title: "Natively unfolded" VPg is essential for Sesbania mosaic virus serine protease activity.
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.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore.
authors.forename: Panayampalli Subbian
authors.initials: PS
authors.lastname: Satheshkumar
authors.forename: Pananghat
authors.initials: P
authors.lastname: Gayathri
authors.forename: Kasaragod
authors.initials: K
authors.lastname: Prasad
authors.forename: Handanahal Subbarao
authors.initials: HS
authors.lastname: Savithri
mesh_terms: D000595:Amino Acid Sequence; D001703:Biophysics / Q000379:methods; D002384:Catalysis; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017353:Gene Deletion; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D016366:Open Reading Frames; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics*; D014780:Viruses / Q000201:enzymology*
chemical_list: D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D010649:Phenylalanine; D012697:Serine Endopeptidases; C078807:nuclear scaffold serine protease