Presentation Title

Structural Analysis of Coxsackievirus B3 by In-line Probing

Advisor Information

William Tapprich

Location

Dr. C.C. and Mabel L. Criss Library

Presentation Type

Poster

Start Date

6-3-2015 9:00 AM

End Date

6-3-2015 10:30 AM

Abstract

Coxsackievirus B3 (CVB3) is a pathogenic Enterovirus of the picornavirus family, with a single-stranded, positive sense RNA genome. Infection can lead to heart disease and pancreatitis. The CVB3 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 CVB3 5’UTR contains 742 bases and seven secondary structure domains. Virulence of CVB3 is found to be associated with domain II. In order to better understand the virus and its pathogenesis, 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 are located. Radiolabeled RNA incubated in an in-line probing folding buffer are visualized by 12% polyacrylamide gel electrophoresis and phosphorimaging. Because highly structured portions of the molecule are less vulnerable to nucleophilic attack, this will also help determine where in the molecule the more structured portions are. Results from in-line probing will enable us to better understand the three-dimensional structure of CVB3, as well as its function.

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Mar 6th, 9:00 AM Mar 6th, 10:30 AM

Structural Analysis of Coxsackievirus B3 by In-line Probing

Dr. C.C. and Mabel L. Criss Library

Coxsackievirus B3 (CVB3) is a pathogenic Enterovirus of the picornavirus family, with a single-stranded, positive sense RNA genome. Infection can lead to heart disease and pancreatitis. The CVB3 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 CVB3 5’UTR contains 742 bases and seven secondary structure domains. Virulence of CVB3 is found to be associated with domain II. In order to better understand the virus and its pathogenesis, 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 are located. Radiolabeled RNA incubated in an in-line probing folding buffer are visualized by 12% polyacrylamide gel electrophoresis and phosphorimaging. Because highly structured portions of the molecule are less vulnerable to nucleophilic attack, this will also help determine where in the molecule the more structured portions are. Results from in-line probing will enable us to better understand the three-dimensional structure of CVB3, as well as its function.