ABSTRACT
The ORF of genome segment 6 (S6) of rice ragged stunt oryzavirus (RRSV) Philippines isolate was cloned and sequenced based on the S6 sequence of the Thailand isolate. Pns6, the 71 kD product of S6 expressed in E. coli, was demonstrated to be a viral non-structural protein of RRSV by Western blotting. The gel mobility shift assays showed that Pns6 had nucleic acid binding activity. Pns6 could interact with single- and double-stranded forms of DNA and RNA, showing a preference for single-stranded nucleic acid and a slight preference for RRSV ssRNA over the rice ssRNA, as demonstrated by both competition and displacement assays. The binding of Pns6 to nucleic acids is strong and sequence non-specific. By using five truncated derivatives of Pns6, it was found that the basic region from amino acid 201 to 273 of Pns6 was the unique nucleic acid binding domain. Subcellular fractionation of leaf tissues of RRSV-infected rice plants and subsequent Western blotting had shown that Pns6 accumulated predominately in the cytoplasmic membrane fraction. The possible role of RRSV Pns6 in virus replication and assembly is discussed.
Subject(s)
Genome, Viral , Nucleic Acids/metabolism , Oryza/virology , Reoviridae/genetics , Amino Acid Sequence , Base Sequence , Blotting, Western , DNA Primers , DNA, Viral/genetics , Electrophoresis, Agar Gel , Escherichia coli/genetics , Molecular Sequence Data , Open Reading Frames , RNA, Viral/genetics , Recombinant Proteins/genetics , Subcellular FractionsABSTRACT
Rice ragged stunt oryzavirus (RRSV) is a member of the genus oryzavirus within the family Reoviridae. Its genome consists of ten segments of dsRNA. The functions of all products encoded by these viral genome segments, except one encoded by S9, have not yet been elucidated. In the present study, the ORF of S 8 of RRSV-Philippines isolate was sequenced and expressed in E. coli. The 67 kD product of S8 could be self-cleaved into two fragments with molecular weights of 43 kD and 26 kD. Western blotting indicated that both 67 kD and 43 kD products were major structural proteins of the virus. It was also found that the 67 kD protein could self aggregate into aggregates with higher sedimentation rate in sucrose gradients during centrifugation. Moreover, the self-aggregation process could be accelerated by the complex of S6 product and genome dsRNAs of RRSV. These results suggest that the S8 products, 67 kD or 43 kD, may be the structural components of the viral inner-capsid.
