N4-acetylcytidine regulates the replication and pathogenicity of enterovirus 71

Figures

• 

  Figure 1.. EV71 contained the ac4C modification and altered the expression pattern of NAT10. (A) UPLC-MS/MS of the EV71 RNA genome. EV71 RNA was extracted from viral supernatants concentrated by ultracentrifugation. The percentage of ac4C among C residues is presented on the y-axis. (B) Anti-ac4C dot blot of EV71 RNA from (A). Methylene blue staining was used as a loading control. The in vitro T7-transcribed EV71 genome and total RNA from EV71-infected Vero cells served as negative and positive controls, respectively. (C) acRIP-qRT-PCR. RNAs from EV71-infected cells, EV71 virions (A), and transcribed by T7 polymerase were incubated with IgG or anti-ac4C antibodies, followed by IP and qRT-PCR. Data are means ± SEMs (n = 3). **P ≤ 0.01, ns: not significant, unpaired Student's t-tests. (D) Mapping of ac4C sites on EV71 RNA by acRIP-Seq. Poly(A)+ RNA, purified from total RNA extracted from EV71-infected Vero cells, was fragmented, and IP was performed using anti-ac4C antibodies, followed by next-generation sequencing. Normalized coverage on acRIP-Seq and input EV71 RNA are presented in red and blue, respectively. The RNA secondary structure was predicted using Mfold software. The red line in…

• 

  Figure 2.. ac4C modifications promoted EV71 replication. (A, C) Viral titers (TCID50/ml). The supernatants of EV71-infected Vero cells with NAT10 knockdown (A) or inhibition by remodelin (C) were collected at the indicated time points postinfection, and EV71 titers were measured as the TCID50. Data are means ± SDs (n = 3). *P ≤ 0.05, **P ≤ 0.01, unpaired Student's t-tests. (B) qRT-PCR was performed to measure EV71 RNA levels in Vero cells in which NAT10 was knocked down at the indicated times postinfection. GAPDH was used as a control. Data are means ± SEMs (n = 3). *P ≤ 0.05, **P ≤ 0.01, unpaired Student's t-tests. (D) Schematic showing the location of ac4C sites in EV71 WT and mutants. The RNA secondary structure was predicted using Mfold software. Yellow solid circles indicate ac4C modification. (E) ac4C levels in EV71 WT and mutants were determined using acRIP-qRT-PCR. EV71 virion RNA was extracted from the supernatants of EV71-infected Vero cells and incubated with anti-ac4C antibodies, followed by IP and qRT-PCR. Data are means ± SEMs (n = 3). **P ≤ 0.01, unpaired Student's t-tests. (F) Growth curves of EV71 WT and ac4C mutants. The supernatants of EV71 WT- or ac4C mutant-infected…

• 

  Figure 3.. ac4C enhanced the translation efficiency of EV71 RNA. (A, B) Western blot analysis of extracts of NAT10-knockdown RD cells infected with EV71 at 12 and 24 hpi (A) or Vero cells infected with EV71 WT and ac4C mutants at 24 hpi (B). GAPDH was used as a loading control. (C, D) Vero cells with or without NAT10 knockdown were infected with EV71 and used to analyze input and ribosome-loaded RNA levels for EV71 (C) and GAPDH (D) at 24 hpi. Data are means ± SEMs (n = 3). **P ≤ 0.01, ns: not significant, unpaired Student's t-tests. (E) Schematic representation of the eGFP reporter vector with insertion of the 5′ UTR of EV71 WT or ac4C mutants. (F) Representative western blots of extracts from RD cells transfected with eGFP reporter vectors (E) at the indicated times post-transfection. β-Actin was used as a loading control. (G) Schematic representation of the in vitro translation template. Yellow solid circles indicate ac4C modification. (H) In vitro translation assays were performed using ac4C(±) RNA template. Gray intensity was quantified using ImageJ. Data are means ± SDs (n = 3). (I–K) Binding of PCBP2, HNRNPK, and FBP2 to RNA from EV71 WT or ac4C mutants. EV71 WT- or ac4C…

• 

  Figure 4.. ac4C enhanced the stability of EV71 RNA. (A) Schematic showing that EV71 3D mutant genomes with different ac4C modification levels were produced by T7 in vitro transcription. Yellow solid circles indicate the ac4C modification. (B–D) EV71 ac4C mutants decreased the stability of EV71 RNA. T7 transcribed WT- and ac4C mutant-3Dmut viral genomes were transfected into Vero cells. qRT-PCR was performed to determine the RNA levels of WT or ac4C mutant 3D-mut viral genomes in Vero cells treated with DMSO (C), remodelin (D), or nothing (B) at the indicated times post-transfection (0 h represents 4 h after transfection). GAPDH was used as a control. Decay graphs were generated by applying the linear regression analysis. Data are means ± SEMs (n = 3). *P ≤ 0.05, **P ≤ 0.01, ns: not significant, two-way ANOVA. (E, F) qRT-PCR was performed to measure the RNA levels of WT-3Dmut (E) or ac4C mutant-3Dmut (F) viral genomes with (ac4C[+]) or without ac4C modification in Vero cells at the indicated times post-transfection (0 h represents 4 h after transfection). GAPDH was used as a control. Data are means ± SEMs (n = 3). **P ≤ 0.01, two-way ANOVA. (G, H) EV71 RNA translation efficiency affected RNA…

• 

  Figure 5.. ac4C promoted the binding of EV71 RNA to 3D. (A, D) Binding of 3D to EV71 RNA following NAT10 inhibition using remodelin. Vero cells overexpressing Flag-3D were treated with DMSO or remodelin and infected with EV71 (A) or EV71 WT and ac4C mutants (D). Crosslinking with formaldehyde and IP using anti-Flag antibodies were performed, followed by quantification using qPCR. Data are means ± SEMs (n = 3). *P ≤ 0.05, **P ≤ 0.01, ns: not significant, unpaired Student's t-test. (B, C) Binding of 3D to EV71 RNA with different ac4C modification levels. Vero cells with Flag-3D overexpression were transfected with EV71 genomes with (ac4CTP/CTP = 1) or without (CTP) ac4C (B) or infected with EV71 WT and ac4C mutants (C). The same treatment as used in (A) and (D). Data are means ± SEMs (n = 3). *P ≤ 0.05, **P ≤ 0.01, unpaired Student's t-test. (E) Schematic of in vitro RNA binding assays near position 331. Yellow solid circles indicate the ac4C modification. (F–K) Binding of 3D and 331 or 350 primer-EV71 RNA pairs in vitro. EV71 RNAs extracted from T7 transcripts (ac4C[±]) or virions were annealed with 331- or 350–1/2/3 primers. His-3D was added, and samples were subjected to IP with anti-His…

• 

  Figure 6.. ac4C mutant virus exhibited reduced pathogenicity in mice. (A) Survival of mice challenged with EV71 WT or ac4C mutant (i.p., 105 PFU). Control mice received DMEM. Seven mice were used per group. *P ≤ 0.05, **P ≤ 0.01, Log-rank (Mantel-Cox) tests. (B) Daily weights of mice from (A). **P ≤ 0.01, two-way ANOVA. (C) Symptoms of mice challenged with EV71 WT or ac4C mutant on day 3 postinfection (i.p., 105 PFU). Symptoms included reduced motility and hind limb paralysis. (D–F) Viral RNA in the limb muscles (D), intestines (E), and brain (F) of AG6 mice infected with EV71, as quantified by qRT-PCR. Data are means ± SEMs (n = 6). *P ≤ 0.05, **P ≤ 0.01, ns: not significant, unpaired Student's t-tests. (G, H) Hematoxylin and eosin staining of the limb muscles (48 and 72 hpi) (G), brain, and intestines (72 hpi) (H). Scale bars, 100 μm. (I) Detection of EV71 particles in the limb muscles, intestines, and brain (72 hpi) using immunohistochemistry. Scale bars, 100 μm.