SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

Figures

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  Fig. 1.. EV71 enhances SIRT1 expression and translocation. (A) A diagram of the EV71 genome structure. EV71 genome contains a single ORF flanked by a 5′UTR and a 3′UTR. The ORF encodes a 250-kDa polyprotein that is processed into P1, P2 and P3 regions, which are further cleaved into mature proteins (VP1 to VP4, 2A to 2C, and 3A to 3Dpol). (B) RD cells were infected with EV71 at a multiplicity of infection (MOI) of 5 for different times. Photographs of infected cells were taken using a digital camera (at 100× magnification). (C–E) RD cells were infected with EV71 at an MOI of 5 for different times (C). RD cells were infected with EV71 for 12 h at different MOI (D). SK-N-SH A372 cells were infected with EV71 at an MOI of 5 for different times (E). The relative amount of SIRT1 and VP1 mRNAs were determined by qRT-PCR (upper panels). SIRT1 and VP1 proteins were detected by western blot analyses using corresponding antibodies (lower panels). (F,G) RD cells (F) and SK-N-SH A372 cells (G) were infected with EV71 at an MOI of 5 for 6 h. Cytoplasm extracts (CE) and nuclear extracts (NE) were prepared. SIRT1, β-actin and lamin A were detected by western blot analyses using corresponding antibodies.…

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  Fig. 2.. SIRT1 is sumoylated with Sumo1 and EV71 facilitates SIRT1 sumoylation. (A) RD cells were harvested and incubated in RIPA buffer for 20 min. Cell lysates were centrifuged at 20,000 g for 15 min to remove cellular debris. IgG, anti-Sumo1 antibody, anti-Sumo2+3 antibody (i.e. an antibody that could recognize both Sumo-2 and Sumo-3) or protein G (IP) was added to supernatants for immunoprecipitation (IP). Sumoylated SIRT1 (Sumo-SIRT1) and SIRT1 were detected with an anti-SIRT1 antibody (IB). (B) RD cells were co-transfected with pcDNA3.1-SIRT1 and plasmid expressing HA–Sumo1, HA–Sumo2 or HA–Sumo3. Cell lysates were prepared and immunoprecipitated with anti-HA antibody. Precipitated sumoylated SIRT1 and SIRT1 were detected with an anti-SIRT1 antibody. (C) RD cells were infected with EV71 for different times. Infected cell lysates were prepared for western blotting using anti-SIRT1 or anti-VP1 antibodies. (D) RD cells were infected with EV71 for different times. Cell lysates were prepared and immunoprecipitated with anti-SIRT1 antibody, and Sumo–SIRT1 was detected with anti-Sumo1 antibody. (E) RD cells were infected with EV71 for different times. Whole-cell lysates (WCL) were prepared…

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  Fig. 3.. SIRT1 inhibits EV71 replication in cytoplasm of infected cells. (A) RD cells were transfected with pcDNA3.1(+)-SIRT1 at 0, 0.5, 1, 1.5 and 2 µg for 24 h and infected with EV71 at an MOI of 5 for 12 h. Cell lysates were prepared. SIRT1, EV71 VP1 and β-actin were detected by western blot analyses with corresponding antibodies. (B) RD cells were transfected with siR-Ctrl, siR-SIRT1#1 or siR-SIRT1#2, for 24 h, and infected with EV71 at an MOI of 5 for 12 h. SIRT1, VP1 and β-actin in cell lysates were detected by western blot analysis. (C) 293T and RD cells were transfected with plasmids expressing wild-type SIRT1 (WT-SIRT1) and SIRT1 with a mutant NLS (mtNLS-SIRT1). The cells were fixed, permeabilized and immunostained with antibody against SIRT1 (a,d,g,j), with Cy3-conjugated goat anti-rabbit-IgG used as a secondary antibody. The nucleus was stained with DAPI (b,e,h,k). The immunofluorescence results were analyzed by confocal laser-scanning microscopy. (D) RD cells were transfected with plasmids expressing mtNLS-SIRT1 at 0, 0.5, 1 and 1.5 µg for 24 h, and infected with EV71 infected at an MOI of 5 for 12 h. VP1 and β-actin in cell lysates were detected by western blot analysis. (E)…

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  Fig. 4.. SIRT1 interacts with EV71 3Dpol both in vivo and in vitro. (A,B) 293T cells were co-transfected with pcDNA3.1(+)-SIRT1 and plasmids pGFP-C1, pGFP-2A, pGFP-2B, pGFP-3AB, pGFP-3C or pGFP-3D (A) or pGFP-3D (B). Cells extracts were prepared for co-immunoprecipitation (IP) using anti-GFP antibody and precipitated with protein G. Interactions between SIRT1 and viral proteins were detected by western blot analyses (IB) using anti-SIRT1 antibody or anti-GFP antibody. (C) 293T cells were co-transfected with pGFP-3D and pcDNA3.1(+)-SIRT1. Cell extracts were prepared for co-immunoprecipitation using anti-SIRT1 antibody and precipitated with protein G. The interaction between SIRT1 and GFP–3Dpol was detected by western blot analyses using anti-GFP antibody or anti-SIRT1 antibody. (D) 293T cells were transfected with pcDNA3.1(+)-SIRT1 for 48 h. Cell lysates were prepared, to which purified GST or GST–3Dpol were added, and were then purified by using a glutathione–Sepharose column and GST-binding buffer. Proteins were pulled down with anti-GST antibody, and interactions between SIRT1 and GST–3Dpol were detected by western blotting with anti-SIRT1 antibody or anti-GST antibody. (E) GST–3Dpol…

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  Fig. 5.. SIRT1 inhibits EV71 genome RNA replication by interacting with 3Dpol. (A) RD cells were infected with EV71 or not (Mock) at an MOI of 5 for 8 h or 12 h, fixed, permeabilized, and immunostained with antibody against 3Dpol (a,e,i) or SIRT1 (b,f,j). Nuclei were stained by DAPI (c,g,k). FITC-conjugated donkey anti-mouse-IgG or Cy3-conjugated goat anti-rabbit-IgG was used as a secondary antibody. Immunofluorescence was detected by confocal laser-scanning microscopy. (B) Schematic diagram of the truncated EV71 3Dpol constructs used in this study, with amino acid numbers indicated. DNA fragments containing mutant EV71 3Dpol genes (3D-NT1, 3D-NT2, 3D-NT3, 3D-NT4 and 3D-NT5) were sub-cloned into plasmid peGFP-C1 to generate plasmids peGFP-C1-3D-NT1 to -NT5, respectively. N-terminal and C-terminal domains are indicated. (C) 293T cells were co-transfected with pcDNA3.1(+)-SIRT1 and peGFP-C1, peGFP-C1-3Dpol and peGFP-C1-3D-NT1–NT5 for 24 h. Cell extracts were prepared for co-immunoprecipitation (IP) assays using anti-GFP antibody and precipitated with protein G. Interactions between SIRT1 and EV71 proteins were determined by western blotting using anti-SIRT1 antibody or anti-GFP antibody.…

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  Fig. 6.. SIRT1 binds directly to EV71 5′UTR, but not 3′UTR. (A–C) Cell extracts of RD, 293T or SK-N-SH cells were prepared and used as inputs, or incubated with no RNA, biotin-16-UTP, non-biotinylated EV71 5′UTR or biotinylated EV71 5′UTR (A). RD cell lysates were prepared and used as input, or were incubated with nonbiotinylated EV71 3′UTR RNA or biotinylated EV71 3′UTR RNA (B). RD cell lysates were prepared and used as input, or were incubated with biotinylated EV71 3′UTR RNA along with different concentrations of non-biotinylated EV71 3′UTR RNA or nonbiotinylated yeast tRNA (C). Protein–RNA pulldown assays were carried out with anti-SIRT1 antibody and precipitated with protein G. Interactions between SIRT1 and EV71 5′UTR were determined by western blotting with anti-SIRT1 antibody. (D) RD cells were infected with EV71 at an MOI of 10 for 12 h. Cell extracts were prepared and used for mRNA RNA extraction (Total RNA), or were used for protein–RNA pulldown assays with anti-SIRT1 antibody, anti-Flag antibody, without antibody or with water and followed by mRNA extraction. Then standard RT-PCR analysis using primers specific to EV71 5′UTR RNA or ribosomal protein S16 (RPS16) was performed.

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  Fig. 7.. SIRT1 represses EV71 IRES activity by binding to stem-loop I, II and III of the 5′UTR. (A) Schematic diagram of EV71 5′UTR secondary structure predicted by M-FOLD software. Numbers indicate the first and last nucleotides in each stem-loop. (B) RD cell lysates were prepared and used as input. Cell lysates were incubated with various truncated constructs of a non-biotinylated EV71 5′UTR or biotinylated EV71 5′UTR. Mixtures were pulled down by streptavidin beads, and SIRT1 in RNA–protein complex was detected by western blotting with anti-SIRT1 antibody. (C) Schematic diagram of the two dicistronic reporter plasmids pRHF and pRHF-5′UTR. pRHF expresses dicistronic mRNA comprising the Renilla luciferase (RLuc) gene at the first cistron and the Firefly luciferase (FLuc) gene at the second cistron (cytomegalovirus, CMV). In pRHF-5′UTR, translation of first cistronic gene (RLuc) is cap-dependent, whereas translation of second cistronic gene (FLuc) depends on IRES activity. A hairpin inserted downstream of first cistron prevents ribosome read-through. (D,E) Cells were co-transfected with pcDNA3.1(+)-SIRT1 and pRHF-5′UTR for 12 h (D) or co-transfected with siR-Ctrl or siR-SIRT1 and pRHF or…

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  Fig. 8.. SIRT1 is colocalized with EV71 RNA in cytoplasm. (A) RD cells were infected or not (Mock) with EV71 at an MOI of 5 for 8 or 12 h. Cells were fixed, permeabilized and immunostained with antibody against EV71 dsRNA (a,e,i) or SIRT1 (b,f,j). FITC-conjugated donkey anti-mouse-IgG or Cy3-conjugated goat anti-rabbit-IgG was used as a secondary antibody. Nuclei were stained with DAPI (c,g,k). m and n show enlargements of h and l. Immunofluorescence was detected using a confocal laser-scanning microscopy. (B) SK-N-SH cells were infected with EV71 at an MOI of 20 for 12 h. Cells were fixed, permeabilized and immunostained with antibody against EV71 dsRNA (a,e) or SIRT1 (b,f). FITC-conjugated donkey anti-mouse-IgG or Cy3-conjugated goat anti-rabbit-IgG was used as a secondary antibody. Nuclei were stained with DAPI (c,g). i shows an enlargement of h. Immunofluorescence was detected with a confocal laser-scanning microscopy. (C) A proposed mechanism underlying the regulation of EV71 replication.