Functional insights into the role of C-terminal disordered domain of Sesbania mosaic virus RNA-dependent RNA polymerase and the coat protein in viral replication in vivo.

The C-terminal disordered domain of sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) interacts with the viral protein P10. The functional significance of this interaction in viral replication was examined by a comparative analysis of genomic and sub-genomic RNA levels (obtained by quantitative real time PCR) in the total RNA extracted from Cyamopsis plants agro-infiltrated with wild-type or mutant forms of SeMV infectious cDNA (icDNA). The sgRNA copy numbers were found to be significantly higher than those of gRNA in the wild-type icDNA transfected plants. Transfection of a mutant icDNA expressing an RdRp lacking the C-terminal disordered domain led to a drastic reduction in the copy numbers of both forms of viral RNA. This could be due to the loss of interaction between the disordered domain of RdRp and P10 and possibly other viral/host proteins that might be required for the assembly of viral replicase. The C-terminal disordered domain also harbours the motif E which is essential for the catalytic function of RdRp. Mutation of the conserved tyrosine within this motif in the full length icDNA resulted in complete inhibition of progeny RNA synthesis in the transfected plants confirming the importance of motif E in the polymerase function in vivo. The role of coat protein (CP) in viral infection was also investigated by agro-infiltration of a CP start codon mutant icDNA which suggested that CP is essential for the encapsidation of viral progeny RNAs at later stages of infection.