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ARS Home » Plains Area » Lincoln, Nebraska » Wheat, Sorghum and Forage Research » Research » Publications at this Location » Publication #124602
Title: MAPPING OF THE P1 PROTEINASE CLEAVAGE SITE IN THE POLYPROTEIN OF WHEAT STREAK MOSAIC TRITIMOVIRUS

Author
item CHOI, I - FORMER USDA-ARS 5440-20
item Horken, Kempton
item Stenger, Drake
item French, Roy

Submitted to: Journal of General Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/26/2001
Publication Date: 3/11/2002
Citation: Choi, I.R., Horken, K.M., Stenger, D.C., French, R.C. 2002. Mapping of the p1 proteinase cleavage site in the polyprotein of wheat streak mosaic tritimovirus. Journal Of General Virology 83:443-450.

Interpretive Summary: Wheat streak mosaic virus (WSMV) produces a single, large protein during infection which is cleaved into smaller functional units by several viral encoded proteinases. This work focused on one of these, the P1 proteinase. The cleavage site was mapped to be between amino acid residues Y352 and G353. The portion of the P1 protein required for enzymatic activity was found to be upstream of the cleavage site and contained several motifs tha are typical of serine proteinases. Identification of the P1 cleavage site allowed the WSMV genome to be manipulated such that a reporter gene (GUS) could be inserted immediately downstream of P1. WSMV tolerated this insertion and active GUS was excised from the virus protein in infected wheat plants. These results indicate that insertion of foreign genes downstream of WSMV P1 will provide an effective means for testing their expression in wheat and other cereals.

Technical Abstract: Monopartite members of the family Potyviridae utilize three viral-encoded proteinases to cleave the viral polyprotein into mature proteins. The amino-terminal region of the viral polyprotein is autolytically cleaved by the P1 proteinase. A domain required for Wheat streak mosaic virus (WSMV) P1 proteinase activity was mapped using a series of templates with nested 3'-truncations or 5'-deletions to program in vitro transcription- translation reactions. The WSMV P1 proteinase cleavage site was mapped to a position downstream of amino acid residue 348 and upstream of amino acid residue 353, with the peptide bond between amino acid residues Y352 and G353 the most probable site of hydrolysis. An alignment of potyvirus polyprotein sequences in the carboxy-terminal region of the P1 domain revealed WSMV P1 contained conserved H257, D267, S303, and FIVXG325-329 residues upstream of the cleavage site that are typical of serine proteinases and shown by others to be required for Tobacco etch virus P1 proteolysis. Insertion of the GUS reporter gene immediately downstream of the P1 cleavage site in a full-length clone of WSMV resulted in systemic infection and GUS expression upon inoculation of plants with in vitro transcripts. When cleaved by P1 at the amino-terminus and NIa proteinase at a site engineered in the carboxy-terminus, active GUS protein expressed by WSMV in infected wheat was shown have electrophoretic mobility similar to wild type GUS protein.