Site-Directed Mutagenesis of Southern Bean Mosaic Virus (Cowpea Strain): in Search of a Viral Protease
type: CreativeWork
description: The second and largest open reading frame within the southern bean mosaic virus (SBMV) genome encodes a 105 kDa polyprotein. Following translation, the polyprotein is cleaved to liberate various proteins necessary for SBMV replication. The elements within polyproteins of the picornavirus superfamily members have a conserved order: Vpg (viral protein, genome-linked)-protease-replicase. Amino acid sequence homologies indicate that the 105 kDa protein of SBMV contains a replicase very similar to those identified in polyproteins of the picorna-like viruses. The presence of a VPg covalently attached to the 5' end of the SBMV genome further suggests that SBMV may be considered a member of the picornavirus superfamily-Serine 558 within the SBMV polyprotein has been proposed to be a catalytic residue of a serine protease. Site-directed mutagenesis was used to create a mutant with a glycine at this position, and coupled in vitro transcription/translation was used to prepare 3H labeled translation products. SDS-PAGE and fluorography were then used to assay for the presence or absence of polyprotein cleavage. Although site-directed mutagenesis was successful in creating the mutant, a possible deletion that complicated the interpretation of the results was identified.
abstract: The second and largest open reading frame within the southern bean mosaic virus (SBMV) genome encodes a 105 kDa polyprotein. Following translation, the polyprotein is cleaved to liberate various proteins necessary for SBMV replication. The elements within polyproteins of the picornavirus superfamily members have a conserved order: Vpg (viral protein, genome-linked)-protease-replicase. Amino acid sequence homologies indicate that the 105 kDa protein of SBMV contains a replicase very similar to those identified in polyproteins of the picorna-like viruses. The presence of a VPg covalently attached to the 5' end of the SBMV genome further suggests that SBMV may be considered a member of the picornavirus superfamily-Serine 558 within the SBMV polyprotein has been proposed to be a catalytic residue of a serine protease. Site-directed mutagenesis was used to create a mutant with a glycine at this position, and coupled in vitro transcription/translation was used to prepare 3H labeled translation products. SDS-PAGE and fluorography were then used to assay for the presence or absence of polyprotein cleavage. Although site-directed mutagenesis was successful in creating the mutant, a possible deletion that complicated the interpretation of the results was identified.
author: Simmons, Alan
keywords: Medical Sciences
date published: 1995-01-01T08:00:00Z
date modified: 2026-07-24T06:07:54Z
provider: Western Kentucky University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Western Kentucky University