Presentation Title

Structural Analysis of Coxsackievirus B3 by In-line Probing

Advisor Information

William Tapprich

Location

UNO Criss Library, Room 112

Presentation Type

Oral Presentation

Start Date

7-3-2014 1:15 PM

End Date

7-3-2014 1:30 PM

Abstract

Coxsackievirus B3 (CVB3) is a pathogenic Enterovirus of the picornavirus family that infects human cells and can lead to myocarditis and pancreatitis. Its positive sense, single-stranded RNA genome consists of 7400 nucleotides with four regions: a 5’ Untranslated Region (5’UTR), an open reading frame, a 3’ Untranslated Region (3’UTR) and a poly-A tail. The folded, three-dimensional structure of the 5’UTR is 742 bases long and contains seven secondary structure domains, including an internal ribosome entry site (IRES) associated with virulence. Because of this, the 5’UTR has been the focus of our research. Single-stranded RNA is able to fold into a variety of conformations, making it vulnerable to spontaneous cleavage under specific conditions. During an “in-line” conformation, a 2’oxygen, a phosphorous center and an adjacent 5’oxygen fold in a way that makes the phosphodiester bond vulnerable to a nucleophilic attack by the 2’oxygen, resulting in cleavage between the phosphorous and the 5’oxygen. In-line probing experiments have been carried out to determine where the sites of cleavage occur. Radiolabeled RNA incubated in a folding buffer are visualized by 12% polyacrylamide gel electrophoresis and phosphorimaging. Previous experiments have determined a theoretical model of the 5’UTR by chemical modification and comparative sequence analysis. In-line probing experiments are being carried out to help authenticate and add detail to the experimental structure. Detailed understanding of the 5’UTR structure is critical for designing antiviral compounds targeting this region of the genome.

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Mar 7th, 1:15 PM Mar 7th, 1:30 PM

Structural Analysis of Coxsackievirus B3 by In-line Probing

UNO Criss Library, Room 112

Coxsackievirus B3 (CVB3) is a pathogenic Enterovirus of the picornavirus family that infects human cells and can lead to myocarditis and pancreatitis. Its positive sense, single-stranded RNA genome consists of 7400 nucleotides with four regions: a 5’ Untranslated Region (5’UTR), an open reading frame, a 3’ Untranslated Region (3’UTR) and a poly-A tail. The folded, three-dimensional structure of the 5’UTR is 742 bases long and contains seven secondary structure domains, including an internal ribosome entry site (IRES) associated with virulence. Because of this, the 5’UTR has been the focus of our research. Single-stranded RNA is able to fold into a variety of conformations, making it vulnerable to spontaneous cleavage under specific conditions. During an “in-line” conformation, a 2’oxygen, a phosphorous center and an adjacent 5’oxygen fold in a way that makes the phosphodiester bond vulnerable to a nucleophilic attack by the 2’oxygen, resulting in cleavage between the phosphorous and the 5’oxygen. In-line probing experiments have been carried out to determine where the sites of cleavage occur. Radiolabeled RNA incubated in a folding buffer are visualized by 12% polyacrylamide gel electrophoresis and phosphorimaging. Previous experiments have determined a theoretical model of the 5’UTR by chemical modification and comparative sequence analysis. In-line probing experiments are being carried out to help authenticate and add detail to the experimental structure. Detailed understanding of the 5’UTR structure is critical for designing antiviral compounds targeting this region of the genome.