THE M(R) 43K MAJOR CAPSID PROTEIN OF RICE RAGGED STUNT ORYZAVIRUS IS A POSTTRANSLATIONALLY PROCESSED PRODUCT OF A M(R) 67,348 POLYPEPTIDE ENCODED BY GENOME SEGMENT-8

The nucleotide sequence of DNA complementary to rice ragged stunt oryz avirus (RRSV) genome segment 8 (S8) of an isolate from Thailand was de termined. RRSV S8 is 1 914 bp in size and contains a single large open reading frame (ORF) spanning nucleotides 23 to 1 810 which is capable of encoding a protein of M(r) 67 348. The N-terminal amino acid seque nce of a similar to 43K virion polypeptide matched to that inferred fo r an internal region of the S8 coding sequence. These data suggest tha t the 43K protein is encoded by S8 and is derived by a proteolytic cle avage. Predicted polypeptide sizes from this possible cleavage of S8 p rotein are 26K and 42K. Polyclonal antibodies raised against a maltose binding protein (MBP)-S8 fusion polypeptide (expressed in Escherichia coli) recognised four RRSV particle associated polypeptides of M(r) 6 7K, 46K, 43K and 26K and all except the 26K polypeptide were also high ly immunoreactive to polyclonal antibodies raised against purified RRS V particles. Cleavage of the MBP-S8 fusion polypeptide with protease F actor X produced the expected 40K MBP and two polypeptides of apparent M(r) 46K and 26K. Antibodies to purified RRSV particles reacted stron gly with the intact fusion protein and the 46K cleavage product but we akly to the 26K product. Furthermore, in vitro transcription and trans lation of the S8 coding region revealed a post-translational self clea vage of the 67K polypeptide to 46K and 26K products. These data indica te that S8 encodes a structural polypeptide, the majority of which is auto-catalytically cleaved to 26K and 46K proteins. The data also sugg est that the 26K protein is the self cleaving protease and that the 46 K product is further processed or undergoes stable conformational chan ges to a similar to 43K major capsid protein.