Rice Tungro Spherical Virus Polyprotein Processing: Identification of a Virus-Encoded Protease and Mutational Analysis of Putative Cleavage Sites

Abstract

Rice tungro spherical virus encodes a large polyprotein containing motifs with sequence similarity to viral serine-like proteases and RNA polymerases. Polyclonal antisera raised against domains of the putative protease and polymerase in fusion with glutathioneS-transferase detected a protein of about 35 kDa and, in very low amounts, a protein of about 70 kDa, respectively, in extracts from infected plants. Inin vitrotranscription/translation systems and inEscherichia coliwe demonstrated a proteolytic activity in the C-terminal region of the polyprotein. This protease rapidly cleaved its polyprotein precursorsin vitro.Mutating a potential cleavage site located N-terminal to the protease domain, Gln2526-Asp2527, diminished processing. The transversion mutation at the putative C-terminal cleavage site of the protease, at Gln2852-Ala2853, led to a delayed and partial processing.