Role of RNA Domain Structure and Orientation in the Coxsackievirus B3 Virulence Phenotype

Figures

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  FIG 1. Sequence alignment of the 5′ UTR from CVB3/GA and CVB3/28. Blue highlighting shows the positions with nucleotide substitutions. Red highlighting shows the boundaries of each 5′ UTR domain in CVB3/28.

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  FIG 2. Secondary structures of CVB3 5′ UTR regions. 5′ UTR RNA from CVB3/28 and CVB3/GA was modified with 1M7 following the SHAPE-MaP protocol. ShapeMapper 2.0 and TurboFold were used to analyze and predict the secondary structures. The nucleotide shading indicates normalized reactivity values of <0.3 (gray), 0.3 to 0.7 (light red), and >0.7 (dark red).

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  FIG 3. Comparison of secondary structures of domain II and III. 5′ UTR RNA from CVB3/28 and CVB3/GA was modified with 1M7 following the SHAPE-MaP protocol. ShapeMapper 2.0 and TurboFold were used to analyze and predict the secondary structures. (a) Secondary structure of superdomain II/III (95 to 224) of the CVB3/GA 5′ UTR. The single-stranded region leading from domain I (88 to 94) to the base of domain II is labeled as the connecting region between the two. The left stem-loop encompasses nucleotides 129 to 139. The right stem-loop encompasses nucleotides 141 to 160 and corresponds to the top stem-loop of domain II (139 to 160) in CVB3/28. Domain III includes positions 184 to 214. (b) Secondary structure of domains II (105 to 181) and III (189 to 210) of the CVB3/28 5′ UTR. LR-1 following the eight-nucleotide single-stranded connection region (90 to 97). LR-2 between domain II and domain III is also indicated. The nucleotide shading indicates normalized reactivity values of <0.3 (gray),0.3 to 0.7 (light red), and >0.7 (dark red).

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  FIG 4. Comparison of domain IV secondary structure. 5′ UTR RNA from CVB3/28 and CVB3/GA was modified with 1M7 following the SHAPE-MaP protocol. ShapeMapper 2.0 and TurboFold were used to analyze and predict the secondary structures. (a) Secondary structure of domain IV of the CVB3/GA 5′ UTR (positions 229 to 465). (b) Secondary structure of domain IV of the CVB3/28 5′ UTR (213 to 456). Stem-loops 4a through 4c of the cruciform structure are labeled. The nucleotide shading indicates normalized reactivity values of <0.3 (gray), 0.3 to 0.7 (light red), and >0.7 (dark red).

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  FIG 5. Comparison of domain V secondary structure. 5′ UTR RNA from CVB3/28 and CVB3/GA was modified with 1M7 following the SHAPE-MaP protocol. ShapeMapper 2.0 and TurboFold were used to analyze and predict the secondary structures. Stem-loop regions (5a to 5d) as well as the intrinsically disordered RNA region are labeled. The nucleotide shading indicates normalized reactivity values of <0.3 (gray), 0.3 to 0.7 (light red), and >0.7 (dark red). (a) Secondary structure of domain V in the CVB3/GA 5′ UTR. The IDRR of CVB3/GA includes nucleotides 562 to 582. (b) Secondary structure of domain V in the CVB3/28 5′ UTR. The IDRR of CVB3/28 includes nucleotides 561 to 581.

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  FIG 6. Tertiary structure models of CVB3 5′ UTRs generated by 3dRNA v2.0. Full-molecule renderings were generated using SHAPE-MaP secondary-structure data to inform 3dRNA v2.0 along with DCA. Tertiary molecules were visualized and colored by domain of interest in PyMOL: red, domain I; orange, domain II; yellow, domain III; green, domain IV; blue, domain V; purple, IDRR; pink, domain VI. Each molecule was oriented to place domain IV of each molecule in the same plane for easier comparison. (a) Tertiary structure of CVB3/GA. Poly(C) regions of domain IV (4a [304 to 307] and 4b [339 to 341]) are in blue. (b) Tertiary structure of CVB3/28. Poly(C) regions in domain IV (4a [304 to 308] and 4b [438 to 439]) are in light green.

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  FIG 7. Tertiary structures of domain II/domain III. Full-molecule renderings were generated using SHAPE-MaP data secondary-structure data to inform 3dRNA 2.0 along with DCA. A single SHAPE-MaP structure was chosen based on the highest statistical quality checks from ShapeMapper 2.0. Individual domains were extracted from the full-molecule models using PyMOL and colored for clarity. (a) Tertiary structures of CVB3/GA superdomain II-III (95 to 224). (b) Tertiary structures of CVB3/28 domain II (105 to 181) and domain III (189 to 209). Domains were oriented to be within the same plane. Domain II is orange; domain III is yellow.

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  FIG 8. Tertiary structures of domain IV. Full-molecule renderings were generated using SHAPE-MaP data secondary-structure data to inform 3dRNA 2.0 along with DCA. A single SHAPE-MaP structure was chosen based on the highest statistical quality checks from ShapeMapper 2.0. Domain IV was again isolated from the full molecule in PyMOL. Each isolated domain IV was oriented to show as many of the vital regions as possible, such as the poly(C) binding regions. (a) CVB3/GA poly(C) regions in 4a (304 to 307) and 4b (339 to 341) are highlighted in blue. (b) CVB3/28 poly(C) regions 4a (303 to 306) and 4b (338 to 340) are also highlighted in blue.

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  FIG 9. Tertiary structures of domain V. Full-molecule renderings were generated using SHAPE-MaP secondary-structure data to inform 3dRNA 2.0 along with DCA. A single SHAPE-MaP structure was chosen based on the highest statistical quality checks from ShapeMapper 2.0. Domain V was isolated from the full molecule in PyMOL. Stem-loops 5a to 5d are labeled. (a) CVB3/GA. the IDRR occupies 562U to 583A. (b) CVB3/28. The IDRR occupies 561U to 581C. Each domain V was oriented to focus on the IDRR (purple) conformation.