Human Cytomegalovirus UL23 Antagonizes the Antiviral Effect of Interferon-γ by Restraining the Expression of Specific IFN-Stimulated Genes
<p>HCMV-encoded protein UL23 inhibits the expression of IFN-inducible genes Apol1, Cmpk2, and Lgals9. (<b>A</b>) UL23 stably expressed HFF cells (HFF-UL23) and control cells (HFF-Vec) were treated with 1000 U/mL IFN-γ for 24 h. Volcanic map showing expression of cellular targets of UL23 in HFF cells after stimulation by IFN-γ. (<b>B</b>) HFF cells infected with HCMV-Towne or HCMV-Towne-ΔUL23 (MOI = 1) for 24 h. Volcanic map showing the effects of UL23 on host gene expression after HCMV infection of HFF cells. (<b>C</b>) Boolean operation analysis showed that HCMV-Towne vs. HCMV-Towne-ΔUL23 differentially expressed genes and Vec-IFN-γ vs. UL23-IFN-γ differentially expressed genes were composed of APOL1, CMPK2, and LGALS9. (<b>D</b>) The effects of ectopic expression of UL23 on the gene expression of APOL1, CMPK2, and LGALS9 with or without IFN-γ (1000 U/mL) stimulation. (<b>E</b>) HCMV or HCMV-ΔUL23 (MOI = 1) was used to infect HFF cells for different time points. The APOL1, CMPK2, and LGALS9 mRNA expressions were measured through RT-qPCR, with GAPDH as the reference. (<b>F</b>) HFF-UL23 and HFF-Vector cells were treated with IFN-γ (1000 U/mL) for 24 h before Western blotting. (<b>G</b>) HCMV-Towne or HCMV-Towne-ΔUL23 (MOI = 1) was used to infect HFF cells for different time points, and total cell lysates were analyzed by immunoblot using the indicated antibodies. Each assay was conducted thrice. Data are shown as mean ± SD. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, NS denotes not significant (<span class="html-italic">p</span> > 0.05). Data are representative of three independent experiments with similar results.</p> "> Figure 2
<p>IFN-inducible proteins APOL1, CMPK2, and LGALS9 inhibit HCMV replication. (<b>A</b>) HFF cells were transfected with the expression constructs of LUC or APOL1, CMPK2, and LGALS9, and protein levels were analyzed by immunoblot. (<b>B</b>) Control and three protein-overexpressing (APOL1, CMPK2, and LGALS9) HFF cells were infected with HCMV-Towne at an MOI of 1. Total RNAs were prepared at 24 h, and the level of IE1 transcripts was measured by RT-qPCR. (<b>C</b>) Cell culture supernatants from cells infected with HCMV-Towne at an MOI of 1 were analyzed at different time points for progeny virus titers using infectious center assays in HFF cells. (<b>D</b>) HFF cells were infected with HCMV-Towne harboring GFP as a reporter. Cells infected at an MOI of 1 were monitored for the spread of the GFP signal. GFP images merged with phase-contrast images were collected at 7 days post-infection. Scale bar, 50 μm. Data are shown as mean ± SD. ** <span class="html-italic">p</span> < 0.01. Data are representative of three independent experiments with similar results.</p> "> Figure 3
<p>A combination of APOL1, CMPK2, and LGALS9 exhibits synergistic anti-HCMV effects. (<b>A</b>) RT-qPCR detection of the synergistic effects of APOL1, CMPK2, and LGALS9 on the expression of the HCMV-Towne IE1 gene (24 h). (<b>B</b>) Virus titer determination APOL1, CMPK2, and LGALS9 synergistic effects on HCMV-Towne replication (7 d). Data are shown as mean ± SD. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, NS denotes not significant (<span class="html-italic">p</span> > 0.05). Data are representative of three independent experiments with similar results.</p> "> Figure 4
<p>UL23 resisted the antiviral effect of IFN-γ by downregulating the expression of APOL1, CMPK2, and LGALS9. (<b>A</b>,<b>B</b>) The effect of HCMV UL23 on the gene expression of APOL1, CMPK2, and LGALS9 with or without IFN-γ (1000 U/mL) stimulation during the HCMV infection (before HCMV infection, HFF cells were pretreated with IFN-γ for 12 h). Total RNAs were prepared at 24 h, the level of genes was measured by RT-qPCR, and total cell lysates were prepared and analyzed by immunoblotting. (<b>C</b>) HFF cells were infected with HCMV-Towne or HCMV-Towne-ΔUL23 at an MOI of 1 and treated with or without 1000 U/mL IFN-γ for 24 h (before HCMV infection, HFF cells were pretreated with IFN-γ for 12 h). Total RNAs were prepared at 24 h, and the level of IE1 transcripts was measured by RT-qPCR. (<b>D</b>) Cell culture supernatants from cells infected with HCMV-Towne or HCMV-Towne-ΔUL23 at an MOI of 1 were analyzed 7 days post-infection for progeny virus titers using infectious center assays in HFF cells. (<b>E</b>) HFF cells were stably transduced to the target gene with lentiviruses containing non-target control shRNA (NT) or shRNAs (APOL1, CMPK2, and LGALS9). (<b>F</b>) Control and stable knockdown HFF cells (APOL1, CMPK2, and LGALS9) were infected with HCMV-Towne at an MOI of 1 and treated with or without 1000 U/mL IFN-γ for 24 h (before HCMV infection, HFF cells were pretreated with IFN-γ for 12 h). Total RNAs were prepared at 24 h, and the level of IE1 transcripts was measured by RT-qPCR. (<b>G</b>) Cell culture supernatants from cells infected with HCMV-Towne at an MOI of 1 were analyzed 7 days post-infection for progeny virus titers using infectious center assays in HFF cells. Data are shown as mean ± SD. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.005, NS denotes not significant (<span class="html-italic">p</span> > 0.05). Data are representative of three independent experiments with similar results.</p> "> Figure 5
<p>A model for the HCMV UL23-mediated suppression of IFN-γ-mediated ISG transcription (APOL1, CMPK2, and LGALS9). During HCMV infection, UL23 disrupts the phosphorylation and nuclear localization of STAT1 and reduces ISG transcription and GAS promoter activation; in the meanwhile, it restrains the inhibitory effects of APOL1, CMPK2, and LGALS9 on HCMV replication.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Virus and Cell Culture
2.2. Reagents and Antibodies
2.3. RNA-Seq and Data Analysis
2.4. Virus Infection and Growth Analysis
2.5. RNA or DNA Isolation and Quantitative Reverse Transcription-PCR (RT-qPCR)
2.6. Western Blotting Assay
2.7. RNA Interference
2.8. Stable Cell Lines
2.9. Statistical Analysis
3. Results
3.1. RNA-Seq Analysis Revealed That HCMV-Encoded UL23 Protein Inhibited the Expression of IFN-Inducible Genes APOL1, CMPK2, and LGALS9
3.2. IFN-γ-Inducible Genes APOL1, CMPK2, and LGALS9 Inhibit HCMV Replication
3.3. The Combination of APOL1, CMPK2, and LGALS9 Exhibits Synergistic Anti-HCMV Effects
3.4. UL23 Limited the Antiviral Effect of IFN-γ by Downregulating the Expression of APOL1, CMPK2, and LGALS9
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
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Wang, H.; Peng, W.; Wang, J.; Zhang, C.; Zhao, W.; Ran, Y.; Yang, X.; Chen, J.; Li, H. Human Cytomegalovirus UL23 Antagonizes the Antiviral Effect of Interferon-γ by Restraining the Expression of Specific IFN-Stimulated Genes. Viruses 2023, 15, 1014. https://doi.org/10.3390/v15041014
Wang H, Peng W, Wang J, Zhang C, Zhao W, Ran Y, Yang X, Chen J, Li H. Human Cytomegalovirus UL23 Antagonizes the Antiviral Effect of Interferon-γ by Restraining the Expression of Specific IFN-Stimulated Genes. Viruses. 2023; 15(4):1014. https://doi.org/10.3390/v15041014
Chicago/Turabian StyleWang, Hankun, Weijian Peng, Jialin Wang, Chunling Zhang, Wangchun Zhao, Yanhong Ran, Xiaoping Yang, Jun Chen, and Hongjian Li. 2023. "Human Cytomegalovirus UL23 Antagonizes the Antiviral Effect of Interferon-γ by Restraining the Expression of Specific IFN-Stimulated Genes" Viruses 15, no. 4: 1014. https://doi.org/10.3390/v15041014