Structure of the 5' nontranslated region of the coxsackievirus b3 genome: Chemical modification and comparative sequence analysis

Abstract

Coxsackievirus B3 (CVB3) is a picornavirus which causes myocarditis and pancreatitis and may play a role in type I diabetes. The viral genome is a single 7,400-nucleotide polyadenylated RNA encoding 11 proteins in a single open reading frame. The 5' end of the viral genome contains a highly structured nontranslated region (5'NTR) which folds to form an internal ribosome entry site (IRES) as well as structures responsible for genome replication, both of which are critical for virulence. A structural model of the CVB3 5'NTR, generated primarily by comparative sequence analysis and energy minimization, shows seven domains (I to VII). While this model provides a preliminary basis for structural analysis, the model lacks comprehensive experimental validation. Here we provide experimental evidence from chemical modification analysis to determine the structure of the CVB3 5'NTR. Chemical probing results show that the theoretical model for the CVB3 5'NTR is largely, but not completely, supported experimentally. In combination with our chemical probing data, we have used the RNASTRUCTURE algorithm and sequence comparison of 105 enterovirus sequences to provide evidence for novel secondary and tertiary interactions. A comprehensive examination of secondary structure is discussed, along with new evidence for tertiary interactions. These include a loop E motif in domain III and a long-range pairing interaction that links domain II to domain V. The results of our work provide mechanistic insight into key functional elements in the cloverleaf and IRES, thereby establishing a base of structural information from which to interpret experiments with CVB3 and other picornaviruses.

FIG. 1.

Secondary structure map of the CVB3 5NTR (65). The sequence is that of CVB3/28 (59). The map shows the proposed secondary structures of the seven predicted structural domains (I to VII).

FIG. 2.

Chemical probing results for domain I (cloverleaf). A. A 12% sequencing gel showing primer extension analysis of modified and unmodified CVB3 RNA. Labels on the left indicate nucleotide positions according to the sequencing tracks (lanes A, C, G, and U), and labels on the right identify positions that are modified. Lane Un, unmodified; lane K, kethoxal; lane D, DMS; lane C, CMCT. B. A 12% sequencing gel to show detailed results for the connecting region between domain I and domain II. Labels on the right indicate nucleotide positions according to the sequencing tracks (lanes U, G, C, and A), and labels on the left identify positions that are modified. Lane Un, unmodified; lane K, kethoxal; lane D, DMS; lane C, CMCT. C. Predicted secondary structure map of domain I, showing modified positions. Filled circles identify strongly modified positions; open circles identify moderately modified positions. Red boxes indicate examples of pairs that are not supported by the comparative sequence analysis results shown in panel D. Green boxes indicate examples of pairs that are supported by phylogentic analysis in panel D. D. Analysis of representative paired positions, showing the number of occurrences of nucleotide identities among 105 enterovirus sequences.

FIG. 3.

Chemical probing results for domain II. A. A 12% sequencing gel showing primer extension analysis of modified and unmodified CVB3 RNA. Labels on the right indicate nucleotide positions according to the sequencing tracks (lanes U, G, C, and A), and labels on the left identify positions that are modified. Lane Un, unmodified; lane K, kethoxal; lane D, DMS; lane C, CMCT. B. Predicted secondary structure map of domain II, showing modified positions. Filled circles identify strongly modified positions; open circles identify moderately modified positions. Red boxes indicate examples of pairs that are not supported by the comparative sequence analysis results shown in panel C. Green boxes indicate examples of pairs that are supported by phylogentic analysis in panel C. C. Analysis of representative paired positions, showing the number of occurrences of nucleotide identities among 105 enterovirus sequences.

FIG. 4.

Chemical probing results for domain III. A. A 12% sequencing gel showing primer extension analysis of modified and unmodified CVB3 RNA. Labels on the left indicate nucleotide positions according to the sequencing tracks (lanes A, C, G, and U), and labels on the right identify positions that are modified. Lane Un, unmodified; lane K, kethoxal; lane D, DMS; lane C, CMCT. B. Predicted secondary structure map of domain III, showing modified positions. Filled circles identify strongly modified positions; open circles identify moderately modified positions. The two potential loop E motifs are indicated by E1 and E2. Red boxes indicate examples of pairs that are not supported by the comparative sequence analysis results shown in panel D. Green boxes indicate examples of pairs that are supported by phylogentic analysis in panel D. C. Diagram of the loop E motif, showing the noncanonical base pairs and the bulged nucleotide. Elements of the loop E motif are numbered: 1, sheared A-G pair; 2, trans-Hoogsteen U-A; 3, bulged A; 4, trans (locally parallel)-Hoogsteen-Hoogsteen A-A. D. Analysis of representative paired positions, showing the number of occurrences of nucleotide identities among 105 enterovirus sequences.

FIG. 5.

Chemical probing results for domain IV. A. A 12% sequencing gel showing primer extension analysis of modified and unmodified CVB3 RNA. Labels on the right indicate nucleotide positions according to the sequencing tracks (lanes U, G, C, and A), and labels on the left identify positions that are modified. Lane Un, unmodified; lane D, DMS; lane C, CMCT. B. Predicted secondary structure map of domain IV, showing modified positions. Filled circles identify strongly modified positions; open circles identify moderately modified positions.

FIG. 6.

Chemical probing results for domain V. A. A 12% sequencing gel showing primer extension analysis of modified and unmodified CVB3 RNA. Labels on the left indicate nucleotide positions according to the sequencing tracks (lanes A, C, G, and U), and labels on the right identify positions that are modified. Lane Un, unmodified; lane K, kethoxal; lane D, DMS; lane C, CMCT. B. Predicted secondary structure map of domain V, showing modified positions. Filled circles identify strongly modified positions; open circles identify moderately modified positions.

FIG. 7.

Chemical probing results for domain VI. A. A 12% sequencing gel showing primer extension analysis of modified and unmodified CVB3 RNA. Labels on the right indicate nucleotide positions according to the sequencing tracks (lanes U and G), and labels on the left identify positions that are modified. Results are shown for CVB3/28. Lane Un, unmodified; lane K, kethoxal; lane D, DMS; lane C, CMCT. B. Predicted secondary structure map of domain VI, showing modified positions. Filled circles identify strongly modified positions; open circles identify moderately modified positions. The pyrimidine-rich sequence (box A) and the AUG (box B) are indicated on the map.

FIG. 8.

Proposed structure model for the CVB3 5′NTR. For each domain, chemical probing results were entered into the RNASTRUCTURE algorithm (43) to generate a proposed structure. This structure was refined using results from the comparative sequence analysis. Strongly modified positions are indicated in red. Weakly modified positions are indicated in green.