RICE TUNGRO SPHERICAL VIRUS POLYPROTEIN PROCESSING - IDENTIFICATION OF A VIRUS-ENCODED PROTEASE AND MUTATIONAL ANALYSIS OF PUTATIVE CLEAVAGE SITES

Rice tungro spherical virus encodes a large polyprotein containing mot ifs with sequence similarity to viral serine-like proteases and RNA po lymerases. Polyclonal antisera raised against domains of the putative protease and polymerase in fusion with glutathione S-transferase detec ted a protein of about 35 kDa and, in very low amounts, a protein of a bout 70 kDa, respectively, in extracts from infected plants. In in vit ro transcription/translation systems and in Escherichia coil we demons trated a proteolytic activity in the C-terminal region of the polyprot ein. This protease rapidly cleaved its polyprotein precursors in vitro . Mutating a potential cleavage site located N-terminal to the proteas e domain, Gln(2526)-Asp(2527), diminished processing. The transversion mutation at the putative C-terminal cleavage site of the protease, at Gln(2852)-Ala(2853), led to a delayed and partial processing. (C) 199 8 Academic Press.