-Flag MAb or control IgG. C The expression of TRIM21 in sgControl and sgTRIM21 HeLa cells was assessed by immunoblot analysis. D HeLa or TRIM21-KO HeLa cells were co-transfected with either 2 μg empty vector or 2 μg plasmid expressing His-UXT-V2, along with 2 μg plasmid expressing Flag-gB and 2 μg plasmid expressing HA-K48. At 24 hpt, cells were harvested and lysed, followed by IP using the anti-Flag Mab or control IgG. For images, one representative experiment out of three is shown.

Article Snippet: Human embryonic kidney 293T (HEK293T) cell line, baby hamster kidney cell line BHK-21, African green monkey kidney cell line Vero and Vero E6, and human cervical epithelial cell line HeLa were purchased from the American Type Culture Collection.

Techniques: Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Transfection, Plasmid Preparation, Expressing, Control, Western Blot

Journal: bioRxiv

Article Title: FLIM Imaging of mCherryTYG Deciphers pH Dynamics and Lifestyles of Salmonella Typhimurium

doi: 10.1101/2025.01.27.635066

Figure Lengend Snippet: Tracking the pH of Salmonella during HeLa cell infection. (A) HeLa cells harboring Lamp1-miRFP703 were infected with Salmonella (red) and imaged using confocal microscopy (left and middle panels) 4 hours post-infection. The right panel shows the corresponding FLIM image. (B) The left and middle panels show a confocal image of HeLa cells infected with Salmonella Typhimurium expressing mCherryTYG, 16h post-infection. The right panel displays the corresponding FLIM image, with the pHi of selected bacteria indicated. (C) Representative fluorescence lifetime histograms of mCherryTYG in Salmonella during HeLa cell infection. (D) Quantification of pH values from panels A and B (insets). Scale bar, 5 μm.

Article Snippet: The human epithelial cell line HeLa (ATCC CCL-2, obtained from the American Type Culture Collection) was used for Salmonella infection experiments.

Techniques: Infection, Confocal Microscopy, Expressing, Bacteria, Fluorescence