Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Primer sequences used for molecular cloning

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Sequencing

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Primer sequences used for qRT-PCR

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Sequencing

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: NAT10 targets LY6E, which mediates ac4C modifications during SINV infection. ( A ) Validation of candidate genes from the RNAseq data using qRT-PCR in NAT10-KD cells infected with SINV. ( B ) Predicted ac4C modification sites for the LY6E pre-mRNA and mature mRNA. ( C ) IP of 293T cells transfected with the NAT10-Myc plasmid and anti-Myc antibody; enriched LY6E mRNA was analyzed using qRT-PCR; the interactions between the NAT10 and LY6E mRNA were also analyzed. ( D ) ( Upper panel ) Immunoblot of the NAT10 immunoprecipitate in panel C. ( Lower panel ) Agarose gel electrophoresis images of the LY6E amplified using qRT-PCR in panel C. ( E ) After incubating with the anti-ac4C antibody and normal rabbit IgG mixed with protein A/G beads at 4°C for 2 h, respectively, incubation was continued with the NAT10-KD Huh7 cell lysate for 2 h. The bound ac4C-modified RNA was eluted and analyzed using qRT-PCR. ( Left panel ) The ac4C-modified RNA was also analyzed using qRT-PCR. ( Right panel ) Agarose gel electrophoresis images of the LY6E amplified using qRT-PCR. Equal amounts of RNA fragments not subjected to immunoprecipitation were used as the input controls. ( F ) ( Upper panel ) Schematic of the 4xS1m aptamer. ( Lower panel ) WT or ac4C site mutated (C–T mut) LY6E mRNA tagged with 4xS1m aptamer was incubated with cell lysates overexpressing NAT10 and separated via streptavidin-conjugated beads. NAT10 in the cell lysate was pulled down, and the LY6E mRNA was detected using an immunoblot. Cells transfected with vectors were used as negative controls. ( G ) ( Upper panel ) Schematic diagram of the dual-luciferase reporter plasmid pmirGLO. ( Lower panel ) Luciferase activity in the NAT10-KD Huh7 ( I ) or A549 (ii) cells transfected with pmirGLO with the WT or ac4C-modifier-site-mutated (C–T mut) the 3′-UTR of the LY6E mRNA. Firefly luciferase activity was normalized to Renilla luciferase activity. ( H, I ) Stability of LY6E mRNA in NAT10-KD Huh7 ( H ) and A549 ( I ) cells after treatment with actinomycin D (5 µg/mL) was analyzed using qRT-PCR at different time points. ( J ) Ly6E mRNA levels were analyzed in Huh7 cells using qRT-PCR at different time points after 24 h of Remodelin treatment with actinomycin D. Blots were quantified with ImageJ software and normalized to control levels. Data are presented as the means ± SEM ( n = 3). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and NS, not significant (A, E, G, H, I, and J, two-way ANOVA with Bonferroni post-test; C, unpaired Student’s t -tests).

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Infection, Biomarker Discovery, Quantitative RT-PCR, Modification, Transfection, Plasmid Preparation, Western Blot, Agarose Gel Electrophoresis, Amplification, Incubation, Immunoprecipitation, Luciferase, Activity Assay, Software, Control

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Predicted ac4C modification sites in the LY6E mRNA determined using PACES ^a

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Modification, Sequencing

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Mutations in the predicted ac4C modification sites of the LY6E mRNA ^a , ^b

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Modification, Sequencing

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: SINV is positively affected by NAT10 as it regulates the stability of the LY6E mRNA. ( A ) qRT-PCR analysis of LY6E mRNA expression in LY6E-KD Huh7 cells. ( B ) qRT-PCR analysis of the SINV RNA expression levels in LY6E-KD Huh7 cells at 24 hpi (MOI = 1). ( C ) Immunoblot analysis of the SINV capsid protein expression in LY6E-KD Huh7 cells at 6, 12, and 24 hpi (MOI = 1). ( D ) Plaque formation assay using the SINV infectious virions obtained from the LY6E-KD Huh7 cell culture medium at 24 hpi (MOI = 1). ( E ) qRT-PCR analysis of the SINV RNA expression levels in LY6E-KD Huh7 cells ectopically expressing LY6E and infected with SINV, 24 hpi (MOI = 1). ( F ) Immunoblot analysis of the SINV capsid protein abundance described in panel ( E ). ( G ) Plaque formation assay using the SINV infectious virions obtained from the culture supernatant described in panel ( E ). ( H ) qRT-PCR analysis of the SINV RNA expression levels in NAT10-KD Huh7 cells ectopically expressing LY6E and infected with SINV, 24 hpi (MOI = 1). ( I ) Immunoblot analysis of the SINV capsid protein abundance as described in panel ( H ). ( J ) Plaque formation assay for the SINV infectious virions obtained from the culture supernatant described in panel ( H ). Blots were quantified with ImageJ software and normalized to control levels. Data are presented as the means ± SEM ( n = 3). * P ≤ 0.05 and *** P ≤ 0.001 (A, B, and D, unpaired Student’s t -tests; E, G, H, and J, one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Quantitative RT-PCR, Expressing, RNA Expression, Western Blot, Plaque Formation Assay, Cell Culture, Infection, Quantitative Proteomics, Software, Control

Journal: Journal of Virology

Article Title: N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA

doi: 10.1128/jvi.01350-23

Figure Lengend Snippet: Working model showing how the loss of NAT10 reduces alphavirus replication. Alphavirus (SINV) infection upregulates NAT10 in host cells and promotes NAT10-mediated ac4C acetylation of LY6E mRNA transcripts, increasing LY6E expression and enhancing alphavirus replication.

Article Snippet: Antibodies against NAT10 (cat. no. 13365-1-AP, 1:2,000, ProteinTech, Wuhan, China), LY6E (cat. no. A09496-2, 1:1,000, Boster, Wuhan, China), dsRNA/J2 (cat. no. 10010200, 1:400, Scicons, Szirak, Hungary), β-tubulin (cat. no. D198906, 1:5,000, Sangon Biotech, Shanghai, China), and ac4C (cat. no. ab25251125, 1:500, Abcam, Cambridge, USA) were also used.

Techniques: Infection, Expressing