Article Figures & Data
Figures
- Fig 1
EV71 VPg binds with 3Dpol in vitro. (A) GST pulldown assay for interactions of EV71 3Dpol with VPg in vitro. Glutathione-Sepharose beads coupled with approximately 5 μg of GST-VPg were incubated with wild-type 3Dpol. After the beads were washed, proteins that bound to the beads were analyzed by 12% SDS-PAGE, followed by staining with Coomassie blue. The positions of 3Dpol and GST-VPg are indicated by arrows on the left. The results shown are representative of at least three independent experiments. Lane M, a standard protein marker. (B) Binding affinity of VPg to wild-type 3Dpol measured by ITC. The binding of VPg to wild-type 3Dpol (at a concentration of 0.1 mM) was determined by ITC by stepwise injection of VPg at a concentration of 0.5 mM. Kd was calculated to be 57.5 μM.
- Fig 2
Overall structure of the EV71 3Dpol-VPg complex. (A) The crystal structure of the EV71 3Dpol-VPg complex is shown as a colored ribbon covered with a transparent molecular surface in two perpendicular views. The finger, palm, and thumb domains are colored light blue, cyan, and green, respectively, and the bound VPg molecule is colored orange. The active center of 3Dpol is indicated by a red arrow. The catalytic base, D238, and the key tyrosine residue, Y3, are shown as colored spheres. (B) Omit map of the bound VPg molecule. The bound VPg molecule is shown as colored sticks and is covered by the omit map at 1.0 σ. The figure is shown in stereo mode.
- Fig 3
Structural details of the interface between EV71 3Dpol and VPg. The structural details of contacts I (A and B) and II (C and D) are shown in the plane (A and C) and ball-and-stick (B and D) modes. Dashed lines indicate hydrogen-bonding and salt bridge interactions. The palm and finger domains of the EV71 3Dpol molecule are colored following the same color scheme described in the legend to Fig. 2, whereas contacts I and II areas are green and red, respectively. The VPg molecules are represented as yellow sticks. The superscripts indicate the origins of the labeled residues.
- Fig 4
GST pulldown assay for EV71 3Dpol and relevant mutants with VPg interactions in vitro. Glutathione-Sepharose beads coupled with approximately 5 μg of GST-VPg (or the relevant mutants) were incubated with either wild-type 3Dpol or the relevant mutants. After the beads were washed, proteins bound to the beads were analyzed by 12% SDS-PAGE, followed by staining with Coomassie blue. (A to E) Interaction between VPg and 3Dpol mutants. The positions of the 3Dpol protein and GST-VPg are indicated by the arrows on the left. The results shown are representative of at least three independent experiments.
- Fig 5
Effect of 3Dpol-VPg interface mutants on VPg uridylylation. (A) The VPg uridylylation assay measured the radioactivity generated by [α-32P]UTP-VPg. (B) The relative activities were calculated by comparing the activities of the EV71 3Dpol mutants with the activity of the WT protein (which was set equal to 100%). Results were obtained from three independent experiments performed in duplicate, and standard deviations (n = 3) are presented.
- Fig 6
Effect of VPg-binding-defective mutants on EV71 3Dpol RNA elongation activity. The RNA elongation assay measured the radioactivity generated by [α-32P]UTP, and the relative activities were calculated by comparing the activities of the EV71 3Dpol mutant proteins with the activity of the WT protein (which was set equal to 100%). D238A and D238H mutants were used as negative controls. Results were obtained from three independent experiments performed in duplicate, and standard deviations (n = 3) are presented. The activity of the WT protein, which was set equal to 100%, is indicated by the dashed line.
- Fig 7
Mutagenesis analysis of the VPg-3Dpol interface on EV71 replication. Results for the mutants of the 3Dpol protein (A) and VPg (B) are shown. (Top) Vero cells were transfected with WT and mutant genome-length RNAs (5 μg) and analyzed for viral VP1 protein expression by IFA 48 h p.t.; (bottom) the plaque morphologies of WT and mutant viruses are also shown. N.D., not detectable.
- Fig 8
Complementation assay for EV71 3Dpol and relevant mutants in trans. (A) Radioactive agent-based VPg uridylylation assay; (B) summary of the results, with the activity of WT EV71 3Dpol being set equal to 100%. Results were obtained from three independent experiments performed in duplicate, and standard deviations (n = 3) are presented.
- Fig 9
Complementation in trans shows that uridylylation occurs in a D238H-concentration-dependent manner. The results of the radioactive agent-based trans complementation assay are summarized, with the activity of 0.5 μM WT EV71 3Dpol being set equal to 100%. Results were obtained from three independent experiments performed in duplicate, and standard deviations (n = 3) are presented.
- Fig 10
Comparison of VPg-binding sites on FMDV, CVB3, and EV71 3Dpol molecules. (A) The structures of 3Dpol of EV71 are covered by a white surface. The VPg proteins bound with FMDV, CVB3, and EV71 RdRp are shown as green, cyan, and purple sticks, respectively. In the surface representation, the 3Dpol residues for VPg binding in FMDV, CVB3, and EV71 are colored red, pale green, and blue, respectively. (B) Sequence alignment of Picornaviridae members. The secondary structure is generated according to the structure of EV71 3Dpol. The VPg-binding sites on EV71, CVB3, and FMDV are highlighted by blue, green, and red frames, respectively, while the residues for 3AB binding and 3B uridylylation in poliovirus (PV) are labeled by red asterisks.
Tables
- Table 1
Data collection and refinement statistics
Parametera Value(s) for: EV71 3Dpol-VPg complexb 3Dpol VPg Ion Solvent Data collection statistics Cell parameters a (Å) 103.9 b (Å) 103.9 c (Å) 131.8 α, β, γ (°) 90.0, 90, 120.0 Space group P3221 Wavelength used (Å) 1.0000 Resolution (Å) 50.00–2.69 (2.74–2.69) No. of all reflections 138,534 (23,580) No. of unique reflections 24,730 (1,225) Completeness (%) 99.5 (97.6) Avg I/σ〈I〉 12.6 (2.2) Rmerge (%) 11.6 (47.5) Refinement statistics No. of reflections used (σF > 0) 22,910 Rwork (%) 21.0 Rfree (%) 26.7 RMSD Bond distance (Å) 0.009 Bond angle (°) 1.263 Average B value (Å2) 58.7 98.5 80.2 67.4 No. of protein atoms 3,698 144 2 327 Resolution (%) in Ramachandran plot Allowed regions 98.5 73.3 Generously allowed regions 1.0 20.0 Disallowed regions 0.5 6.7 ↵a Rmerge = ΣhΣl |Iih − 〈Ih〉|/ΣhΣI 〈Ih〉, where I is the intensity of a reflection and 〈Ih〉 is the mean of the observations Iih of reflection h. Rwork = Σ(||Fp(obs)| − |Fp(calc)||)/Σ|Fp(obs)|, where obs and calc are the observed and calculated values, respectively. Rfree is an R factor for a preselected subset (5%) of reflections that was not included in the refinement.
↵b Numbers in parentheses are the corresponding values for the highest-resolution shell.
- Table 2
Hydrogen bonds and salt bridge mediating the interaction between EV71 3Dpol and VPg
Site VPg 3Dpol Distance (Å) Residue Atom Residue Atom Hydrogen bonds Ala2 N Leu322 O 2.82 Tyr3 N Asn323 Oδ1 3.10 Ala15 N Lys315 O 2.70 Arg17 N Thr313 O 2.93 Arg17 Nε Tyr73 OH 3.15 Arg17 Nθ1 Phe314 O 2.84 Gly1 O Gln84 Nε2 2.70 Ala15 O Lys315 N 2.38 Salt bridge Gly1 N Asp320 Oδ1 3.93 - Table 3
Summary of mutagenesis analysisa
Mutation IFA result Plaque formation % uridylation % RNA elongation GST pulldown assay result WT ++++ Yes 100 100 ++++ D238H − No 0 0 ++++ Y73A ++++ 85 >95 +/− H80A ++++ 70 >95 +/− Q84A ++++ 150 >95 ++ Q87A 110 >95 ++ I311A − No 40 98 +/− K312A ++++ Yes >100 >100 ++++ T313A +/− No <5 >100 +/− F314A − No <5 >100 +/− K315A +++ Yes >100 >100 ++++ G316A ++++ Yes >100 90 ++++ I317A − No <5 >95 +/− L319A − No 20 >95 − D320A − No 5 >95 − E321A ++++ Yes 200 >95 ++++ L322A − No 75 >95 ++++ N323A +++ Yes 120 >95 +/− Y335A − No 15 >95 +/− F337A − No 40 >95 +/− I339A 90 >95 ++++ D340A 130 >95 ++ E343A 120 >95 ++ ↵a Summary of mutagenesis analysis of 3Dpol-VPg interface on EV71 replication. The replication levels of WT and mutant RNAs are categorized into nonreplicative (−), weak (+/−), medium (++), and strong (++++) on the basis of the IFA results. The binding affinities between 3Dpol mutants with VPg are summarized as not observed (−), weak (+/−), medium (++), and strong (++++).
