Structural basis for cloverleaf RNA-initiated viral genome replication

Figures

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  Figure 1.. Enterovirus cloverleaf RNA forms an H-type four-way junction. (A) Schematic representation of an enterovirus genome. The genome contains one open reading frame (ORF), flanked by a highly structured 5′ untranslated region (UTR) and a 3′ UTR with a poly(A) tail. The 5′ UTR contains the cloverleaf and internal ribosome entry site (IRES) structures and is covalently linked to VPg (viral protein genome-linked). The cloverleaf structure is located at the 5′ terminus of the 5′ UTR immediately followed by the 3′ poly-rC region (hatched box). (B) Design of tRNA-fused coxsackievirus B3 (CVB3) cloverleaf. CVB3 cloverleaf (nucleotides 2–87) is inserted into the anticodon loop of human tRNALys scaffold sequence to create tRNA-CLCVB3. The nucleotide numbers in cloverleaf are also shown on the secondary structure in parenthesis in blue. Cloverleaf consists of stem A and stem–loops B, C and D. The PCBP2 binding site in stem–loop B is colored in yellow, and the 3CDpro binding site is colored in green. The pyrimidine mismatch region in stem–loop D is boxed. (C) Crystal structure of CVB3 cloverleaf fused with a tRNA-scaffold. Cloverleaf RNA is colored by domain: stem A, orange; stem–loop B, blue;…

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  Figure 2.. Cloverleaf RNA is stabilized by the A•C-U base triple. (A) Sequence alignment of enterovirus cloverleaf RNA. The cloverleaf sequences from coxsackievirus B3 and A2 (CVB3, AY752944.2; CVA, NC_038306.1), poliovirus (PV, V01149.1), human enterovirus (HEV-B, NC_001472.1), echovirus (EV-E3, AB647326.1) and human rhinovirus 2 and 14 (HRV2, AB647326.1; HRV14, K02121.1) are aligned. Conserved nucleotides are shown in bold. The overall sequence identity among the seven RNAs is 55%. Structural assignment of the cloverleaf domains (stem A and stem–loops B, C and D) is shown at the top of the alignment. The positions of the PCBP2 binding sites (I and II), 3CDpro binding site, pyrimidine mismatch and the A•C-U base triple are indicated. (B) Superposition of CVB3 and PV cloverleaf structures. The CVB3 (blue) and PV (green) cloverleafs can be superimposed with an r.m.s.d. of 1.99 Å over 64 atom pairs (C3’). The 5′ and 3′ ends of nucleotides are numbered for PV cloverleaf. (C) Conservation of enterovirus cloverleaf. Nucleotide conservation was calculated by R-scape from a total of 153 enterovirus sequences and nucleotides are colored based on the conservation score, red, >97% conservation;…

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  Figure 3.. Small-angle X-ray scattering (SAXS) data analysis. (A) Scattering curves of PV wild-type and A39U mutant cloverleaf. Experimental scattering patterns for PV wild-type cloverleaf (0.95 mg/ml, blue) and A39U mutant cloverleaf (0.89 mg/ml, red) are shown. In the Guinier plot (inset), the Guinier region (qRG < 1.3) is delimited by the gray vertical lines. The red solid lines are the linear regression fit in the Guinier region, and the residuals are colored green. The radius of gyration (RG) determined from the Guinier plot is 36.2 and 36.9 Å for wild-type and A39U cloverleaf, respectively. (B) Pairwise distance distribution function P(r) analysis of PV wild-type and A39U mutant cloverleaf. The pairwise distance distribution function P(r) calculated from the SAXS curves shows that the A39U mutant displays an increased population in the 50–80 Å interatomic distance range compared to the wild-type, consistent with a conformational change within cloverleaf. The radius of gyration (RG) and maximal distance (Dmax) determined from the pairwise distance distribution function are 38.0 and 128 Å for wild-type and 38.2 and 124 Å for A39U mutant. In comparison, the RG and Dmax values calculated…

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  Figure 4.. Isothermal calorimetry of PCBP2 domains with poly-rC containing RNAs. (A) Interaction of PCBP2 KH1/2 domain with PV cloverleaf RNA. Isolated stem–loop B, WT cloverleaf (tRNA-CLPV) and A39U mutant cloverleaf (tRNA-CLA39U) were titrated into PCBP2 KH1/2 domain. Thermograms and fitted binding curves are shown. Data were fit with single binding-site model. Titrations were repeated at least twice. (B) Interaction of PCBP2 KH3 domain with PV cloverleaf RNA. Isolated stem–loop B, WT cloverleaf and A39U mutant cloverleaf were titrated into PCBP2 KH3 domain. The data were fit with single binding-site model. Note KH3 domain binds A39U mutant cloverleaf with higher affinity than WT cloverleaf, suggesting that stem–loop B site is not accessible in WT cloverleaf. (C) Interaction of PCBP2 domains with PV 3′poly-rC site. The linear ssRNA containing the 3′poly-rC sequence was titrated into either KH1/2 or KH3 domains. The binding affinities to the linear ssRNA are similar for both PCBP2 domains. Note that the KH3 domain binds 3′poly-rC with 2:1 (protein/RNA) stoichiometry. Summary of parameters are listed in Table S1.

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  Figure 5.. Model of the ternary complex containing cloverleaf RNA, PCBP2, and 3CDpro. (A) Locations of PCBP2 and 3CDpro binding sites in cloverleaf RNA. Cloverleaf RNA is shown as ribbon diagram in a transparent surface and colored as in Figure 1. The nucleotides indicated in PCBP2 binding in stem–loop B (blue surface) and 3CDpro binding in stem–loop D (green surface) are depicted with darker colors in PV cloverleaf. The 3′ poly-rC site adjacent to stem A (PCBP2 binding site II) is also indicated. (B, C) Model of the cloverleaf-PCBP2 complex. The cloverleaf-PCBP2 (KH3 domain) model was generated using the Nova-2 KH3 domain structure complexed with a 20 nt RNA hairpin (PDB code 1EC6). The nova-2 RNA sequence that interacts with the KH3 domain (11AUCAC15, blue) corresponds to 22UCCCA26 and 22ACCCA26 in CVB3 and PV cloverleaf, respectively (B). The cloverleaf stem–loop B and nova-2 RNA hairpins were overlaid by the stem (green box) to generate a complete cloverleaf structure. Superposed cloverleaf and Nova-2 nucleotides are shown in blue and pink, respectively. For clarity, the PCBP2 KH3 domain is omitted. The cloverleaf-PCBP2 (KH3 domain) complex (C). The KH3 domain residues that directly…

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  Figure 6.. Model of 3CDpro-enhanced PCBP2 interaction with cloverleaf. Enterovirus genome contains the 5′ cloverleaf and the 3′ poly-A tail. In the absence of 3CDpro, multiple PCBP2 molecules can bind the two poly-rC sites of cloverleaf with low affinity. Following viral protein translation, 3CDpro binds stem–loop D and ‘unlocks’ the A•C-U base triple. This allows a hinge-like motion between the two arms of the H configuration, and exposes the PCBP2-binding site on stem–loop B. Additionally, 3CDpro binding to stem–loop D may restrict the flexibility of the 3′ C-rich region (PCBP2-binding site II). This orients the 3′ C-rich region such that a single PCBP2 molecule can bind to both poly-rC sites simultaneously, increasing the binding affinity of PCBP2 to cloverleaf. The resulting cloverleaf-PCBP2-3CDpro ternary complex then facilitates genome circularization by recruiting the poly(A) binding protein associated with the 3′ poly(A) tail, and subsequent initiation of viral RNA synthesis.