Abstract
Polyprotein processing is a major strategy used by many plant and animal viruses to maximize the number of protein products obtainable from a single open reading frame. In Sesbania mosaic virus, open reading frame-2 codes for a polyprotein that is cleaved into different functional proteins in cis by the N-terminal serine protease domain. The soluble protease domain lacking 70-amino-acid residues from the N terminus (deltaN70Pro, where Pro is protease) was not active in trans. Interestingly, the protease domain exhibited trans-catalytic activity when VPg (viral protein genome-linked) was present at the 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 showed that the deltaN70Pro-VPg fusion protein had a characteristic secondary structure with a 230 nm positive CD peak. Mutation of Trp-43 in the VPg domain to phenylalanine abrogated the positive peak with concomitant loss in cis- and trans-proteolytic activity of the deltaN70Pro domain. Further, deletion of VPg domain from the polyprotein completely abolished proteolytic processing. The results suggested 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 Sesbania mosaic virus, and probably by this mechanism, the polyprotein processing could be regulated in planta.
Highlights
Sesbania mosaic virus (SeMV)1 belongs to the genus Sobemovirus, which infects Sesbania grandiflora [1]
The results suggested a novel mechanism of activation of the protease, wherein the interaction between the natively unfolded viral protein genome-linked (VPg) and the protease domains via aromatic amino acid residues alters the conformation of the individual domains and the active site of the protease
VPg is an activator of protease in Sesbania mosaic virus, and probably by this mechanism, the polyprotein processing could be regulated in planta
Summary
SeMV, Sesbania mosaic virus; VPg, viral protein genome-linked; Pro, protease; Ni-NTA, nickel-nitrilotriacetic acid; RdRP, RNA-dependent RNA polymerase; IF, initiation factor. The Pro domain is responsible for the proteolytic maturation of the polyprotein and cleaves between E-T and E-S amino acid residues. Expression of the protease along with the N-terminal extension corresponding to the VPg sequence enhanced its proteolytic activity [5]. When present at the C terminus of the protease domain (⌬N70Pro-VPg), VPg modulates both cis- and trans-catalytic activities of the protease by inducing a. The results presented suggested that the natively unfolded VPg regulates the proteolytic maturation of the polyprotein in sobemoviruses
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