Abstract
We describe methods to express the nucleoprotein (NP) of Lassa fever virus (LASV) in E. coli, to purify and crystallize it using the sitting-drop vapor diffusion method. The crystals were screened using Rigaku micro-007 X-ray generator and a dataset was collected at a resolution of 2.36 Å. The crystals belong to space group P3, with the unit cell parameters a = b = 176.35 Å, c = 56.40 Å, α = β = 90°, and γ = 120°. Using the X-ray diffraction method, we constructed a three-dimensional structure of the LASV NP that should aid in the development of novel therapeutic strategies against this virus, for which vaccine and effective treatment modalities are currently unavailable.
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Charrel RN, de Lamballerie X, Emonet S (2008) Phylogeny of the genus Arenavirus. Curr Opin Microbiol 11(4):362–368
Khan SH, Goba A, Chu M, Roth C, Healing T, Marx A, Fair J, Guttieri MC, Ferro P, Imes T, Monagin C, Garry RF, Bausch DG, Mano River Union Lassa Fever Network (2008) New opportunities for field research on the pathogenesis and treatment of Lassa fever. Antiviral Res 78(1):103–115
Kitching A, Addiman S, Cathcart S, Bischop L, Krahé D, Nicholas M, Coakley J, Lloyd G, Brooks T, Morgan D, Turbitt D. (2009) A fatal case of Lassa fever in London, January 2009 Euro Surveill. 14(6). pii: 19117.
Haas WH, Breuer T, Pfaff G, Schmitz H, Köhler P, Asper M, Emmerich P, Drosten C, Gölnitz U, Fleischer K, Günther S (2003) Imported Lassa fever in Germany: surveillance and management of contact persons. Clin Infect Dis 36(10):1254–1258
Illick MM, Branco LM, Fair JN, Illick KA, Matschiner A, Schoepp R, Garry RF, Guttieri MC (2008) Uncoupling GP1 and GP2 expression in the Lassa virus glycoprotein complex: implications for GP1 ectodomain shedding. Virol J 5:161
Lenz O, ter Meulen J, Klenk HD, Seidah NG, Garten W (2001) The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P. Proc Natl Acad Sci U S A 98(22):12701–12705
Eichler R, Strecker T, Kolesnikova L, ter Meulen J, Weissenhorn W, Becker S, Klenk HD, Garten W, Lenz O (2004) Characterization of the Lassa virus matrix protein Z: electron microscopic study of virus-like particles and interaction with the nucleoprotein (NP). Virus Res 100(2):249–255
Mahanty S, Hutchinson K, Agarwal S, McRae M, Rollin PE, Pulendran B (2003) Cutting edge: impairment of dendritic cells and adaptive immunity by Ebola and Lassa viruses. J Immunol 170(6):2797–2801
Qi X, Lan S, Wang W, Schelde LM, Dong H, Wallat GD, Ly H, Liang Y, Dong C (2010) Cap binding and immune evasion revealed by Lassa nucleoprotein structure. Nature 468(7325):779–783
Huang Q, Shao J, Lan S, Zhou Y, Xing J, Dong C, Liang Y, Ly H (2015) In vitro and in vivo characterizations of pichinde viral nucleoprotein exoribonuclease functions. J Virol 89(13):6595–6607
Hastie KM, Kimberlin CR, Zandonatti MA, MacRae IJ, Saphire EO (2011) Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 3′ to 5′ exonuclease activity essential for immune suppression. PNAS 108(6):2396–2401
Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor University Press, Cold Spring Harbor, NY
Acknowledgments
We thank Dr. Louise Major for providing the pLou3 plasmid, Dr. Catherine Botting for assistance with mass spectroscopy, Dr. Zaitsev Slava for helping with data collection, and Dr. Bjoern Meyer for prompt editorial assistance. This work was supported in part by the NIAID/NIH through the new-direction awards mechanism of the SERCEB grant (U54-AI057157) to YL and HL, by the NIAID/NIH R01 AI083409 to YL, R01 AI093580 and R56 AI091805 to HL, and the Medical Research Council grant (G1100110/1) to CJD.
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Qi, X., Wang, W., Dong, H., Liang, Y., Dong, C., Ly, H. (2018). Expression and X-Ray Structural Determination of the Nucleoprotein of Lassa Fever Virus. In: Salvato, M. (eds) Hemorrhagic Fever Viruses. Methods in Molecular Biology, vol 1604. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6981-4_12
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DOI: https://doi.org/10.1007/978-1-4939-6981-4_12
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