Fig 1.
iTRAQ analysis of SVA-infected ST cells.
ST cells were mock- or SVA-infected at MOI 0.1 and harvested at 12 and 24 hpi for proteomic analysis. (A) Cluster analysis of the 50 upregulated and 20 downregulated genes with the greatest fold change values. Red indicates increased (infected/mock) gene expression, and blue indicates decreased expression. (B) Validation of iTRAQ data by qRT-PCR analyses. Mx2, IFIT1, ISG15, UBE2L6, and RSAD2 mRNA levels were upregulated, and ANXA6, STRBP, and SWAP70 mRNA levels were downregulated. Values are presented as means ± SD from three independent experiments.
Fig 2.
Effects of differentially expressed proteins on SVA replication.
(A) Host proteins involved in SVA replication. (B) Western blot of transfected BHK-21 cells challenged with SVA for 16 h. (C) Viability of cells overexpressing the indicated genes. Values are presented as the mean ± SD from three independent experiments.
Fig 3.
UBE2L6 overexpression promotes SVA replication.
(A) Western blot of pUBE2L6-HA and/or empty vector (Vec) transfected BHK-21 cells challenged with SVA for 16 h. VP1 and UBE2L6 were probed with anti-VP1 and anti-HA antibodies. (B) Viral RNA levels were quantitated using qRT-PCR. (C) Virus yields in infected cell supernatants presented as TCID50 per milliliter. (D) IFA of SVA infected cells. All samples run in triplicate. *, P < 0.05; **, P < 0.01.
Fig 4.
Knockout of UBE2L6 suppresses SVA replication.
(A) Sequences in UBE2L6 targeted by sgRNAs 1–3. AGG (green) is the protospacer adjacent motif (PAM). The highlighted base (blue) indicates an indel. (B) Sanger sequencing showing the indel in UBE2L6. (C) Western blot analysis of UBE2L6 expression in the BHK-UBE2L6-KO and BHK-Wt cells. β-actin was used as a loading control. (D) Viability assay of BHK-UBE2L6-KO and BHK-Wt cells. 2× 104 cells were seeded into wells of 96 well plates and viability were measured at 0 h, 24 h and 40 h. (E-F) BHK-UBE2L6-KO and Wt cells were transfected with pCAGGS-UBE2L6-HA or empty vector then infected with SVA (MOI 0.01) for 16 h. Levels of VP1 (E) and virus titers (F) were determined by Western blot and TCID50 respectively. Data are expressed as the means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01.
Fig 5.
(A) Immunoprecipitation and Western blot of BHK-21 cells cotransfected with Flag-tagged SVA proteins and HA-tagged UBE2L6; proteins immunoprecipitated with rabbit anti-HA. (B) Western blot showing that the UBE2L6-3D interaction is specific. p3D-Flag and pUBE2L6-HA cotransfected cell lysates were collected, and then co-IP was performed using mouse anti-Flag. (C) Colocalization of UBE2L6 and viral 3D. Confocal microscopy of BHK-21 cells transfected with pUBE2L6-HA then infected with SVA (MOI 0.01) for 16 h. SVA 3D (green), pUBE2L6-HA (red), and nuclei (blue). Experiments were performed in triplicate.
Fig 6.
SVA 3D is ubiquitinated during infection.
BHK-21 cells (1 × 107) were infected with SVA (MOI 1) and harvested at 7 h post infection before immunoprecipitation with an anti-3D antibody. IP and IB analyses were performed with the indicated antibodies. (A) Western blot of infected cell lysates. (B) Immunoprecipitation with anti-3D antibody, and Western blotting with anti-Ub, and anti-3D antibodies, of infected cells lysates.
Fig 7.
UBE2L6 mediates ubiquitination of 3D.
(A) pUBE2L6 ubiquitinates 3D in a concentration-dependent manner. HEK-293T cells, cotransfected with p3D-Flag and pUBE2L6-Myc (0, 1, or 2 μg), were subjected to co-IP with anti-Flag (MAb), then Western blotting with anti-HA, anti-Flag and anti-Myc MAbs. (B) The cysteine at position 86 in UBE2L6, is necessary for 3D ubiquitination by UBE2L6. Cell lysates from HEK-293T cells cotransfected with p3D-Flag and pUBE2L6-Myc, pUBE2L6 (C86S), pUBE2L6 (C98S), or pUBE2L6 (C102S), together with pHA-Ub were subjected to IP and immunoblotting. (C, D) The residues K48 and K63 in 3D are necessary for UBE2L6-mediated polyubiquitination of 3D. HEK293T cells cotransfected with p3D-Flag, pUBE2L6-Myc, pHA-Ub, or pHA-Ub-K48 (only K48 of the seven Lys residues remained), pHA-Ub-K63 (only K63 of the seven Lys residues remained), pHA-Ub-K48R (pHA-Ub with mutation of K48 to R) or pHA-Ub-K63R (pHA-Ub with mutation of K63 to R), were subjected to IP and immunoblotting as described above. (E) HEK293T cells were cotransfected with p3D-Flag and pUBE2L6-Myc or vector for 24 h followed by treatment with 10 μM MG132 or DMSO for 6 h. Thirty hours later, the cell extracts were subjected to IP and immunoblotting by using antibodies to endogenous Ub, K48 and K63.
Fig 8.
Ubiquitination affects the stability of 3D.
(A) Western blot for 3D protein in BHK-21 cells cotransfected with pUBE2L6-Flag or empty vector, and p3D-Flag for 24 h then treated with cycloheximide (CHX). Band intensity was quantified using ImageJ, β-actin was used as loading control. The relative quantities of 3D are shown as percentages of 3D at 0 h. (B) Western blot for 3D protein in BHK-21 cells cotransfected with empty vector, pUBE2L6-Myc, or pUBE2L6(C86S), together with p3D-Flag for 24 h then treated with cycloheximide. (C) BHK-Wt cells or BHK-UBE2L6-KO cells were transfected with p3D-Flag for 24 h, designated 0 h, and then treated with CHX or MG132, as indicated, for up to 12 h. Cells were harvested and analyzed by Western blot with anti-Flag, anti-UBE2L6 and anti-β-Actin antibodies. (D, E) BHK21 cells cotransfected with empty vector, pUBE2L6-Myc, or pUBE2L6(C86S), p3D-Flag, together with pUb-HA (D) or without pUb-HA (E) for 24 h then treated with cycloheximide. And the levels of ubiquitination of 3D were evaluated by IP and immunoblotting. Data are expressed as the means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01.
Fig 9.
Lys169 and Lys321 of SVA 3D are major ubiquitination sites.
(A) Ubiquitination motifs and the conservation of Ub sites. The height of each letter corresponds to the frequency of that amino acid residue at that position. The central K indicates ubiquitinated Lys. (B) Amino acid sequence of SVA 3D. The lysine (K) residues in the potential ubiquitination sites are shown in red, and the K residues corresponding to those in our mutants are underlined. (C) Predicted three-dimensional structure of 3D, SVA strain SVV-CH-SD, and the positions of mutated K residues. (D) Co-IP and Western blotting of HEK-293T cells cotransfected with pUBE2L6-Myc and p3D-FlagWt, K161R, K169R, K321R, K366R or K418R, together with pHA-Ub. Co-IP was done using anti-Flag MAb, and Western blotting was done with anti-HA, anti-Flag, and anti-Myc MAbs. (E) Alignment of 3D sequences from different viruses of Picornaviridae: Senecavirus A (SVA) (GenBank accession no. MH779611), coxsackievirus B3 (CVB3) (GenBank accession no. M16572), Encephalomyocarditis virus (EMCV) (GenBank accession no. HM641897), coxsackievirus A9 (CVA9) (GenBank accession no. D00627), coxsackievirus A16 (CVA16) (GenBank accession no. AY790926), enterovirus 71 (EV71) (GenBank accession no. AB204852) and poliovirus (PVB2) (GenBank accession no. KT353719). The selected K169 and K321 lysine residues are in green.
Fig 10.
Construction and ubiquitination detection of mutant viruses.
(A) Construction scheme of full-length cDNA clones containing mutations in 3D and two rescued SVA strains. (B) BHK-21 cells infected with 100 TCID50 parental rSVA or mutants were used for one-step growth curves. (C) Plaque morphology in BHK-21 cells of rescued viruses. (D) Co-IP and Western blotting of infected BHK-21 cells. The densitometric analysis of Ub/3D or 3D/β-actin protein levels are depicted as relative intensities. Values are presented as the mean ± SD from three independent experiments. *, P < 0.05; **, P < 0.01.
Fig 11.
Model of UBE2L6-mediated promotion of SVA replication.
SVA infection upregulates the expression of UBE2L6, an E2 ubiquitin-binding enzyme, and UBE2L6 interacts with viral protein 3D and mediates K48/K63 chains to improve the stability of SVA 3D. In UBE2L6, the mutation C86S results in the loss of UBE2L6-mediated ubiquitination of 3D, and in 3D, the mutations K169R and K321R also result in unubiquitinated 3D, as these are the sites of UBE2L6-mediated ubiquitination. Any of these mutations result in inhibition of SVA replication.