Structure and function of Sesbania mosaic virus serine protease domain

Abstract

Poly-protein processing is a common strategy used by many viruses to generate different functional products from a single protein. Viral proteases play a crucial role in this maturation process. 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-VPg, VPg-P10 and P10-P8 at E{325}T{326}, E {402}T{403}and E{498}S{499}sites, respectively. Thus, the protease has both E-T and E-S specificities. The polyprotein has a domain arrangement of Protease-VPg-P10-P8, which is cleaved by the protease. The purified serine protease was not active in trans. Interestingly, the protease domain exhibited trans-catalytic activity when VPg (viral protein genome-linked) was present at its C-terminus. Bioinformatic analysis of VPg primary structure suggested that it could be a disordered protein. Biophysical studies validated this observation and VPg resembled "natively unfolded" proteins. CD spectral analysis of DN70Pro-VPg fusion protein showed a positive CD peak at 230 nm, suggestive of some aromatic interaction between protease and VPg. Mutation of W43 in the VPg domain to phenylalanine/alanine abrogated the positive peak with concomitant loss in cis and trans proteolytic activities of the DN70Pro domain. Further, deletion of VPg domain from the polyprotein completely abolished proteolytic processing. The results suggest a novel mechanism of activation of the protease, wherein the interaction between the natively unfolded VPg and the protease domains via aromatic amino acid residues alters the conformation of the individual domains and the active site of the protease. Thus, VPg is an activator of protease in SeMV and probably by this mechanism the polyprotein processing could be regulated in planta. The three dimensional structure of the SeMV protease has revealed the residues involved in substrate binding (H298, T279, N308, R309), catalysis (H181, D216 and S284) and interaction with VPg (F269, W271, Y315, Y319). Mutational analysis of the residues forming the substrate binding pocket suggest that residues H298, T279 and R308 are absolutely required for the protease activity whereas N308 may not be so crucial. Further to delineate the interacting partners of W43, mutants of the above mentioned aromatic residues were generated. Results suggest that F269 and W271 residues of protease domain are involved in aromatic stacking interaction with VPg.