Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A –D Validation of UHRF1 as a restriction factor across coronavirus genera. Control and UHRF1 -knockout A549-ACE2 cells were infected with alphacoronaviruses (HCoV-NL63, MOI 1, 24 h; SADS-CoV, MOI 1, 24 h) and betacoronavirus (SARS-CoV-2, MOI 0.1, 24 h). UHRF1 -knockout HeLa cells were challenged with betacoronavirus (HCoV-OC43, MOI 1, 24 h), gammacoronavirus (IBV, MOI 1, 24 h), and deltacoronavirus (PDCoV, MOI 0.3, 24 h). Infection efficiency was analyzed by flow cytometry for the percentage of N-positive cells. E –I Proviral effect of UHRF1 on unrelated RNA viruses. UHRF1 -knockout A549 cells were infected with ZIKV (MOI 1, 24 h), SINV (MOI 3, 24 h), VSV (MOI 1, 15 h), H1N1 (MOI 1, 24 h), and EMCV (MOI 0.1, 10 h). Infection efficiency was determined by flow cytometry for the percentage of viral-positive cells. Error bars represent standard deviations from three independent experiments ( n = 3), and each was performed in duplicate. Unpaired, two-sided t-test; mean ± s.d.; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns not significant.

Article Snippet: Control and UHRF1 -knockout A549 cells were detached with TrypLE and incubated with primary antibodies against APN (Invitrogen #14-0138-82, 1 μg/ml), heparan sulfate (10E4) (USBiological #H1890, 1 μg/ml), ACE2 (Sino Biological #10108-RP01, 1:250), DC-SIGN (Biolegend #330102, 1 μg/ml), or TIM-1 (Biolegend #354002, 1 μg/ml) at 4 °C for 25 minutes.

Techniques: Biomarker Discovery, Control, Knock-Out, Infection, Flow Cytometry

Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A –C Pseudovirus infection assays. Control and UHRF1 -knockout A549-ACE2 cells were infected with VSV-based pseudoviruses. D –F Virus binding and internalization assays. Cells were incubated with HCoV-229E (MOI 10). Bound or internalized virions were quantified by qRT-PCR or analyzed by confocal microscopy. Representative images from three independent experiments were shown. G , H Western blotting analysis of gene expression in gene-knockout A549 or HeLa cells. Representative images from three independent experiments were shown. I Infection efficiency of HCoV-229E (MOI 0.5, 24 h) in control and UHRF1 -knockout HeLa cells, determined by flow cytometry. J . Relative APN mRNA levels and infection efficiency of HCoV-229E (MOI 1, 12 h) in control and UHRF1 -knockout primary human bronchial epithelial cells (HBEC). mRNA levels were analyzed by qRT-PCR, and infectivity was determined by flow cytometry. K Temporal expression of APN in UHRF1 -knockout A549 cells. Cell lysates were collected at different days post-transduction of sgRNA-expressing lentivirus and analyzed by western blotting. Representative images from three independent experiments were shown. L Surface expression of APN analyzed by flow cytometry in A549 cells edited with control or UHRF1 sgRNA. M Relative APN mRNA levels analyzed by qRT-PCR in A549 cells edited with control or UHRF1 sgRNA. N Relative APN mRNA levels analyzed by qRT-PCR in A549 cells treated with UHRF1 inhibitor UF146 for 2 days. O Control and two APN -knockout A549 clonal cell lines were edited with control or UHRF1 sgRNA, and infected with HCoV-229E (MOI 0.5, 24 h). Infectivity was determined by flow cytometry. P UHRF1 -knockout cells were pre-treated with 5 μg/ml APN-blocking antibody or isotype control for 1 h, then infected with HCoV-229E (MOI 0.5, 24 h) in the presence of antibody. qRT-PCR was performed to determine the relative levels of HCoV-229E N gene. Error bars represent standard deviations from three independent experiments ( n = 3), and each performed in duplicate. Unpaired, two-sided t-test ( A – C , I , J , M ); two-way ANOVA with Sidak’s test ( D, O ); one-way ANOVA with Sidak’s test ( N , P ); mean ± s.d.; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Control and UHRF1 -knockout A549 cells were detached with TrypLE and incubated with primary antibodies against APN (Invitrogen #14-0138-82, 1 μg/ml), heparan sulfate (10E4) (USBiological #H1890, 1 μg/ml), ACE2 (Sino Biological #10108-RP01, 1:250), DC-SIGN (Biolegend #330102, 1 μg/ml), or TIM-1 (Biolegend #354002, 1 μg/ml) at 4 °C for 25 minutes.

Techniques: Infection, Control, Knock-Out, Virus, Binding Assay, Incubation, Quantitative RT-PCR, Confocal Microscopy, Western Blot, Gene Expression, Gene Knockout, Flow Cytometry, Expressing, Transduction, Blocking Assay

Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A UHRF1 does not regulate the expression of exogenous HA-tagged APN and ACE2 from transiently transfected plasmids. Representative Western blotting images from three independent experiments were shown. B Bisulfite sequencing of CpG sites in APN proximal promoter from control and UHRF1 -knockout A549 cells. The methylation (Meth) rate was calculated as the ratio of methylated sites to the total number of sites tested. C A549 cells were treated with 5-AZA for 3 days, and relative APN mRNA levels were determined by qRT-PCR. D , E Huh7 stably expressing DNMT3A were generated by lentivirus transduction. Relative APN mRNA levels were determined by qRT-PCR ( D ), and infectivity was detected by flow cytometry after infection with HCoV-229E (MOI 0.01, 24 h) ( E ). F , G In vitro methylation and dual-luciferase reporter assays. The methylation status of luciferase reporter plasmid was verified by HpaII/MspI digestion ( F ). Representative image from three independent experiments was shown ( F ). The luciferase activity of unmethylated (Unmeth) or methylated (Meth) luciferase reporter plasmid co-transfected with internal control pRL-TK was measured at 24 h post-transfection. Results were normalized to the unmethylated plasmid ( G ). H Electrophoretic mobility shift assay (EMSA). Nuclear extracts were incubated with biotin-labeled unmethylated or methylated APN promoter probe to detect DNA-protein complexes. Representative images from three independent experiments were shown. I , J Chromatin immunoprecipitation (ChIP) assay with c-Maf expression. qPCR was performed to detect c-Maf binding to the transcription factor (TF) binding site ( I ) or CpG island ( J ) of the APN proximal promoter. K Schematic diagram of UHRF1 truncations. L , M UHRF1 -knockout A549 stably expressing wild-type or truncated UHRF1 were established by lentivirus transduction and verified by western blotting ( L ). Representative images from three independent experiments were shown ( L ). Cells were infected with HCoV-229E (MOI 0.5, 24 h) at day 10 post-transduction, and the infectivity was determined by flow cytometry ( M ). Error bars represent standard deviations from three independent experiments ( n = 3), and each performed in duplicate. One-way ANOVA with Sidak’s test ( C , M ); unpaired, two-sided t-test ( D , E , G , I – J ); mean ± s.d.; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Control and UHRF1 -knockout A549 cells were detached with TrypLE and incubated with primary antibodies against APN (Invitrogen #14-0138-82, 1 μg/ml), heparan sulfate (10E4) (USBiological #H1890, 1 μg/ml), ACE2 (Sino Biological #10108-RP01, 1:250), DC-SIGN (Biolegend #330102, 1 μg/ml), or TIM-1 (Biolegend #354002, 1 μg/ml) at 4 °C for 25 minutes.

Techniques: Expressing, Transfection, Western Blot, Methylation Sequencing, Control, Knock-Out, Methylation, Quantitative RT-PCR, Stable Transfection, Generated, Transduction, Infection, Flow Cytometry, In Vitro, Luciferase, Plasmid Preparation, Activity Assay, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Chromatin Immunoprecipitation, Binding Assay

Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A Volcano plot of RNA-seq analysis. Total cellular RNA was extracted from control and UHRF1 -knockout A549-ACE2 cells and subjected to RNA-seq. Genes with an absolute Log 2 fold change >2 and adjusted P -value < 0.05 were considered as differentially expressed. Differential expression analysis was performed using DESeq2 with a two-sided Wald test. P -values were adjusted for multiple comparisons using the Benjamini-Hochberg method. B Schematic of focused CRISPR activation screening. A sub-library targeting 2172 of the 2210 upregulated genes identified from RNA-seq analysis of UHRF1 -knockout cells, with ~4 sgRNAs per gene, was generated and transduced into A549-ACE2-dCas9 cells. Cells were infected with HCoV-229E-mGreen (MOI 0.5, 24 h) or SARS-CoV-2 transcription- and replication-competent virus-like particles in which the N gene is replaced by the reporter GFP (trVLP-GFP) (MOI 0.5, 24 h). Infected reporter-positive cells were sorted for genomic DNA extraction and sgRNA sequence analysis. Created in BioRender. Wang, P. (2025) https://BioRender.com/35yt09k . C , D Genes identified from CRISPR screens for HCoV-229E ( C ) and SARS-CoV-2 ( D ). Genes were analyzed by MAGeCK software and sorted based on -log 10 (MAGeCK score) and P -values. The algorithm employs a one-sided test to identify genes under positive selection, and P -values were adjusted for multiple testing using the Benjamini-Hochberg method.

Article Snippet: Control and UHRF1 -knockout A549 cells were detached with TrypLE and incubated with primary antibodies against APN (Invitrogen #14-0138-82, 1 μg/ml), heparan sulfate (10E4) (USBiological #H1890, 1 μg/ml), ACE2 (Sino Biological #10108-RP01, 1:250), DC-SIGN (Biolegend #330102, 1 μg/ml), or TIM-1 (Biolegend #354002, 1 μg/ml) at 4 °C for 25 minutes.

Techniques: RNA Sequencing, Control, Knock-Out, Quantitative Proteomics, CRISPR, Activation Assay, Generated, Infection, Virus, DNA Extraction, Sequencing, Software, Selection

Journal: Science Advances

Article Title: Epitranscriptomic m 5 C methylation of SARS-CoV-2 RNA regulates viral replication and the virulence of progeny viruses in the new infection

doi: 10.1126/sciadv.adn9519

Figure Lengend Snippet: ( A ) Timeline of Ad5-hACE2–mediated SARS-CoV-2 WT strain infection mice model ( Nsun2 +/+ mice and Nsun2 +/− mice). ( B ) Body weight changes of SARS-CoV-2 WT strain–infected Nsun2 +/+ mice ( n = 5) and Nsun2 +/− mice ( n = 5). ( C ) qPCR analysis of RNA levels of N and E genes in lungs from Ad5-hACE2–mediated SARS-CoV-2 WT strain–infected Nsun2 +/+ mice ( n = 5) or Nsun2 +/− mice ( n = 5). ( D ) The hematoxylin and eosin (H&E) stain of lungs of uninfected mice group and Ad5-hACE2–mediated SARS-CoV-2 WT strain–infected mice group ( Nsun2 +/+ mice and Nsun2 +/− mice). ( E ) Immunofluorescence analysis of Ad5-hACE2–mediated SARS-CoV-2 WT strain–infected mice group ( Nsun2 +/+ mice and Nsun2 +/− mice). SARS-CoV-2 N (green). ACE2 (red). 4′,6-diamidino-2-phenylindole (DAPI, blue). ( F ) Timeline of SARS-CoV-2 Beta variant (B.1.351) infection mice model ( Ifnar −/− Nsun2 +/+ mice and Ifnar −/− Nsun2 +/− mice). ( G ) Body weight changes of SARS-CoV-2 Beta variant (B.1.351)–infected Ifnar −/− Nsun2 +/+ mice ( n = 5) and Ifnar −/− Nsun2 +/− mice ( n = 5). ( H ) qPCR analysis of RNA levels of N and E genes in lungs from SARS-CoV-2 Beta variant (B.1.351)–infected Ifnar −/− Nsun2 +/+ mice ( n = 5) and Ifnar −/− Nsun2 +/− mice ( n = 5). ( I ) The H&E stain of lungs of SARS-CoV-2 Beta variant (B.1.351)–infected Ifnar −/− Nsun2 +/+ mice and Ifnar −/− Nsun2 +/− mice. ( J ) Immunofluorescence analysis of lungs of SARS-CoV-2 Beta variant (B.1.351)–infected Ifnar −/− Nsun2 +/+ mice and Ifnar −/− Nsun2 +/− mice. SARS-CoV-2 N (red). DAPI (blue). ** P < 0.01, *** P < 0.001.

Article Snippet: The antibodies used were as follows: rabbit anti-NSUN2 (Proteintech, 20854-1-AP), rabbit anti-METTL3 (Proteintech, 15073-1-AP), mouse anti–SARS-CoV-2 N (Sino Biological, 40143-MM05), rabbit anti-human ACE2 (Sino Biological, 10108-RP01), and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Proteintech, 60004-1-Ig).

Techniques: Infection, Staining, Immunofluorescence, Variant Assay