Journal: Virulence

Article Title: Human cytomegalovirus UL23 inhibits immune cell migration and blocks antiviral immune cell responses by reducing the expression of chemokines CCL2 and CCL5

doi: 10.1080/21505594.2025.2500493

Figure Lengend Snippet: UL23 inhibits the expression of chemokines CCL2 and CCL5 during HCMV infection. (a) HFF cells (1×10 5 ) were infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 0.1,1,3) for 12 h before qPCR analysis. RT-PCR shows the expression levels of the UL23 mRNA. (b-c) HFF cells (1×10 5 ) were infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for different time points before qPCR and ELISA analysis. RT-PCR shows the expression of the UL23 mRNA. (d-e) UL23 stably expressed HFF cells (HFF-UL23) and control cells (HFF-PCDH) (1×10 5 ) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for 12 h before qPCR and ELISA analysis. RT-PCR shows the expression of the UL23 and IE1 mRNA. Each assay was conducted thrice. (f, g) HFF cells (1×10 5 ) infected with untreated or UV-inactivated HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for 12 h before qPCR and ELISA analysis. RT-PCR shows the expression of the UL23 and IE1 mRNA. (h) UL23 stably expressed HFF cells (HFF-UL23) and control cells (HFF-PCDH) (1×10 5 ) were infected with HSV-1 (MOI = 1), RSV (MOI = 1) for 12 h before RT-qPCR and ELISA analysis. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01 (unpaired, two-tailed Student’s t-test). Data are representative of three independent experiments with similar results.

Article Snippet: For the neutralizing experiment, the culture medium was harvested and incubated with anti-CCL2 Ab (MAB679-SP, R&D Systems), anti-CCL5 Ab (12000–1-AP, Proteintech) or control normal IgG (#3900S, Cell signalling) for 1 h to neutralize CCL2 and CCL5, then added to the conditioned media in the lower chamber.

Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Stable Transfection, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Virulence

Article Title: Human cytomegalovirus UL23 inhibits immune cell migration and blocks antiviral immune cell responses by reducing the expression of chemokines CCL2 and CCL5

doi: 10.1080/21505594.2025.2500493

Figure Lengend Snippet: UL23 down-regulates the expression of chemokines CCL2 and CCL5 by inhibiting IRF-3 phosphorylation. (a)HFF cells (1×10 5 ) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for the indicated times. Immunoblots were performed with the indicated antibodies. (b) UL23 stably expressed HFF cells (HFF-UL23) and control cells (HFF-PCDH) (1×10 5 ) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for 12 h. Immunoblots were performed with the indicated antibodies. (c) HFF cells (1×10 5 ) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for 12 h, and then subjected to subcellular fractionation. Cytoplasmic and nuclear extracts were examined by immunoblotting using the indicated antibodies. (d) HFF cells (1×10 4 ) were seeded in 96 wells plates and treated with B×795 at the indicated concentrations for 48 h. Cell viability was evaluated using CCK8 assay. (e) HFF cells (1×10 5 ) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 at an MOI of 1 in the absence or presence of B×795(2.5 μM). At 12 h, total cell lysates were prepared to immunoblot with indicated antibodies. (f) HFF cells (1×10 5 ) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 at an MOI of 1 in the absence or presence of B×795(2.5 μM). At 12 h, total cell supernatants were prepared to ELISA.

Article Snippet: For the neutralizing experiment, the culture medium was harvested and incubated with anti-CCL2 Ab (MAB679-SP, R&D Systems), anti-CCL5 Ab (12000–1-AP, Proteintech) or control normal IgG (#3900S, Cell signalling) for 1 h to neutralize CCL2 and CCL5, then added to the conditioned media in the lower chamber.

Techniques: Expressing, Phospho-proteomics, Infection, Western Blot, Stable Transfection, Control, Fractionation, CCK-8 Assay, Enzyme-linked Immunosorbent Assay

Journal: Virulence

Article Title: Human cytomegalovirus UL23 inhibits immune cell migration and blocks antiviral immune cell responses by reducing the expression of chemokines CCL2 and CCL5

doi: 10.1080/21505594.2025.2500493

Figure Lengend Snippet: UL23 inhibited immune cell migration by down-regulating the expression of chemokines CCL2 and CCL5. (a) Pattern diagram of migration. (b) Cell culture supernatants from the indicated times-incubated HCMV-Towne or HCMV-ΔUL23-infected (MOI = 1) or mock-infected HFF cells were used to perform a migration assay. THP-1/Jurkat/PBMC cells (1 × 10 5 ) were added into the upper chamber of the transwell plates and allowed to migrate for 12 h into the lower chamber. Subsequently, cell numbers in the lower chamber were counted and migration percentage were calculated as described in materials and methods. (c) THP-1/Jurkat/PBMC cells (1×10 5 ) migration assay by the supernatants from HFF-UL23 or HFF-PCDH cells were mock infected or infected with HCMV-ΔUL23 (MOI = 1) for 24 h. After migrating for 12 h into the lower chamber, the number of cells migrating into the lower chamber was counted. (d) THP-1/Jurkat/PBMC cells (1×10 5 ) migration assay by the supernatants from HFF cells were un-infected (mock) or infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) and treated without or with 2 μM B×795 for 12 h. After migrating for 12 h into the lower chamber, the number of cells migrating into the lower chamber was counted. (E)HFF cells (1×10 5 ) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for 24 h and the culture supernatants were harvested. The culture supernatant was preincubated with anti-CCL2 and anti-CCL5 antibodies or IgG isotype control antibodies at a concentration of 1 μg/mL at 37°C for 1 h. After incubation, the supernatant was added to the lower chamber of the transwell plate, and 1 × 10 5 THP-1/Jurkat/PBMC cells were added to the upper chamber. After migrating for 12 h into the lower chamber, the number of cells migrating into the lower chamber was counted.

Article Snippet: For the neutralizing experiment, the culture medium was harvested and incubated with anti-CCL2 Ab (MAB679-SP, R&D Systems), anti-CCL5 Ab (12000–1-AP, Proteintech) or control normal IgG (#3900S, Cell signalling) for 1 h to neutralize CCL2 and CCL5, then added to the conditioned media in the lower chamber.

Techniques: Migration, Expressing, Cell Culture, Incubation, Infection, Control, Concentration Assay

Journal: Virulence

Article Title: Human cytomegalovirus UL23 inhibits immune cell migration and blocks antiviral immune cell responses by reducing the expression of chemokines CCL2 and CCL5

doi: 10.1080/21505594.2025.2500493

Figure Lengend Snippet: UL23 resists the antiviral cellular immune responses. (a) Pattern diagram of migration-co-culture. (b) HFF cells (5×10 4 ) were seeded in the lower chamber of the transwell plates and infected with Towne or ΔUL23 (MOI = 1). THP-1/Jurkat/PBMC cells were added into the upper chamber of the transwell plates immediately upon removal of the virus. Activated Jurkat cells and PBMC cells were co-stimulated with PMA and Ionomycin (PMA concentration was 10 ng/mL and Ionomycin concentration was 500 ng/mL). Upper chambers were then removed after 24 h of migration. 4 days post coculture, the virus particles were then harvested for measurements of the viral titres with standard plaque assays. (c) HFF cells (5×10 4 ) were seeded in the lower chamber of the transwell plates and infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1). THP-1/Jurkat/PBMC cells were added into the upper chamber of the transwell plates immediately upon removal of the virus. Upper chambers were then removed after 24 h of migration. 4 days post coculture, RNA was harvested and IE1 gene expression was measured by qPCR. (d-e) HFF cells (5×10 4 ) were seeded in the lower chamber of the transwell plates and infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1). Then added anti-CCL2 and anti-CCL5 antibodies or IgG isotype control antibodies (1 μg/mL) into the culture medium. THP-1/Jurkat/PBMC cells were added into the upper chamber of the transwell plates. Upper chambers were then removed after 24 h of migration. 4 days post coculture, the virus particles were then harvested for measurements of the viral titres with standard plaque assays. RNA was harvested and IE1 gene expression were determined as described before.

Article Snippet: For the neutralizing experiment, the culture medium was harvested and incubated with anti-CCL2 Ab (MAB679-SP, R&D Systems), anti-CCL5 Ab (12000–1-AP, Proteintech) or control normal IgG (#3900S, Cell signalling) for 1 h to neutralize CCL2 and CCL5, then added to the conditioned media in the lower chamber.

Techniques: Migration, Co-Culture Assay, Infection, Virus, Concentration Assay, Gene Expression, Control

Journal: Virulence

Article Title: Human cytomegalovirus UL23 inhibits immune cell migration and blocks antiviral immune cell responses by reducing the expression of chemokines CCL2 and CCL5

doi: 10.1080/21505594.2025.2500493

Figure Lengend Snippet: The mode of HCMV UL23-mediated immune evasion. During the period of HCMV infection, UL23 decreases the expression of CCL 2 and CCL5 by restraining the IRF-3 phosphorylation and inhibits the migration of immune cells, thereby weakening the ability of the immune system to clear the virus.

Article Snippet: For the neutralizing experiment, the culture medium was harvested and incubated with anti-CCL2 Ab (MAB679-SP, R&D Systems), anti-CCL5 Ab (12000–1-AP, Proteintech) or control normal IgG (#3900S, Cell signalling) for 1 h to neutralize CCL2 and CCL5, then added to the conditioned media in the lower chamber.

Techniques: Infection, Expressing, Phospho-proteomics, Migration, Virus