Sesbania mosaic virus (SeMV) polyprotein was shown to undergo proteolytic processing when expressed in E. coli. Mutational analysis of the proposed catalytic triad residues (H181, D216, and S284) present in the N-terminal serine protease domain of the polyprotein showed that the protease was indeed responsible for this processing. Analysis of the cleavage site mutants confirmed the cleavage between protease-viral protein genome linked (VPg) and VPg-RNA-dependent RNA polymerase (RdRP) at E(325)-T(326) and E(402)-T(403) sites, respectively. An additional suboptimal cleavage at E(498)-S(499) site was also identified which resulted in the further processing of RdRP to 10- and 52-kDa proteins. Thus, the protease has both E-T and E-S specificities. The polyprotein has a domain arrangement of protease-VPg-p10-RdRP, which is cleaved by the protease. The purified serine protease was also active in trans and cleaved the polyprotein at the same specific sites. These results demonstrate that the serine protease domain is responsible for the processing of SeMV polyprotein both in cis and in trans.