Cis-proteolytic activity of a recombinant nuclear inclusion a (NIa) proteinase from Sugarcane Streak Mosaic Virus, a member of the genus Poacevirus in the family Potyviridae

Abstract

The complete genome of Sugarcane streak mosaic virus-Andhra Pradesh isolate (SCSMV-AP), a member belonging to the genus Poacevirus of the family Potyviridae was previously sequenced. Among the non-structural proteins, nuclear inclusion protein a proteinase (NIa-Pro) is a multifunctional protein that plays an important role in the life cycle of Potyviridae members. In this study, N-terminal hexahistidine (His6)-tagged NIa-Pro gene of SCSMV-AP was amplified by RT-PCR, cloned, and expressed in Escherichia coli. Recombinant NIa-Pro was purified by Ni-NTA affinity chromatography and used to raise polyclonal antibodies. cis-Proteolytic activity of NIa-Pro was confirmed in vitro using a recombinant polyprotein substrate containing the virus protein genome-linked (VPg) and NIa cleavage site. Mutant VPgNIa constructs were generated by site-directed mutagenesis to investigate the role of conserved amino acid residues H242, D277 and H204 in the catalytic process of NIa proteinase. Such an analysis revealed that H242 and D277 constitute two of the predicted amino acid residues of the catalytic traid and H204 is probably a crucial amino acid at P1 position and confirmed the predicted unusual cleavage site (H/A) between VPg and NIa of SCSMV polyprotein.