No abstract

Sequence analysis revealed that rice dwarf phytoreovirus segment S12 is 1066 nucleotides long with a small out-of-phase, overlapping open reading frame (ORF) as well as a major ORF. The large ORF (positions 42 to 980) encodes 312 amino acids, while the small one (bases 313 to 591) encodes 92 amino acids with an additional in-frame AUG codon (positions 337-339) 24 nucleotides downstream from the first one. Transcripts from a full-length cDNA directed the in vitro synthesis of three polypeptides of 33 (considered to be translated from the long ORF), 8, and 7 kDa. Alteration of each of the two ATG codons on the small ORF demonstrated their involvement in the generation of the 8- and 7-kDa polypeptides. Although it is still unknown whether these proteins are expressed in vivo, the small ORF is shown to be conserved in S9s of two other members of the genus Phytoreovirus, rice gall dwarf virus and wound tumor virus, suggesting its common, important function.

Genomic segment S12 of rice dwarf virus and segment S9 of wound tumor virus, both members of the genus Phytoreovirus, have small out-of-phase overlapping open reading frames (ORFs). Western blot (immunoblot) analysis revealed that rice dwarf virus S12 mRNA specified translation products from the large ORF and two overlapping small ORFs both in rice plant hosts and in Spodoptera frugiperda insect cells. These results provide the first example of a tricistronic mRNA for a segmented double-stranded RNA virus. Similarly, wound tumor virus S9 mRNA was found to direct the synthesis of protein products from both the large ORF and small out-of-frame ORF in S. frugiperda cells. Results of site-specific and deletion mutagenesis studies were consistent with a leaky scanning translation mechanism for the synthesis of the small ORFs.

The complete nucleotide sequence of rice dwarf phytoreovirus genome segment 2 (S2) was determined to be 3,512 nucleotides long with one open reading frame initiating at nucleotide 15 and terminating at nucleotide 3363. The encoded polypeptide was predicted to have 1,116 residues with a relative molecular weight of 123 kD. Comparison of S2 of two isolates showed they had identical lengths and 97 and 98.3% nucleotide and amino acid sequence identities, respectively. A search of the Swiss-Prot data base (R 22.0) failed to find any proteins with significant homology to the S2-encoded protein. Determination of the nucleotide sequence of the S2 has completed the sequence determination of the genome of rice dwarf virus. Homology searches made for proteins encoded by each of the genomic segments showed that the polypeptide encoded by SI 1 has similarity to histone HI protein and VP6 of blue tongue virus, indicating it might possess nucleic acid binding properties.

The 46K outer capsid protein encoded by RNA segment $8 and the 42K polypeptide, previously thought to be the segment S9-encoded structural protein, were isolated from a rice dwarf phytoreovirus purified preparation, and then analysed by peptide mapping and electroblot-ELISA. Staphylococcus aureus V8 protease peptide mapping patterns of the 42K and 46K proteins were similar. Two monoclonal antibodies (MAbs), obtained after immunization with virus particles dissociated by 0.1% SDS, were each specific for both the 42K and 46K proteins. Furthermore, the MAbs bound common peptide fragments which were generated by digestion of the 42K and 46K proteins with V8 protease or proteinase K. These results strongly suggest that the 42K protein is not a gene product of $9 but a product overlapping with the 46K outer capsid protein. Whether the two proteins are functionally distinct remains to be determined.Rice dwarf phytoreovirus (RDV) (Boccardo & Milne, 1984) is considered to contain a genome composed of t2 segmented dsRNAs and seven structural proteins. The genomic RNAs are termed S1 to Sl2 in increasing order of mobility in a polyacrylamide gel. It was reported by Nakata et al. (1978) that the seven constituent proteins were encoded by S1, $2, $3, $5, $7, $8 and $9 based both on their potential coding capacities as monocistronic mRNAs and on the sizes of the polypeptides detected in the virus particles.