The nucleoprotein of severe fever with thrombocytopenia syndrome virus processes a stable hexameric ring to facilitate RNA encapsidation

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV), a member of the Phlebovirus genus from the Bunyaviridae family endemic to China, is the causative agent of life-threatening severe fever with thrombocytopenia syndrome (SFTS), which features high fever and hemorrhage. Similar to other negative-sense RNA viruses, SFTSV encodes a nucleocapsid protein (NP) that is essential for viral replication. NP facilitates viral RNA encapsidation and is responsible for the formation of ribonucleoprotein complex. However, recent studies have indicated that NP from Phlebovirus members behaves in inhomogeneous oligomerization states. In the present study, we report the crystal structure of SFTSV NP at 2.8 ? resolution and demonstrate the mechanism by which it processes a ringshaped hexameric form to accomplish RNA encapsidation. Key residues essential for oligomerization are identifi ed through mutational analysis and identifi ed to have a signifi cant impact on RNA binding, which suggests that correct formation of highly ordered oligomers is a critical step in RNA encapsidation. The fi ndings of this work provide new insights into the discovery of new antiviral reagents for Phlebovirus infection.

Keywords

SFTSV / nucleoprotein / oligomer / RNP as-KEYWORDS / sembly / crystal structure

References

[1] Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., Mc-Coy, A.J., Moriarty, N.W., Read, R.J., Sacchettini, J.C., Sauter, N.K., and Terwilliger, T.C. (2002). PHENIX: building new software for au-tomated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 58, 1948-1954 .10.1107/S0907444902016657
[2] Albertini, A.A., Wernimont, A.K., Muziol, T., Ravelli, R.B., Clapier, C.R., Schoehn, G., Weissenhorn, W., and Ruigrok, R.W. (2006). Crystal structure of the rabies virus nucleoprotein-RNA complex. Science 313, 360-363 .10.1126/science.1125280
[3] Baker, N.A., Sept, D., Joseph, S., Holst, M.J., and McCammon, J.A. (2001). Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A 98, 10037-10041 .10.1073/pnas.181342398
[4] Chen, Q., Wang, Q., Xiong, L., and Lou, Z. (2011). A structural view of the conserved domain of rice stress-responsive NAC1. Protein Cell 2, 55-63 .10.1007/s13238-011-1010-9
[5] Cheng, G. (2011). Innovator of in vitro virus culture!aDr. Chen-HsiangHuang. Protein Cell 2, 782-783 .10.1007/s13238-011-1110-6
[6] DeLano, W. (2002). The PyMOL molecular graphics system. De-Lano Scientific, San Carlos , CA, USA.
[7] Deng, B., Zhang, S., Geng, Y., Zhang, Y., Wang, Y., Yao, W., Wen, Y., Cui, W., Zhou, Y., Gu, Q., . (2012). Cytokine and chemokine levels in patients with severe Fever with thrombocytopenia syn-drome virus. PLoS One 7, e41365. 10.1371/journal.pone.0041365
[8] Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for mo-lecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126-2132 .10.1107/S0907444904019158
[9] Ferron, F., Li, Z., Danek, E.I., Luo, D., Wong, Y., Coutard, B., Lantez, V., Charrel, R., Canard, B., Walz, T., . (2011). The hexamer struc-ture of Rift Valley fever virus nucleoprotein suggests a mechanism for its assembly into ribonucleoprotein complexes. PLoS Pathog 7, e1002030. 10.1371/journal.ppat.1002030
[10] Gai, Z.T., Zhang, Y., Liang, M.F., Jin, C., Zhang, S., Zhu, C.B., Li, C., Li, X.Y., Zhang, Q.F., Bian, P.F., . (2012). Clinical Progress and Risk Factors for Death in Severe Fever with Thrombocytopenia Syndrome Patients. J Infect Dis 206, 1095-1102 .10.1093/infdis/jis472
[11] Green, T.J., Rowse, M., Tsao, J., Kang, J., Ge, P., Zhou, Z.H., and Luo, M. (2011). Access to RNA encapsidated in the nucleocapsid of ve-sicular stomatitis virus. J Virol 85, 2714-2722 .10.1128/JVI.01927-10
[12] Green, T.J., Zhang, X., Wertz, G.W., and Luo, M. (2006). Structure of the vesicular stomatitis virus nucleoprotein-RNA complex. Science 313, 357-360 .10.1126/science.1126953
[13] Guo, Y., Wang, W., Ji, W., Deng, M., Sun, Y., Zhou, H., Yang, C., Deng, F., Wang, H., Hu, Z., . (2012). Crimean-Congo hemorrhagic fever virus nucleoprotein reveals endonuclease activity in bunyavi-ruses. Proc Natl Acad Sci U S A 109, 5046-5051 .10.1073/pnas.1200808109
[14] Hastie, K.M., Liu, T., Li, S., King, L.B., Ngo, N., Zandonatti, M.A., Woods, V.L., Jr., de la Torre, J.C., and Saphire, E.O. (2011). Crystal structure of the Lassa virus nucleoprotein-RNA complex reveals a gating mechanism for RNA binding. Proc Natl Acad Sci U S A 108, 19365-19370 .10.1073/pnas.1108515108
[15] Jiao, Y., Zeng, X., Guo, X., Qi, X., Zhang, X., Shi, Z., Zhou, M., Bao, C., Zhang, W., Xu, Y., . (2011). Preparation and evaluation of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for detection of total antibodies in human and animal sera by double-antigen sandwich enzyme-linked immuno-sorbent assay. J Clin Microbiol 50, 372-377 .10.1128/JCM.01319-11
[16] Krissinel, E., and Henrick, K. (2007). Inference of macromolecular as-semblies from crystalline state. J Mol Biol 372, 774-797 .10.1016/j.jmb.2007.05.022
[17] Laskowski, R., MacArthur, M., Moss, D., and Thornton, J. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26, 283-291 .10.1107/S0021889892009944
[18] McCoy, A., Grosse-Kunstleve, R., Adams, P., Winn, M., Storoni, L., and Read, R. (2007). Phaser crystallographic software. J Appl Cryst 40, 658-674 .10.1107/S0021889807021206
[19] Ng, A.K., Zhang, H., Tan, K., Li, Z., Liu, J.H., Chan, P.K., Li, S.M., Chan, W.Y., Au, S.W., Joachimiak, A., . (2008). Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design. Faseb J 22, 3638-3647 .10.1096/fj.08-112110
[20] Nichol, S.T., Beaty, B.J., and Elliott, R.M. (2005). Family Bunyaviridae. In Virus taxonomy: classification and nomenclature of viruses, C.M . Fauquet, M.A. Mayo, J. Maniloff, U. Desselberger, and L.A. Ball, eds. (San Diego, CA, Elsevier Academic Press), pp. 695-716 .
[21] Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. In Macromolecular Crystallog-raphy, part A, C.W. Carter Jr., and R.M. Sweet, eds. (Academic Press) , pp. 307-326 .10.1016/S0076-6879(97)76066-X
[22] Qi, X., Lan, S., Wang, W., Schelde, L.M., Dong, H., Wallat, G.D., Ly, H., Liang, Y., and Dong, C. (2011). Cap binding and immune evasion revealed by Lassa nucleoprotein structure. Nature 468, 779-783 .10.1038/nature09605
[23] Raymond, D.D., Piper, M.E., Gerrard, S.R., and Smith, J.L. (2010). Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation. Proc Natl Acad Sci U S A 107, 11769-11774 .10.1073/pnas.1001760107
[24] Raymond, D.D., Piper, M.E., Gerrard, S.R., and Smith, J.L. (2012). Phleboviruses encapsidate their genomes by sequestering RNA bases. Proc Natl Acad Sci U S A 109, 19208-19213 .10.1073/pnas.1213553109
[25] Ren, L., Qin, X., Cao, X., Wang, L., Bai, F., Bai, G., and Shen, Y. (2011). Structural insight into substrate specificity of human intestinal maltase-glucoamylase. Protein Cell 2, 827-836 .10.1007/s13238-011-1105-3
[26] Rudolph, M.G., Kraus, I., Dickmanns, A., Eickmann, M., Garten, W., and Ficner, R. (2003). Crystal structure of the borna disease virus nucleoprotein. Structure 11, 1219-1226 .10.1016/j.str.2003.08.011
[27] Ruigrok, R.W., Crepin, T., and Kolakofsky, D. (2011). Nucleoproteins and nucleocapsids of negative-strand RNA viruses. Curr Opin Mi-crobiol 14, 504-510 .10.1016/j.mib.2011.07.011
[28] Stone, R. (2010). Infectious diseases. Rival teams identify a virus be-hind deaths in central China. Science 330, 20-21 .10.1126/science.330.6000.20
[29] Sun, Y., Guo, Y., and Lou, Z. (2012a). A versatile building block: The structures and functions of negative-sense single-stranded RNAvirus nucleocapsid proteins. Protein Cell 3, 893-902 .10.1007/s13238-012-2087-5
[30] Sun, Y., Jin, C., Zhan, F., Wang, X., Liang, M., Zhang, Q., Ding, S., Guan, X., Huo, X., Li, C., . (2012b). Host cytokine storm is as-sociated with disease severity of severe Fever with thrombocytope-nia syndrome. J Infect Dis 206, 1085-1094 .10.1093/infdis/jis452
[31] Xuan, C., Shi, Y., Qi, J., Zhang, W., Xiao, H., and Gao, G.F. (2011). Structural vaccinology: structure-based design of influenza A virus hemagglutinin subtype-specific subunit vaccines. Protein Cell 2, 997-1005 .10.1007/s13238-011-1134-y
[32] Ye, Q., Krug, R.M., and Tao, Y.J. (2006). The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444, 1078-1082 .10.1038/nature05379
[33] Yu, X.J., Liang, M.F., Zhang, S.Y., Liu, Y., Li, J.D., Sun, Y.L., Zhang, L., Zhang, Q.F., Popov, V.L., Li, C., . (2011). Fever with thrombocy-topenia associated with a novel bunyavirus in China. N Engl J Med 364, 1523-1532 10.1056/NEJMoa1010095