Presentation Title

Probing RNA Structure in the 5' Untranslated Region of Coxsackievirus B3 Genomic RNA

Advisor Information

William Tapprich

Location

UNO Criss Library, Room 231

Presentation Type

Oral Presentation

Start Date

2-3-2018 11:00 AM

End Date

2-3-2018 11:15 AM

Abstract

Coxsackievirus B3 (CVB3) is a cardiovirulent enterovirus that utilizes a 5’ untranslated region (5’UTR) to complete critical viral processes. This includes an internal ribosome entry site (IRES) responsible for cap-independent translation. Ample evidence supports the hypothesis that the 5’UTR with its IRES is an important virulence determinant for the virus. We are investigating the structure of the 5’UTR from RNA genomes derived from naturally occurring virulent and avirulent viruses. Chimeric constructs and site-directed mutations of these genomes are part of the analysis. Our structural studies include queries of the RNA itself as well as the RNA complexed with host proteins such as polyrC binding protein 2 (PCBP2). We explore RNA structure in solution using base-specific modifying agents such as dimethyl sulfate as well as backbone probes through selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). Our results have resulted in a detailed secondary structure model for the 5’UTR, including those domains involved in the IRES. Comparing the structure of 5’UTR sequences from virulent and avirulent genomes, including chimeric constructs, shows there are key structural differences that correlate to the virulence phenotype. These studies also identify a critical virulence determinant in domain II of the 5’UTR. Chemical probing of 5’UTR molecules with bound PCBP2 shows that regions in the cloverleaf (domain I) and domain IV are protected from modification, indicative of direct protein interaction in these regions. Interestingly, other regions, particularly in the capping stem-loops of domain IV, show increased accessibility to chemical probes, indicative of conformational changes in RNA structure in response to protein binding. These accessibility increases are concentration dependent. In summary, our structural studies are defining the structure and structural dynamics of the 5’UTR from an important enterovirus.

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Mar 2nd, 11:00 AM Mar 2nd, 11:15 AM

Probing RNA Structure in the 5' Untranslated Region of Coxsackievirus B3 Genomic RNA

UNO Criss Library, Room 231

Coxsackievirus B3 (CVB3) is a cardiovirulent enterovirus that utilizes a 5’ untranslated region (5’UTR) to complete critical viral processes. This includes an internal ribosome entry site (IRES) responsible for cap-independent translation. Ample evidence supports the hypothesis that the 5’UTR with its IRES is an important virulence determinant for the virus. We are investigating the structure of the 5’UTR from RNA genomes derived from naturally occurring virulent and avirulent viruses. Chimeric constructs and site-directed mutations of these genomes are part of the analysis. Our structural studies include queries of the RNA itself as well as the RNA complexed with host proteins such as polyrC binding protein 2 (PCBP2). We explore RNA structure in solution using base-specific modifying agents such as dimethyl sulfate as well as backbone probes through selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). Our results have resulted in a detailed secondary structure model for the 5’UTR, including those domains involved in the IRES. Comparing the structure of 5’UTR sequences from virulent and avirulent genomes, including chimeric constructs, shows there are key structural differences that correlate to the virulence phenotype. These studies also identify a critical virulence determinant in domain II of the 5’UTR. Chemical probing of 5’UTR molecules with bound PCBP2 shows that regions in the cloverleaf (domain I) and domain IV are protected from modification, indicative of direct protein interaction in these regions. Interestingly, other regions, particularly in the capping stem-loops of domain IV, show increased accessibility to chemical probes, indicative of conformational changes in RNA structure in response to protein binding. These accessibility increases are concentration dependent. In summary, our structural studies are defining the structure and structural dynamics of the 5’UTR from an important enterovirus.