Site Directed Mutagenesis of a Putative Protease Cleavage Site Within the 105 Kilodation Protein of Southern Bean Mosaic Virus

There are four open reading frames (ORF) in three positive-sense translational phases of Southern Bean Mosaic Virus (SBMV). ORF2 codes for a 105 KDa polyprotein which is proposed to be autocatalytically cleaved into smaller functional proteins. Amino acid sequence homology between the SBMV and poliovirus, foot and mouth disease virus, and cowpea mosaic virus reveal that SBMV has a similar genomic organization to picornaviruses with a conserved order: Vpg (Viral protein, genome-linked )-protease-replicase. Two Gln-Ser (QS) amino acid pairs within the 105 KDa polyprotein, C75= 930-936, C60= 1305-1310, are proposed to be the cleavage sites based on 1) similar structural arrangement around the two QS pairs to that of known cleavage sites of picornaviruses and potyviruses; 2) computer prediction of the cleavage products from the SBMV RNA sequence; 3) observed in vitro translation products. In order to investigate the role of the C60 QS pair in the proteolytic processing of the 105 KDa polyprotein, a mutagenic oligo was designed to create various amino acid substitutions in the QS pair. A coupled in vitro transcription/translation system was used to produce the protein products of both wildtype and mutants. During the procedure, tritiated leucine was incorporated into the protein products. The protein products were separated by SDS-PAGE and the cleavage patterns were detected after autoradiography. Two substitutions of the C60 QS pair-Pro-Val (PS) in mutant6, Gin-Pro (QP) in mutant19-reduced the yield of the 60 KDa protein by different levels. This result supports the hypothesis that the C60 QS is a cleavage site.

Name thesis:degree_name

Master of Science

Discipline thesis:degree_discipline

Department of Biology

Year

1995

Author dc:creator

• Shi, Jingyu

• Medical Sciences

Repository record dc:identifier

https://digitalcommons.wku.edu/theses/884

OAI identifier oai:identifier

oai:digitalcommons.wku.edu:theses-1887