Characterization of RNA dependent RNA polymerase (RdRp) of Sesbania mosaic virus a member of genus Sobemovirus

Sesbania mosaic virus (SeMV) belongs to the genus sobemovirus and it infects sesbania grandiflora pers agathi. SeMV is a single stranded positive sense RNA virus, with 5\’ end of the genome covalently-linked viral protein called VPg (viral protein genome linked). The 3\’ end of the genomic RNA lacks a poly (A) tail and does not have t-RNA like structure. The genome of SeMV has three overlapping frames, a small ORF1 at 5\’ end (movement protein) and ORF3 at 3\’ terminus (coat protein). The central part of the genome has two overlapping ORFs, ORF2a and ORF2ab both coding for polyproteins, protease –Vpg-p10-p8 and protease-Vpg-RdRp respectively. ORF2b (RdRp) is expressed by -1 ribosomal frameshifting mechanism. Replication of sobemoviruses and the role of cis- and transacting elements are poorly understood. In order to elucidate the mechanism of replication, we cloned and overexpressed RdRp of SeMV in E. coli. His-tagged full length RdRp was found to be insoluble. Efforts to make the protein soluble under different conditions and by deletion of C-terminal hydrophobic residues were unsuccessful. Therefore RdRp was cloned as GST fusion protein. GST RdRp was soluble however; the purification was hampered by its inability to bind GSH beads. Hence, total crude E.coli lysate expressing GST-RdRp was used for functional analysis. We observed that the GST-RdRp was functionally active and it requires genomic RNA as template for polymerase function