Structural basis for 3C and 3CD recruitment by enteroviral genomes during negative-strand RNA synthesis

Figures

• 

  Fig. 1. Overall crystal structures of intact CL-3C and sD-3C complexes. a Schematics showing the CL-mediated assembly of the replication initiation complex during (−)-strand RNA synthesis of enteroviral genome replication. b The crystal structure of intact CVB3 CL in complex with the viral 3C was solved at 2.69 Å resolution, which showed two copies of 3C monomers (designated 3C1 and 3C2) bound per RNA molecule. c The crystal structure of the sD-3C complex solved at 2.41 Å resolution shows two copies of 3C monomers bound per sD molecule, identical to that in the intact CL-3C complex. d The secondary structure of the CL showing the key features of CL-3C interactions within the sD stem-loop structure. e The superposition of the sD-3C complex with the corresponding region of the CL-3C complex (all atoms RMSD = 0.64 Å). f Two copies of 3C bind the sD stem-loop as monomers with no significant interactions between themselves except the K12 residue within the hydrogen-bonding distance ( ~ 3 Å) closer to the protein-RNA interface. g Representative chromatograms for the 21.4 kDa 3C protein (red) superimposed with a molecular size marker (blue), suggesting no dimerization of the 3C in solution. h…

• 

  Fig. 2. Specific interactions involved in 3C binding with CL’s sD subdomain. a Structure of CL-bound 3C showing the RNA binding interface that includes N-terminal α-helix and L1 and L2 loops (colored yellow). b The crystal structure of the CL-3C complex elucidates the positions of each 3C molecule interacting with the sD stem-loop region, which mainly interacts with nucleotides upstream of the Py•Py helix (cyan) and 3C2 with nucleotides downstream of the Py•Py helix. Each 3C molecule uses the same RNA-binding surface (colored wheat for 3C1 and colored yellow for 3C2 for clarity). c The specific residues of the 3C1 (left) and 3C2 (right) involved in RNA binding interactions. The sD stem-loop has been represented as a molecular surface (wheat) with a Py•Py helix (cyan). The 3C1 residues interact with nucleotides (d) downstream and (e) upstream of the Py•Py helix. f Similarly, the 3C2 residues interact with nucleotides upstream and (g) downstream of the Py•Py helix. h The molecular surface of 3C molecules according to the electrostatics of each residue (blue = positive; red = negative; white = neutral charge) shows how two 3C molecules establish a positively charged surface for binding sD…

• 

  Fig. 3. ITC-based binding studies of CL and 3C interactions across enteroviruses. a Representations of all CVB3 CL constructs used for ITC measurements showing the positions of mutations or insertions within the sD loop. The nucleotides colored in black indicate point mutations or insertions. The red, green, and blue colored nucleotides represent the corresponding simultaneous mutations or insertions. b The apparent Kds for binding wild-type CVB3 3C with CL mutants as measured by ITC. The data with * were adopted from Das et al.27 for facile comparisons. c Alignment of 3C-bound sD and protein-free sD crystal structures showing altered conformations of the loop and bulge regions. The 3C-free sD loop forms a GNRA-type tetraloop due to the G66C mutation27, but the protein-bound WT loop forms a UNCG-type tetraloop. d Superposition of the 3C-bound sD and a hypothetical sD model (green) with the non-canonical Py•Py region replaced by canonical pairs via U72A, U73G, and U74A mutations. e The apparent Kds for binding wild-type CVB3 CL with 3C mutant constructs. The horizontal dotted line in b and d indicates the Kd for wild-type CL binding with wild-type 3C for facile comparison. The apparent…

• 

  Fig. 4. Interactions of enteroviral 3CD with CLs. a Schematics of a CVB3 CL construct used for BLI measurements of 3C, 3D, and 3CD proteins. The 3' overhang sequence in the CL construct hybridizes with a 5' biotinylated complementary DNA oligo, allowing the construct to immobilize in streptavidin biosensors. The saturated BLI responses (wavelength shift) generated by the interactions of sensor-immobilized CL for various concentrations of (b) CVB3 3C and (c) 3CD (filled circles) were fitted with a binding isotherm (dotted curve). The reported error in Kds represents the standard error of the fitting. See Supplementary Fig. 13 and the methods for details. d A model of CVB3 3CD predicted by the AlphFold3, showing 3C and 3D domains connected by a flexible linker. e Superposition of the AlphaFold3-predicted 3CD model with each 3C molecule of the CL-3C complex crystal structure, showing no apparent 3D domain interaction with the CL. f The alignment of the previously determined PV1 3CD crystal structure44 with each 3C molecule of the CVB3 CL-3C complex crystal structure shows potential contacts of the linker region in both 3CD molecules with the CL. g The apparent Kd for a mutant CVB3 3CD with…