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United States Department of Agriculture

Agricultural Research Service

Title: Determination of the P1 Proteinase Cleavage Site and Development of An Improved Gene Expression Vector Based on Wheat Streak Mosaic Virus

Authors
item Horken, Kempton
item Choi, Il-Ryong - FORMER USDA-ARS 5440-20-
item Stenger, Drake
item French, Roy

Submitted to: American Society for Virology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: March 22, 2001
Publication Date: July 28, 2001
Citation: Horken, K.M., Choi, I., Stenger, D.C., French, R.C. 2001. Determination of the p1 proteinase cleavage site and development of an improved gene expression vector based on wheat streak mosaic virus. American Society For Virology Meeting. Not published by meeting

Technical Abstract: The P1 proteinase domain and cleavage site in the wheat streak mosaic virus (WSMV)polyprotein were mapped using a series of amino- and carboxy-terminal truncations generated by coupled in vitro transcription-translation. Fine mapping identified the WSMV P1 proteinase self-cleavage site between amino acid residues Y-352 & G-353, so that mature WSMV P1 protein is considerably ylarger than P1 of tobacco etch virus(TEV).Polyprotein sequence alignment o three WSMV strains,a related tritimovirus (brome streak mosaic virus),and TEV revealed similarly spaced H,D, and S residues (essential for TEV P1 proteinase activity)& additional conserved motifs relative to P1 cleavage site. An infectious clone of WSMV was engineered to contain a unique Sal I site immediately downstream of the Y-352 codon(pWSMV-S1RN).GUS was inserted at this site(pWSMV-GUS-S1RN)such that GUS protein would be excised from the polyprotein by P1 proteinase at Y-352,& at the carboxy-end at an engineered NIa proteinase cleavage site. Expression and stability of GUS a this position was better than when GUS was inserted between WSMV NIb & CP. Inoculation of spring wheat (cv. Bobwhite) plants up to 60 days old resulted in systemic expression of GUS in both leaves & heads,indicating a broad plant age window for foreign gene expression by this gene vector. Additional gene expression vectors based on pWSMV-GUS-S1RN were constructed,with GUS at the P1 position and additional foreign gene inserts (~ 1kb in size)located between the NIb and CP cistrons. WSMV GUS-bearing gene vectors retained infectivity in wheat,yielded GUS expression patterns in roots & leaves similar to pWSMV-GUS-S1RN, & maintained the second insert for at least 30 days as assayed by RT-PCR. Thus,the WSMV gene vector is capable of expressing a reporter gene and a second gene of interest.

Last Modified: 12/21/2014
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