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ATCC human cervical epithelial cell line hela

UXT-V2 inhibits HSV-2 replication. A–C <t>HeLa</t> 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.

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

Journal: Virologica Sinica

doi: 10.1016/j.virs.2025.08.004

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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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.

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.

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.

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.

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

C. trachomatis infection induces ISG15 synthesis in epithelial cells. (A and C) Time course of infection of HeLa cells with C. trachomatis (A and C, MOI = 1) or with increasing MOI (B, 42 hpi). After infection, ISG15 transcript ( C ) and protein ( A-B ) levels were examined by RT-qPCR and Western blot, respectively. ( D ) HeLa cells were incubated with IFNα (10 ng/mL) or C. trachomatis (MOI = 1) for 30 h followed by immunostaining. ( E ) HeLa cells were incubated with C. trachomatis (MOI = 1) for 42 h or IFN-I (IFNα/β, 10 ng/mL for each) for 24 h before determining the expression of ISG15. ( F ) Primary epithelial cells from patients were incubated with C. trachomatis at the indicated MOI. ISG15 expression was examined 42 h post-infection (left and middle panels) or at the indicated times post-infection (right panel). The Western blots and immunofluorescence data are representative of at least two independent experiments. All other data represent three independent experiments. One-way ANOVA test (time course) and unpaired t -test (dose course in primary cells) were performed, and the p-value of the comparison with uninfected control is shown.

Journal: mBio

Article Title: Chlamydia -driven ISG15 expression dampens the immune response of epithelial cells independently of ISGylation

doi: 10.1128/mbio.02401-24

Figure Lengend Snippet: C. trachomatis infection induces ISG15 synthesis in epithelial cells. (A and C) Time course of infection of HeLa cells with C. trachomatis (A and C, MOI = 1) or with increasing MOI (B, 42 hpi). After infection, ISG15 transcript ( C ) and protein ( A-B ) levels were examined by RT-qPCR and Western blot, respectively. ( D ) HeLa cells were incubated with IFNα (10 ng/mL) or C. trachomatis (MOI = 1) for 30 h followed by immunostaining. ( E ) HeLa cells were incubated with C. trachomatis (MOI = 1) for 42 h or IFN-I (IFNα/β, 10 ng/mL for each) for 24 h before determining the expression of ISG15. ( F ) Primary epithelial cells from patients were incubated with C. trachomatis at the indicated MOI. ISG15 expression was examined 42 h post-infection (left and middle panels) or at the indicated times post-infection (right panel). The Western blots and immunofluorescence data are representative of at least two independent experiments. All other data represent three independent experiments. One-way ANOVA test (time course) and unpaired t -test (dose course in primary cells) were performed, and the p-value of the comparison with uninfected control is shown.

Article Snippet: The human cervical epithelial HeLa cell line was from ATCC (CCL-2).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Incubation, Immunostaining, Expressing, Immunofluorescence, Comparison, Control

Autocrine IFN signaling is not implicated in Chlamydia -induced ISG15 expression by epithelial cells. (A) Two clones of ISG15-KO HeLa cells and wild-type cells were infected with C. trachomatis (MOI = 1) for 42 h or incubated with IFNα (10 ng/mL) for 24 h. After treatment, the transcript levels of the ISGs including IFIT3 , RIG1 , ISG56, and MXA normalized to actin were determined and expressed relative to non-infected WT cells. ( B ) HeLa cells were infected with C. trachomatis (MOI = 1) for 42 h. The expression of ISGs were normalized to actin and are expressed relative to non-infected cells. ( C ) U5A epithelial cells with mutated IFNa/b receptor and the parental 2fTGH cells were infected by C. trachomatis at the indicated MOI, and the ISG15 transcript level normalized to actin was determined at 42 hpi. The data represent three independent experiments. Unpaired t test in A and one-way ANOVA test in B were performed. The p-values of the comparison with WT cells ( A ), Chlamydia -induced ISG15 ( B ), or as indicated are shown.

Journal: mBio

Article Title: Chlamydia -driven ISG15 expression dampens the immune response of epithelial cells independently of ISGylation

doi: 10.1128/mbio.02401-24

Figure Lengend Snippet: Autocrine IFN signaling is not implicated in Chlamydia -induced ISG15 expression by epithelial cells. (A) Two clones of ISG15-KO HeLa cells and wild-type cells were infected with C. trachomatis (MOI = 1) for 42 h or incubated with IFNα (10 ng/mL) for 24 h. After treatment, the transcript levels of the ISGs including IFIT3 , RIG1 , ISG56, and MXA normalized to actin were determined and expressed relative to non-infected WT cells. ( B ) HeLa cells were infected with C. trachomatis (MOI = 1) for 42 h. The expression of ISGs were normalized to actin and are expressed relative to non-infected cells. ( C ) U5A epithelial cells with mutated IFNa/b receptor and the parental 2fTGH cells were infected by C. trachomatis at the indicated MOI, and the ISG15 transcript level normalized to actin was determined at 42 hpi. The data represent three independent experiments. Unpaired t test in A and one-way ANOVA test in B were performed. The p-values of the comparison with WT cells ( A ), Chlamydia -induced ISG15 ( B ), or as indicated are shown.

Article Snippet: The human cervical epithelial HeLa cell line was from ATCC (CCL-2).

Techniques: Expressing, Clone Assay, Infection, Incubation, Comparison

The TBK1/Sting/IRF3 pathway lies upstream of ISG15 synthesis by epithelial cells in response to Chlamydia infection. ( A-B ) The indicated genes were silenced by siRNA (30 nM) for 48 h before Chlamydia infection. At 42 h post-infection, ISG15 expression was examined either by RT-qPCR ( A ) or by immunoblot ( B ). ( C ) HeLa cells were incubated with c-di-AMP at the indicated concentration for 24 h before quantification of the ISG15 levels in whole cell lysates by Western blot. ( D ) c-di-AMP (10 µM) was introduced into HeLa cells using transfection reagent at 37°C for the indicated times. The cells were then washed and were incubated for an additional 24 h before examining ISG15 level in whole cell lysates by Western blot. Incubation of HeLa cells with IFNα (10 ng/mL) for 24 h was used as positive control (C and D). ( E-F ) siRNA against cGAS or irrelevant oligonucleotides were transfected into HeLa cells for 48 h before Chlamydia infection. At 42 h post-infection, the expression of cGAS (E, left panel), ISG15 (E, middle and right panels), IL6 and IL8 ( F ), and actin were measured. All data represent three independent experiments. The p-values of unpaired t test with the condition “control siRNA” ( A ) or between the indicated conditions ( E, F ) are shown.

Journal: mBio

Article Title: Chlamydia -driven ISG15 expression dampens the immune response of epithelial cells independently of ISGylation

doi: 10.1128/mbio.02401-24

Figure Lengend Snippet: The TBK1/Sting/IRF3 pathway lies upstream of ISG15 synthesis by epithelial cells in response to Chlamydia infection. ( A-B ) The indicated genes were silenced by siRNA (30 nM) for 48 h before Chlamydia infection. At 42 h post-infection, ISG15 expression was examined either by RT-qPCR ( A ) or by immunoblot ( B ). ( C ) HeLa cells were incubated with c-di-AMP at the indicated concentration for 24 h before quantification of the ISG15 levels in whole cell lysates by Western blot. ( D ) c-di-AMP (10 µM) was introduced into HeLa cells using transfection reagent at 37°C for the indicated times. The cells were then washed and were incubated for an additional 24 h before examining ISG15 level in whole cell lysates by Western blot. Incubation of HeLa cells with IFNα (10 ng/mL) for 24 h was used as positive control (C and D). ( E-F ) siRNA against cGAS or irrelevant oligonucleotides were transfected into HeLa cells for 48 h before Chlamydia infection. At 42 h post-infection, the expression of cGAS (E, left panel), ISG15 (E, middle and right panels), IL6 and IL8 ( F ), and actin were measured. All data represent three independent experiments. The p-values of unpaired t test with the condition “control siRNA” ( A ) or between the indicated conditions ( E, F ) are shown.

Article Snippet: The human cervical epithelial HeLa cell line was from ATCC (CCL-2).

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Incubation, Concentration Assay, Transfection, Positive Control, Control

ISG15 regulates inflammation from an intracellular location and independently of ISGylation. ( A ) Culture medium was collected at different times post-infection for ISG15 detection by dot blot. First row: 0, 3, 6, and 9 hpi; second row: 12, 18, 24, and 30 hpi; third row: 36, 42, and 48 hpi; fourth row: 25, 50, 100, and 200 ng/mL of rhISG15. ( B ) ISG15-KO HeLa cells were pre-treated with the indicated concentration of rhISG15 for 30 min before Chlamydia infection (MOI = 1). The cells were further incubated for 42 h in the presence of rhISG15 before RNA extraction and quantification of IL6 and IL8 expression by RT-qPCR. ( C ) HeLa cells were incubated with C. trachomatis (MOI = 1) for 42 h or with IFN-α/β (10 ng/mL) for 24 h, lysed, and the amount of ISG15 (free form & conjugates) was analyzed by immunoblot on whole cell lysates. ( D ) Primary cervical epithelial cells were treated with IFNα (10 ng/mL) for 24 h or with C. trachomatis (MOI = 10) for 42 h followed by ISG15 detection. Top panels of C and D show overexposed images of the upper part of the membranes to visualize ISGylated proteins. ( E ) ISG15-KO cells (C2 and C4) or complemented cells (with ISG15 WT or ΔGG) were infected or not with C. trachomatis (MOI = 1) for 42 h before measuring the transcription of IL6 and IL8 . The panel on the left shows ISG15 expression in the complemented cells. The images displayed are representative of two independent experiments in A and of three independent experiments in D using primary cells from three individuals. All other data correspond to three independent experiments. The p-values of unpaired t test between the indicated groups are shown.

Journal: mBio

Article Title: Chlamydia -driven ISG15 expression dampens the immune response of epithelial cells independently of ISGylation

doi: 10.1128/mbio.02401-24

Figure Lengend Snippet: ISG15 regulates inflammation from an intracellular location and independently of ISGylation. ( A ) Culture medium was collected at different times post-infection for ISG15 detection by dot blot. First row: 0, 3, 6, and 9 hpi; second row: 12, 18, 24, and 30 hpi; third row: 36, 42, and 48 hpi; fourth row: 25, 50, 100, and 200 ng/mL of rhISG15. ( B ) ISG15-KO HeLa cells were pre-treated with the indicated concentration of rhISG15 for 30 min before Chlamydia infection (MOI = 1). The cells were further incubated for 42 h in the presence of rhISG15 before RNA extraction and quantification of IL6 and IL8 expression by RT-qPCR. ( C ) HeLa cells were incubated with C. trachomatis (MOI = 1) for 42 h or with IFN-α/β (10 ng/mL) for 24 h, lysed, and the amount of ISG15 (free form & conjugates) was analyzed by immunoblot on whole cell lysates. ( D ) Primary cervical epithelial cells were treated with IFNα (10 ng/mL) for 24 h or with C. trachomatis (MOI = 10) for 42 h followed by ISG15 detection. Top panels of C and D show overexposed images of the upper part of the membranes to visualize ISGylated proteins. ( E ) ISG15-KO cells (C2 and C4) or complemented cells (with ISG15 WT or ΔGG) were infected or not with C. trachomatis (MOI = 1) for 42 h before measuring the transcription of IL6 and IL8 . The panel on the left shows ISG15 expression in the complemented cells. The images displayed are representative of two independent experiments in A and of three independent experiments in D using primary cells from three individuals. All other data correspond to three independent experiments. The p-values of unpaired t test between the indicated groups are shown.

Article Snippet: The human cervical epithelial HeLa cell line was from ATCC (CCL-2).

Techniques: Infection, Dot Blot, Concentration Assay, Incubation, RNA Extraction, Expressing, Quantitative RT-PCR, Western Blot

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