Crystal structure of a highly conserved enteroviral 5′ cloverleaf RNA replication element

Figures

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  Fig. 1. Enteroviral genome organization and overall structure of CVB3 5′CL in complex with Fab BL3-6. a Schematic of an enterovirus genome depicting the location of the 5′CL and its proposed secondary structure with putative binding sites for viral 3CD and host PCBP proteins. b Secondary structure of CVB3 5′CL2a crystallization construct based on previous biochemical analysis, where the Fab BL3-6 binding epitope 5′-GAAACAC-3′ motif replaces the L2 loop of the subdomain sB. The nucleotides colored in gray represent the mutations or insertions compared to the wild-type sequence. c The crystal structure of the CVB3 5′CL2a cocrystallized with the Fab BL3-6 and solved at 1.9 Å resolution. The Fab is obscured in rotated views of the RNA structure for clarity. Figure panels and corresponding labels are colored analogously for facile comparison.

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  Fig. 2. Structural features of the CVB3 5′CL subdomains. a The crystal-derived secondary structure of the CVB3 5′CL2a construct. The dotted line indicates tertiary interactions between the L3 loop A40 and the P4 helix Py-Py region. b The tertiary interactions between the sA and sB subdomains. c Structure of the trinucleotide loop within the sC subdomain. d Superposition of the 5′-ccAUUgg-3′ and the 5′-ccUCUgg-3′ stem-loops (RMSD = 0.44 Å) from the crystal structures of CVB3 5′CL2a (red triloop with purple stem) and Agrobacterium tRNALeu (yellow, PDB code: 5AH5)60. Only the loop nucleotides are shown in detail for clarity. e Structure of the dinucleotide bulge within the sD subdomain P4 helix. Black dashed lines and red spheres in (b) and (e) reflect heteroatoms within hydrogen bonding distances and water molecules, respectively. Gray mesh represents the 2|Fo | -|Fc | electron density map at contour level 1σ and carve radius 1.8 Å.

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  Fig. 3. Comparison of the crystal and previous NMR structures of the 5′CL sD subdomain. a The crystal structure of the Py-Py base-pairs within the P4 helix of CVB3 5′CL2a. b The phosphate-to-phosphate distance between the base-pairs across the P4 helix. c Superposition of the CVB3 5′CL2a subdomain sD crystal structure (cyan) with previously reported NMR structures of the sD subdomains for CVB3 5′CL (blue) and an enteroviral consensus sequence (magenta). d Superposition of only the Py-Py region for the three structures as described in (c). e The major groove width within the P4 helix. The dashed black lines depict the distances between the Pi and Pi+6 across the helix. f The GNRA-type tetraloop structure observed in the crystallization 5′CL2a construct. g The UNCG-type tetraloop, as observed in the previous NMR structure of the isolated sD subdomain of CVB3 5′CL38. Black dashed lines (a), (b), and (e) represents heteroatoms within hydrogen bonding distances. Gray mesh in (c) and (d) represents the 2|Fo | - |Fc | electron density map at contour level 1σ and carve radius 1.8 Å.

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  Fig. 4. Structure of CVB3 5′CL sD subdomain and its binding interactions with 3 C protein. a Representative ITC profile for the WT 5′CL. The heat is released upon successive injections of 2 µl of CVB3 3C (~400 µM) to the RNA solution (~10 µM) in the calorimetry cell. b, c The binding curves from the ITC data for the binding of the 3C with various crystallization RNA constructs (see Supplementary Fig. 13, 14, and 15 for details). d Structure of the CVB3 5′CL2a, showing the tertiary interactions between the sC and sD subdomains, especially the docking of the sC-loop A40 into the Py-Py region of the P4 helix within the sD subdomain. e The details of A-minor type tertiary interactions between the A40 and the C56•U73 base-pair within the Py-Py region. f An A40U mutation model showing disruption of the A-minor interactions between the sC-loop and the Py-Py helix. g The binding curves from the ITC data for the binding of the 3C with the A40U mutant of the parent 5′CL2 construct. Black dashed lines in (d), (e), and (f) represent heteroatoms within hydrogen bonding distances. Gray mesh in d and e represents the 2|Fo | - |Fc | electron density map at contour level 1σ and carve radius 1.8 Å.

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  Fig. 5. Consensus structural model of the enteroviral 5′CLs. a Proposed secondary structural model of consensus enteroviral 5′CL (also see Supplementary Fig. 20 for details). The 5′CLs are one of the most conserved RNA elements among enteroviruses (b) The three-dimensional consensus fold of the 5′CLs in enteroviral genomes based on the crystal structure of CVB3 5′CL showing binding modes of viral 3CD and host PCBP proteins. The red dashed lines represent tertiary interactions and depict coaxial stacking of the corresponding subdomains.