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.

This is a preview of subscription content, log in to check access.

References

  1. 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.

    CAS  PubMed  Article  Google Scholar 

  2. 2.

    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.

    CAS  PubMed  Google Scholar 

  3. 3.

    International committee on taxonomy of viruses, 2014. http://ictvonlineorg/virustaxonomyasp.

  4. 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.

  5. 5.

    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.

    CAS  PubMed  Article  Google Scholar 

  6. 6.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    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.

    CAS  PubMed  Article  Google Scholar 

  8. 8.

    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.

    CAS  PubMed  Google Scholar 

  9. 9.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    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.

    PubMed  Article  Google Scholar 

  11. 11.

    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.

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    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.

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    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.

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    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.

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    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.

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    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.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    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.

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    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.

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    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.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    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.

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    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.

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    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.

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    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.

    CAS  PubMed  Article  Google Scholar 

  27. 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. 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.

    CAS  Google Scholar 

  29. 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.

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    Sambrook, J. and Russell, D.W., Molecular Cloning–a Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2006.

    Google Scholar 

  31. 31.

    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.

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Boonham, N. and Barker, I., Virus strain discrimination using recombinant antibodies, Dis. Markers, 2000, vol. 16, nos. 1–2, pp. 95–97.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    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.

    CAS  PubMed  Article  Google Scholar 

  34. 34.

    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.

    CAS  PubMed  Article  Google Scholar 

  35. 35.

    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.

    CAS  PubMed  Article  Google Scholar 

  36. 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.

    CAS  PubMed  Google Scholar 

  37. 37.

    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.

    PubMed  Article  Google Scholar 

  38. 38.

    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.

    CAS  PubMed  Google Scholar 

  39. 39.

    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.

    CAS  PubMed  Google Scholar 

  40. 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.

    CAS  Article  Google Scholar 

  41. 41.

    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.

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    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.

    CAS  Article  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Ch. V. Chandrika Ray.

Additional information

The article is published in the original.

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Keywords

  • Sugarcane streak mosaic virus
  • Poacevirus
  • NIa proteinase
  • polyclonal antiserum
  • cis-proteolytic activity