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Monopartite members of the family Potyviridae utilize three virus-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 P1 proteinase activity of Wheat streak mosaic virus (WSMV) 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 Y 352 and G 353 the most probable site of hydrolysis. An alignment of potyvirus polyprotein sequences in the carboxyterminal region of the P1 domain revealed WSMV P1 contained conserved H 257, D 267, S 303 and FIVXG 325-329 residues upstream of the cleavage site that are typical of serine proteinases and shown by others to be required for P1 proteolysis in Tobacco etch virus. 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 had electrophoretic mobility similar to wild-type GUS protein.