Structural analysis of the Coxsackievirus B3 genome by in-line probing

Advisor Information

William Tapprich

Location

Milo Bail Student Center Council Room

Presentation Type

Oral Presentation

Start Date

8-3-2013 10:15 AM

End Date

8-3-2013 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 the 5’UTR. 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 subjected to in-line probing is visualized by 10% polyacrylamide gel electrophoresis and phosphorimaging. Because highly structured portions of the molecule are less vulnerable to nucleophilic attack, in-line probing analysis will determine the location of more structured regions. Results from in-line probing will enable us to better understand the threedimensional structure of the CVB3 genome, as well as its function.

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Mar 8th, 10:15 AM Mar 8th, 10:30 AM

Structural analysis of the Coxsackievirus B3 genome by in-line probing

Milo Bail Student Center Council Room

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 the 5’UTR. 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 subjected to in-line probing is visualized by 10% polyacrylamide gel electrophoresis and phosphorimaging. Because highly structured portions of the molecule are less vulnerable to nucleophilic attack, in-line probing analysis will determine the location of more structured regions. Results from in-line probing will enable us to better understand the threedimensional structure of the CVB3 genome, as well as its function.