The M(r) 43K major capsid protein of rice ragged stunt oryzavirus is a post-translationally processed product of a M(r) 67,348 polypeptide encoded by genome segment 8.
The nucleotide sequence of DNA complementary to rice ragged stunt oryzavirus (RRSV) genome segment 8 (S8) of an isolate from Thailand was determined. RRSVS8 is 1914 bp in size and contains a single large open reading frame (ORF) spanning nucleotides 23 to 1810 which is capable of encoding a protein of M(r) 67,348. The N-terminal amino acid sequence of a approximately 43K virion polypeptide matched to that inferred for an internal region of the S8 coding sequence. These data suggest that the 43K protein is encoded by S8 and is derived by a proteolytic cleavage. Predicted polypeptide sizes from this possible cleavage of S8 protein 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) 67K, 46K, 43K and 26K and all except the 26K polypeptide were also highly immunoreactive to polyclonal antibodies raised against purified RRSV particles. Cleavage of the MBP-S8 fusion polypeptide with protease Factor X produced the expected 40K MBP and two polypeptides of apparent M(r) 46K and 26K. Antibodies to purified RRSV particles reacted strongly with the intact fusion protein and the 46K cleavage product but weakly to the 26K product. Furthermore, in vitro transcription and translation of the S8 coding region revealed a post-translational self cleavage of the 67K polypeptide to 46K and 26K products. These data indicate that S8 encodes a structural polypeptide, the majority of which is auto-catalytically cleaved to 26K and 46K proteins. The data also suggest that the 26K protein is the self cleaving protease and that the 46K product is further processed or undergoes stable conformational changes to a approximately 43K major capsid protein.
CSIRO Division of Plant Industry, Canberra, Australia.
N M
NM
Upadhyaya
E
E
Zinkowsky
W
W
Kositratana
P M
PM
Waterhouse
D000595:Amino Acid Sequence; D000906:Antibodies; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D016679:Genome, Viral; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D012275:Oryza / Q000821:virology*; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012087:Reoviridae / Q000235:genetics* / Q000378:metabolism*; D013785:Thailand; D014771:Virion / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D017931:DNA Primers; D011993:Recombinant Fusion Proteins