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
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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.
KeywordsSugarcane streak mosaic virus Poacevirus NIa proteinase polyclonal antiserum cis-proteolytic activity
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- 1.Hema, M., Joseph, J., Gopinath, K., Sreenivasulu, P., and Savithri, H.S., Molecular characterization and interviral relationships of a flexuous filamentous virus causing mosaic disease of sugarcane (Saccharum officinarum L.) in India, Arch. Virol., 1999, vol. 144, no. 3, pp. 479–490.PubMedCrossRefGoogle Scholar
- 3.International committee on taxonomy of viruses, 2014. http://ictvonlineorg/virustaxonomyasp.Google Scholar
- 4.Subbareddy, Ch.V., Complete genome sequence analysis of Sugarcane streak mosaic virus (SStMV), development of duplex IC-RT-PCR for detection and discrimination of SStMV and Sugarcane mosaic virus (SCMV) and generation of SStMV-free sugarcane (Saccharum spp. Hybrid) by in vitro meristem tip culture technology, Ph. D. Thesis., Tirupati: Sri Venkateswara Univ., 2009.Google Scholar
- 27.Adams, M.J., Zerbini, F.M., French, R., Rabenstein, F., Stenger, D.C., and Valkonen, J.P.T., Potyviridae, in Virus Taxonomy, Adams, A.M.Q., Wada, M.J., Carstens, E.B., and Lefkowitz, E.J., Eds., Oxford: Elsevier, 2011, pp. 1069–1090.Google Scholar
- 28.Hwang, D.C., Kim, D.H., Kang, B.H., Song, B.D., and Choi, K.Y., Molecular cloning, expression, and purification of nuclear inclusion A proteinase from Tobacco vein mottling virus, Mol. Cell, 2000, vol. 10, no. 2, pp. 148–155.Google Scholar
- 29.Parks, T.D., Howard, E.D., Wolpert, T.J., Arp, D.J., and Dougherty, W.G., Expression and purification of a recombinant Tobacco etch virus NIa proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form, Virology, 1995, vol. 210, no. 1, pp. 194–201.PubMedCrossRefGoogle Scholar
- 30.Sambrook, J. and Russell, D.W., Molecular Cloning–a Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2006.Google Scholar
- 36.Schaad, M.C., Haldeman-Cahill, R., Cronin, S., and Carrington, J.C., Analysis of the VPg-proteinase (NIa) encoded by tobacco etch potyvirus: Effects of mutations on subcellular transport, proteolytic processing, and genome amplification, J. Virol., 1996, vol. 70, no. 10, pp. 7039–7048.PubMedGoogle Scholar
- 40.Nallamsetty, S., Kapust, R.B., Tözsér, J., Cherry, S., Tropea, J.E., Copeland, T.D., and Waugh, D.S., Efficient site-specific processing of fusion proteins by Tobacco vein mottling virus protease in vivo and in vitro, Protein Expression Purif., 2004, vol. 38, no. 1, pp. 108–115.CrossRefGoogle Scholar