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Heterologous expression and isolation of influenza A virus nuclear export protein NEP

Biochemistry (Moscow) volume 82, pages 1529–1537 (2017)Cite this article

Abstract

Influenza A virus nuclear export protein NEP (NS2, 14.4 kDa) plays a key role in various steps of the virus life cycle. Highly purified protein preparations are required for structural and functional studies. In this study, we designed a series of Escherichia coli plasmid constructs for highly efficient expression of the NEP gene under control of the constitutive trp promoter. An efficient method for extraction of NEP from inclusion bodies based on dodecyl sulfate treatment was developed. Preparations of purified NEP with either N-or C-terminal (His)6-tag were obtained using Ni-NTA agarose affinity chromatography with yield of more than 20 mg per liter of culture. According to CD data, the secondary structure of the proteins matched that of natural NEP. A high propensity of NEP to aggregate over a wide range of conditions was observed.

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Abbreviations

a.a.:

amino acid residue

bp:

base pairs

IPTG:

isopropyl-β-D-thiogalactopyranoside

NEP:

nuclear export protein

NEP-C:

NEP with C-terminal (His)6-tag

NEP-N:

NEP with N-terminal (His)6-tag

PCR:

polymerase chain reaction

References

  1. Darapaneni, V., Prabhaker, V. K., and Kukol, A. (2009) Large-scale analysis of influenza A virus sequences reveals potential drug target sites of non-structural proteins, J. Gen. Virol., 90, 2124–2133.

    CAS  Article  PubMed  Google Scholar 

  2. Manz, B., Schwemmle, M., and Brunotte, L. (2013) Adaptation of avian influenza A virus polymerase in mammals to overcome the host species barrier, J. Virol., 87, 7200–7209.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Odagiri, T., and Tobita, K. (1990) Mutation in NS2, a nonstructural protein of influenza A virus, extragenically causes aberrant replication and expression of the PA gene and leads to generation of defective interfering particles, Proc. Natl. Acad. Sci. USA, 87, 5988–5992.

    CAS  PubMed  Google Scholar 

  4. Yasuda, J., Nakada, S., Kato, A., Toyoda, T., and Ishihama, A. (1993) Molecular assembly of influenza virus: association of the NS2 protein with virion matrix, Virology, 196, 249–255.

    CAS  Article  PubMed  Google Scholar 

  5. Iwatsuki-Horimoto, K., Horimoto, T., Fujii, Y., and Kawaoka, Y. (2004) Generation of influenza A virus NS2 (NEP) mutants with an altered nuclear export signal sequence, J. Virol., 78, 10149–10155.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Brunotte, L., Flies, J., and Bolte, H. (2014) The nuclear export protein of H5N1 influenza A viruses recruits matrix 1 (M1) protein to the viral ribonucleoprotein to mediate nuclear export, J. Biol. Chem., 289, 20067–20077.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Watanabe, K., Shimizu, T., Noda, S., Tsukahara, F., Maru, Y., and Kobayashi, N. (2014) Nuclear export of the influenza virus ribonucleoprotein complex: interaction of Hsc70 with viral proteins M1 and NS2, FEBS Open Bio, 4, 683–688.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Gorai, T., Goto, H., Noda, T., Watanabe, T., KozukaHata, H., Oyama, M., Takano, R., Neumann, G., Watanabe, S., and Kawaoka, Y. (2012) F1Fo-ATPase, Ftype proton-translocating ATPase, at the plasma membrane is critical for efficient influenza virus budding, Proc. Natl. Acad. Sci. USA, 109, 4615–4620.

    CAS  Google Scholar 

  9. Akarsu, H., Burmeister, W. P., Petosa, C., Petit, I., Muller, C. W., Ruigrok, R. W., and Baudin, F. (2003) Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2), EMBO J., 22, 4646–4655.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Akarsu, H., Iwatsuki-Horimoto, K., Noda, T., Kawakami, E., Katsura, H., Baudin, F., Horimoto, T., and Kawaoka, Y. (2011) Structure-based design of NS2 mutants for attenuated influenza A virus vaccines, Virus Res., 155, 240–248.

    CAS  Article  PubMed  Google Scholar 

  11. Lommer, B. S., and Luo, M. (2002) Structural plasticity in influenza virus protein NS2 (NEP), J. Biol. Chem., 277, 7108–7117.

    CAS  Article  PubMed  Google Scholar 

  12. Salahuddin, P., and Khan, A. U. (2010) Structural and functional analysis of NS1 and NS2 proteins of H1N1 subtype, Genomics Prot. Bioinform., 8, 190–199.

    CAS  Article  Google Scholar 

  13. Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M., Perez, D. R., Donis, R., Hoffmann, E., Hobom, G., and Kawaoka, Y. (1999) Generation of influenza A viruses entirely from cloned cDNAs, Proc. Natl. Acad. Sci. USA, 96, 9345–9350.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Ward, A. C., Castelli, L. A., Lucantoni, A. C., White, J. F., Azad, A. A., and Macreadie, I. G. (1995) Expression and analysis of the NS2 protein of influenza A virus, Arch. Virol., 140, 2067–2073.

    CAS  Article  PubMed  Google Scholar 

  15. Hu, Y., Liu, X., Zhang, A., Zhou, H., Liu, Z., Chen, H., and Jin, M. (2015) CHD3 facilitates vRNP nuclear export by interacting with NES1 of influenza A virus NS2, Cell Mol. Life Sci., 72, 971–982.

    CAS  Article  PubMed  Google Scholar 

  16. Tawaratsumida, K., Phan, V., Hrincius, E. R., High, A. A., Webby, R., Redecke, V., and Hacker, H. (2014) Quantitative proteomic analysis of the influenza A virus nonstructural proteins NS1 and NS2 during natural cell infection identifies PACT as an NS1 target protein and antiviral host factor, J. Virol., 88, 9038–9048.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Reuther, P., Giese, S., Gotz, V., Kilb, N., Manz, B., Brunotte, L., and Schwemmle, M. (2014) Adaptive mutations in the nuclear export protein of human-derived H5N1 strains facilitate a polymerase activity-enhancing conformation, J. Virol., 88, 263–271.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Shimizu, T., Takizawa, N., Watanabe, K., Nagata, K., and Kobayashi, N. (2011) Crucial role of the influenza virus NS2 (NEP) C-terminal domain in M1 binding and nuclear export of vRNP, FEBS Lett., 585, 41–46.

    CAS  Article  PubMed  Google Scholar 

  19. Inglis, S. C., Barrett, T., Brown, C. M., and Almond, J. W. (1979) The smallest genome RNA segment of influenza virus contains two genes that may overlap, Proc. Natl. Acad. Sci. USA, 76, 3790–3794.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Greenspan, D., Krystal, M., Nakada, S., Arnheiter, H., Lyles, D. S., and Palese, P. (1985) Expression of influenza virus NS2 nonstructural protein in bacteria and localization of NS2 in infected eucaryotic cells, J. Virol., 54, 833–843.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Koroleva, O. N., Dubrovin, E. V., Yaminsky, I. V., and Drutsa, V. L. (2016) Effect of DNA bending on transcriptional interference in the systems of closely spaced convergent promoters, Biochim. Biophys. Acta, 1860, 2086–2096.

    CAS  Article  PubMed  Google Scholar 

  22. Koroleva, O. N., Drutsa, V. L., Dolinnaya, N. G., Tsytovich, A. V., and Shabarova, Z. A. (1984) DNA-like duplexes containing repetitive sequences. VII. Chemicoenzymatic synthesis of polymers with fragment of natural promoters, Mol. Biol. (Moscow), 18, 146–160.

    CAS  Google Scholar 

  23. Maniatis, T., Fritsch E., and Sambroock, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory Press, N.Y.

    Google Scholar 

  24. Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680–685.

    CAS  Article  PubMed  Google Scholar 

  25. Bornhorst, J. A., and Falke, J. J. (2000) Purification of proteins using polyhistidine affinity tags, Methods Enzymol., 326, 245–254.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Hemsley, A., Arnheim, N., Toney, M. D., Cortopassi, G., and Galas, D. J. (1989) A simple method for site-directed mutagenesis using the polymerase chain reaction, Nucleic Acids Res., 17, 6545–6551.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Sivashanmugam, A., Murray, V., Cui, C., Zhang, Y., Wang, J., and Li, Q. (2009) Practical protocols for production of very high yields of recombinant proteins using Escherichia coli, Protein Sci., 18, 936–948.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Bass, S. H., and Yansura, D. G. (2000) Application of the E. coli trp promoter, Mol. Biotechnol., 16, 253–260.

    CAS  Article  PubMed  Google Scholar 

  29. Wang, X., Zhou, B., Hu, W., Zhao, Q., and Lin, Z. (2015) Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8, Microb. Cell Fact., 14,88.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Greenfield, N. J., and Fasman, G. D. (1969) Computed circular dichroism spectra for the evaluation of protein conformation, Biochemistry, 8, 4108–4116.

    CAS  Article  PubMed  Google Scholar 

  31. Richards, F. M., and Knowles, J. R. (1968) Glutaraldehyde as a protein cross-linkage reagent, J. Mol. Biol., 37, 231–233.

    CAS  Article  PubMed  Google Scholar 

  32. Kowalczyk, M., and Bardowski, J. (2003) Overproduction and purification of the CcpA protein from Lactococcus lactis, Acta Biochim. Pol., 50, 455–459.

    CAS  PubMed  Google Scholar 

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Author information

Affiliations

  1. Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics, 119991, Moscow, Russia

    A. O. Golovko

  2. Lomonosov Moscow State University, Faculty of Chemistry, 119991, Moscow, Russia

    O. N. Koroleva

  3. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 11991, Moscow, Russia

    V. L. Drutsa

Authors
  1. A. O. Golovko
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  2. O. N. Koroleva
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  3. V. L. Drutsa
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Corresponding author

Correspondence to O. N. Koroleva.

Additional information

Original Russian Text © A. O. Golovko, O. N. Koroleva, V. L. Drutsa, 2017, published in Biokhimiya, 2017, Vol. 82, No. 12, pp. 1907-1916.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM17-334, November 6, 2017.

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Golovko, A.O., Koroleva, O.N. & Drutsa, V.L. Heterologous expression and isolation of influenza A virus nuclear export protein NEP. Biochemistry Moscow 82, 1529–1537 (2017). https://doi.org/10.1134/S0006297917120124

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  • DOI: https://doi.org/10.1134/S0006297917120124

Keywords

  • influenza A virus
  • nuclear export protein (NEP)
  • affinity chromatography
  • protein aggregation