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.
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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.
Xu, D.L., Zhou, G.H., Xie, Y.J., Mock, R., and Li, R., Complete nucleotide sequence and taxonomy of Sugarcane streak mosaic virus, member of a novel genus in the family Potyviridae, Virus Genes, 2010, vol. 40, no. 3, pp. 432–439.
International committee on taxonomy of viruses, 2014. http://ictvonlineorg/virustaxonomyasp.
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.
Tatineni, S., Ziems, A.D., Wegulo, S.N., and French, R., Triticum mosaic virus: A distinct member of the family Potyviridae with an unusually long leader sequence, Phytopathology, 2009, vol. 99, no. 8, pp. 943–950.
Carrington, J.C., Freed, D.D., and Sanders, T.C., Autocatalytic processing of the potyvirus helper component proteinase in Escherichia coli and in vitro, J. Virol., 1989, vol. 63, no. 10, pp. 4459–4463.
Mavankal, G. and Rhoads, R.E., In vitro cleavage at or near the N-terminus of the helper component protein in the Tobacco vein mottling virus polyprotein, Virology, 1991, vol. 185, no. 2, pp. 721–731.
Anindya, R. and Savithri, H.S., Potyviral NIa proteinase, a proteinase with novel deoxyribonuclease activity, J. Biol. Chem., 2004, vol. 279, no. 31, pp. 32159–32169.
Carrington, J.C., Cary, S.M., and Dougherty, W.G., Mutational analysis of Tobacco etch virus polyprotein processing: cis- and trans-proteolytic activities of polyproteins containing the 49-kilodalton proteinase, J. Virol., 1988, vol. 62, no. 7, pp. 2313–2320.
Daròs, J.A. and Carrington, J.C., RNA binding activity of NIa proteinase of tobacco etch potyvirus, Virology, 1997, vol. 237, no. 2, pp. 327–336.
Urcuqui-Inchima, S., Haenni, A.L., and Bernardi, F., Potyvirus proteins: A wealth of functions, Virus Res., 2001, vol. 74, nos. 1–2, pp. 157–175.
Riechmann, J.L., Laín, S., and García, J.A., Highlights and prospects of potyvirus molecular biology, J. Gen. Virol., 1992, vol. 73, no. 1, pp. 1–16.
Murphy, J.F., Rhoads, R.E., Hunt, A.G., and Shaw, J.G., The VGg of Tobacco etch virus RNA is the 49-kDa proteinase or the N-terminal 24-kDa part of the proteinase, Virology, 1990, vol. 178, no. 1, pp. 285–288.
Parks, T.D. and Dougherty, W.G., Substrate recognition by the NIa proteinase involves multiple domains: characterization using genetically engineered hybrid proteinase molecules, Virology, 1991, vol. 182, no. 1, pp. 17–27.
Carrington, J.C. and Dougherty, W.G., Small nuclear inclusion protein encoded by a plant potyvirus genome is a proteinase, J. Virol., 1987, vol. 61, no. 8, pp. 2540–2548.
Gao, L., Shen, W., Yan, P., Tuo, D., Li, X., and Zhou, P., Nia-pro of Papaya ring spot virus interacts with papaya methionine sulfoxide reductase B, Virology, 2012, vol. 434, no. 1, pp. 78–87.
Bazan, J.F. and Fletterick, R.J., Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications, Proc. Natl. Acad. Sci. USA, 1988, vol. 85, no. 21, pp. 7872–7876.
Dougherty, W.G., Carrington, J.C., Cary, S.M., and Parks, T.D., Biochemical and mutational analysis of a plant virus polyprotein cleavage site, EMBO J., 1988, vol. 7, no. 5, pp. 1281–1287.
Jebasingh, T., Pandaranayaka, E.P., Mahalakshmi, A., Kasin Yadunandam, A., Krishnaswamy, S., and Usha, R., Expression, purification and molecular modelling of the NIa proteinase of Cardamom mosaic virus, J. Biomol. Struct. Dyn., 2013, vol. 31, no. 6, pp. 602–611.
Phan, J., Zdanov, A., Evdokimov, A.G., Tropea, J.E., Peters, H.K., Kapust, R.B., Li, M., Wlodawer, A., and Waugh, D.S., Structural basis for the substrate specificity of Tobacco etch virus proteinase, J. Biol. Chem., 2002, vol. 277, no. 52, pp. 50564–50572.
Sun, P., Austin, B.P., Tözsér, J., and Waugh, D.S., Structural determinants of Tobacco vein mottling virus protease substrate specificity, Protein Sci., 2010, vol. 19, no. 11, pp. 2240–2251.
Dougherty, W.G. and Parks, T.D., Post-translational processing of the Tobacco etch virus 49-kDa small nuclear inclusion polyprotein: identification of an internal cleavage site and delimitation of VPg and proteinase domains, Virology, 1991, vol. 183, no. 2, pp. 449–456.
Kim, D.H., Hwang, D.C., Kang, B.H., Lew, J., Han, J., Song, B.D., and Choi, K.Y., Effects of internal cleavages and mutations in the C-terminal region of NIa proteinase of turnip mosaic potyvirus on the catalytic activity, Virology, 1996, vol. 226, no. 2, pp. 183–190.
Dougherty, W.G. and Semler, B.L., Expression of virus-encoded proteinases: Functional and structural similarities with cellular enzymes, Microbiol. Rev., 1993, vol. 57, no. 4, pp. 781–822.
Joseph, J. and Savithri, H.S., Mutational analysis of the NIa proteinase from pepper vein banding potyvirus, Arch. Virol., 2000, vol. 145, no. 12, pp. 2493–2502.
Adams, M.J., Antoniw, J.F., and Beaudoin, F., Overview and analysis of the polyprotein cleavage sites in the family Potyviridae, Mol. Plant Pathol., 2005, vol. 6, no. 4, pp. 471–487.
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.
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.
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.
Sambrook, J. and Russell, D.W., Molecular Cloning–a Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2006.
Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, vol. 227, no. 5259, pp. 680–685.
Boonham, N. and Barker, I., Virus strain discrimination using recombinant antibodies, Dis. Markers, 2000, vol. 16, nos. 1–2, pp. 95–97.
Hust, M., Maiss, E., Jacobsen, H.J., and Reinard, T., The production of a genusspecific recombinant antibody (scFv) using a recombinant potyvirus protease, J. Virol. Methods, 2002, vol. 106, no. 2, pp. 225–233.
Rouis, S., Lafaye, P., Jaoua-Aydi, L., Sghaier, Z., Ayadi, H., and Gargouri-Bouzid, R., Cloning and expression of functional single-chain Fv antibodies directed against NIa and coat proteins of Potato virus Y, J. Virol. Methods, 2006, vol. 137, no. 1, pp. 1–6.
Gargouri-Bouzid, R., Jaoua, L., Rouis, S., Saïdi, M.N., Bouaziz, D., and Ellouz, R., PVY-resistant transgenic potato plants expressing an anti-NIa protein scFv antibody, Mol. Biotechnol., 2006, vol. 33, no. 2, pp. 133–140.
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.
García, J.A., Laín, S., Cervera, M.T., Riechmann, J.L., and Martín, M.T., Mutational analysis of plum pox potyvirus polyprotein processing by the NIa proteinase in Escherichia coli, J. Gen. Virol., 1990, vol. 71, no. 12, pp. 2773–2779.
Kim, D.H., Park, Y.S., Kim, S.S., Lew, J., Nam, H.G., and Choi, K.Y., Expression, purification, and identification of a novel self-cleavage site of the Nla C-terminal 27-kDa protease of turnip mosaic potyvirus C5, Virology, 1995, vol. 213, no. 12, pp. 517–525.
Mathur, C. and Savithri, H.S., Novel ATPase activity of the polyprotein intermediate, Viral Protein genomelinked- Nuclear Inclusion-a protease, of pepper vein banding potyvirus, Biochem. Biophys. Res. Commun., 2012, vol. 427, no. 1, pp. 113–118.
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.
Parks, T.D., Leuther, K.K., Howard, E.D., Johnston, S.A., and Dougherty, W.G., Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase, Anal. Biochem., 1994, vol. 216, no. 2, pp. 413–487.
Zheng, N., Pérezjde, J., Zhang, Z., Domínguez, E., Garcia, J.A., and Xie, Q., Specific and efficient cleavage of fusion proteins by recombinant Plum pox virus NIa protease, Protein Expression Purif., 2008, vol. 57, no. 2, pp. 153–162.
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Chandrika Ray, C.V., Hema, M. 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 . Mol. Genet. Microbiol. Virol. 31, 102–108 (2016). https://doi.org/10.3103/S0891416816020038
- Sugarcane streak mosaic virus
- NIa proteinase
- polyclonal antiserum
- cis-proteolytic activity