Molecular cloning, sequencing, functional analysis and expression in E. coli of major core protein gene (S3) of rice...

The complete nucleotide sequence of major core protein gene (segment S3) of rice dwarf virus (RDV) Chinese isolate was determined after cDNA cloning from the viral genomic RNA. Sequence analysis showed that the cloned fragment is 3195 bp in length and contains a single open reading frame (ORF), encoding the major core protein (P3) which M(r) of 114 K. The nucleotide and deduced amino acid sequences of S3 of this isolate share significant homology (94.1% and 97%, respectively) with those of S3 of the Japanese isolate. At the amino acid level, P3 of RDV Chinese isolate shares significant homology with P3 of rice gall dwarf virus (RGDV), significant regional homology with the rotavirus penetration, and homology with spheroidin of amsacta entomopoxvirus (SPH), which is the major protein of the occlusion body, with clp-like ATP-dependent protease binding subunit and with ATP-dependent protease ATP-binding subunit. Amino acid sequence analysis also showed that P3 contains RNA-dependent RNA polymerase (RDRP) motif-like elements such as DXXXD, SGXXXXXXN, GDD and ENXXXY. These results may suggest that P3 is a multifunctional protein which plays very important roles in the virus structure formation, virus replication and penetration processes. The full length cDNA sequence of RDV S3 and a partial one which covers nt 1004-3195 were cloned into bacterial expression vector pTrcHisB for expression. The full length cDNA sequence failed to be expressed in E. coli, but the partial sequence was successfully expressed there as confirmed by the Western blot analysis. Further analysis of RDV P3 is under way.