Abstract
The genome of rice dwarf phytoreovirus (RDV) is composed of 12 double-stranded RNA segments, of which segment S6 encodes a non-structural protein Pns6 identified as the movement protein. In this report, Pns6 with a 6-histidine tag at the N-terminal was expressed in E. coli after induction under low temperature (18°C) and low concentration (0.4 mmol/L and 0.2 mmol/L) of IPTG, and then purified by Ni-chelated affinity chromatography. Stability analysis indicated that the expressed HisPns6 protein was stable at 37°C after 24 h treatment. This recombinant protein was then used to make monoclonal antibody. Total 18 hybridoma clones were obtained. The specificity of antibodies was tested by Western blot using native Pns6 extracted from RDV-infected rice leaves, and 15 positive clones were confirmed. Map** of the antigenic sites of Pns6 using antibodies showed that the most sensitive antigen determinant is located in the C-terminal region (the 296th–509th amino acids) of Pns6, which is confirms bioinformatics analysis.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Holmes I H, Boccardo G, Estes M K, et al. Family reoviridae. In: classification and nomenclature of viruses. Sixth Report of the International Committee on Taxonomy Viruses. Arch Virol Suppl, 1995, 10: 208–239
Zhu Y, Hemmings A M, Iwasaki K, et al. Details of the arrangement of the outer capsid of rice dwarf phytoreovirus, as visualized by two-dimensional crystallography. J Virol, 1997, 71: 8899–8901 1:CAS:528:DyaK2sXmvV2htbw%3D, 9343255
Zhu S, Gao F, Cao X, et al. The rice dwarf virus P2 protein interacts with ent-kaurene oxidases in vivo, leading to reduced biosynthesis of gibberellins and rice dwarf symptoms. Plant Physiol, 2005, 139: 1935–1945 10.1104/pp.105.072306, 1:CAS:528:DC%2BD2MXhtlGmsb%2FK, 16299167
Zhou F, Pu Y, Wei T, et al. The P2 capsid protein of the nonenveloped rice dwarf phytoreovirus induces membrane fusion in insect host cells. Proc Natl Acad Sci USA, 2007, 104: 19547–19552 10.1073/pnas.0708946104, 1:CAS:528:DC%2BD1c**tlansg%3D%3D, 18042708
Hagiwara K, Higashi T, Namba K, et al. Assembly of single-shelled cores and double-shelled virus-like particles after baculovirus expression of major structural proteins P3, P7 and P8 of Rice dwarf virus. J Gen Virol, 2003, 84: 981–984 10.1099/vir.0.18904-0, 1:CAS:528:DC%2BD3sXjtVajsr4%3D, 12655100
Ueda S, Masuta C, Uyeda I. Hypothesis on particle structure and assembly of rice dwarf phytoreovirus: Interactions among multiple structural proteins. J Gen Virol, 1997, 78: 3135–3140 1:CAS:528:DyaK2sXnslGntrw%3D, 9400961
Zhong B, Kikuchi A, Moriyasu Y, et al. A minor outer capsid protein, P9, of Rice dwarf virus. Arch Virol, 2003, 148: 2275–2280 10.1007/s00705-003-0160-3, 1:CAS:528:DC%2BD3sXosVajsb4%3D, 14579184
Yin Z, Ji X, Wu Y, et al. Minor outer capsid protein P9 of rice dwarf virus shows transcriptional activation in vivo. J Agri Sci Tech, 2007, 9: 61–65
Wei T, Kikuchi A, Suzuki N, et al. Pns4 of rice dwarf virus is a phosphoprotein, is localized around the viroplasm matrix, and forms minitubules. Arch Virol, 2006, 151: 1701–1712 10.1007/s00705-006-0757-4, 1:CAS:528:DC%2BD28XotVyntr8%3D, 16609816
Wei T, Shimizu T, Hagiwara K, et al. Pns12 protein of Rice dwarf virus is essential for formation of viroplasms and nucleation of viral-assembly complexes. J Gen Virol, 2006, 87: 429–438 10.1099/vir.0.81425-0, 1:CAS:528:DC%2BD28XhtV2jt78%3D, 16432031
Xu H, Li Y, Mao Z, et al. Rice dwarf phytoreovirus segment S11 encodes a nucleic acid binding protein. Virology, 1998, 240: 267–272 10.1006/viro.1997.8945, 1:CAS:528:DyaK1cXmtFCitg%3D%3D, 9454700
Li Y, Bao Y, Wei C, et al. Rice dwarf phytoreovirus segment S6-encoded nonstructural protein has a cell-to-cell movement function. J Virol, 2004, 78: 5382–5389 10.1128/JVI.78.10.5382-5389.2004, 1:CAS:528:DC%2BD2cXjvFeqtr8%3D, 15113917
Cao X, Zhou P, Zhang X, et al. Identification of an RNA silencing suppressor from a plant double-stranded RNA virus. J Virol, 2005, 79: 13018–13027 10.1128/JVI.79.20.13018-13027.2005, 1:CAS:528:DC%2BD2MXhtFWisbvP, 16189004
Waigmann E, Ueki S, Trutnyeva K, et al. The ins and outs of nondestructive cell-to-cell and systemic movement of plant viruses. Crit Rev Plant Sci, 2004, 23: 195–250 10.1080/07352680490452807, 1:CAS:528:DC%2BD2cXlvFWrtL0%3D
Morozov S Y, Solovyev A G. Triple gene block: modular design of a multifunctional machine for plant virus movement. J Gen Virol, 2003, 84: 1351–1366 10.1099/vir.0.18922-0, 1:CAS:528:DC%2BD3sXks1Gmtrs%3D, 12771402
Kalinina N O, Rakitina D A, Yelina N E, et al. RNA-binding properties of the 63 kDa protein encoded by the triple gene block of poa semilatent hordeivirus. J Gen Virol, 2001, 82: 2569–2578 1:CAS:528:DC%2BD3MXnsFOit7c%3D, 11562549
Kalinina N O, Rakitina D V, Solovyev A G, et al. RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block. Virology, 2002, 296: 321–329 10.1006/viro.2001.1328, 1:CAS:528:DC%2BD38XksFOgtLk%3D, 12069530
Donald R G, Lawrence D M, Jackson A O. The barley stripe mosaic virus 58-kilodalton beta(b) protein is a multifunctional RNA binding protein. J Virol, 1997, 71: 1538–1546 1:CAS:528:DyaK2sXls12jtg%3D%3D, 8995680
Ma Z, Yang H, Tian B. Expression of non-structural protein S6 of rice dwarf virus in E. coli and preparation of its antiserum. Virol Sin, 2003, 18: 496–499 1:CAS:528:DC%2BD2MXjtVOgt7s%3D
Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual (2nd ed). New York: Cold Spring Harbor Laboratory Press, 1989
Duan M R, Nan J, Liang Y H, et al. DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein. Nucleic Acids Res, 2007, 35:1145–1154 10.1093/nar/gkm001, 1:CAS:528:DC%2BD2sXjslOgtrc%3D, 17264121
Zhou F, Wu G, Deng W, et al. Interaction of rice dwarf virus outer capsid P8 protein with rice glycolate oxidase mediates relocalization of P8. FEBS Lett, 2007, 581: 34–40 10.1016/j.febslet.2006.11.073, 1:CAS:528:DC%2BD2sXht1Wrug%3D%3D, 17174956
Brill L M, Dechongkit S, DeLaBarre B, et al. Dimerization of recombinant tobacco mosaic virus movement protein. J Virol, 2004, 78: 3372–3377 10.1128/JVI.78.5.3372-3377.2004, 1:CAS:528:DC%2BD2c**s1Klt7c%3D, 15016859
Kalinina N O, Fedorkin O N, Samuilova O V, et al. Expression and biochemical analyses of the recombinant potato virus X 25K movement protein. FEBS Lett, 1996, 397: 75–78 10.1016/S0014-5793(96)01138-6, 1:CAS:528:DyaK28XmvFaqsL4%3D, 8941717
Author information
Authors and Affiliations
Corresponding author
Additional information
Supported by the Special Commonweal Industry Research Program of Agriculture (Grant no. nyhyzx07-051), and the National High Technology Research and Development Program of China (Grant No. 2007AA021505) to Prof. LI Yi
Rights and permissions
About this article
Cite this article
Ji, X., Wei, C. & Li, Y. Expression of rice dwarf phytoreovirus Pns6 and the specificity analysis of its monoclonal antibodies. SCI CHINA SER C 52, 958–964 (2009). https://doi.org/10.1007/s11427-009-0129-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11427-009-0129-x