Interference of the Expression of Non-structural Protein Pns11 of Rice dwarf virus (RDV) Inhibits Viral Replication in Leafhopper (Nephotettix cincticeps) Vector
Abstract：Rice dwarf virus (RDV) is mainly transmitted by leafhopper (Nephotettix cincticeps) in a persistent-propagative manner. The genome of RDV consists of 12 dsRNA segments, encoding at least 7 structural proteins (P1, P2, P3, P5, P7, P8 and P9) and 5 non-structural proteins (Pns4, Pns6, Pns10, Pns11 and Pns12). Pns11 of RDV is known to be a gene silencing suppressor in plant, and participates in the formation of viroplasm for viral replication and assembly of progeny virions in the cultural cells of N. cincticeps. However, the definite function of Pns11 in viroplasm in insect vector is still unknown yet. In the present study, RNA interference (RNAi) was firstly applied via delivering the synthesized dsRNAs in vitro from Pns11 gene into cultural cells of N. cincticeps. Immuno-fluorescence assay showed that when Pns11 was knocked down, the antigens of Pns11 were significantly reduced, and RDV was restricted within 1~2 cultural cells. The infectivity evaluated with the fluorescent antibody focus counting method showed the level of the viral infection decreased about 5 times. These results indicated that RNAi targeting on Pns11 gene significantly reduced the expression of Pns11, leading the RDV accumulation and infection, was blocked in the cultural cells. Thus, Pns11 was assumed to associate with viral replication. Then, microinjection of dsRNAs from Pns11 into the body of N. cincticeps was performed. qRT-PCR test showed the knockdown of Pns11 significantly decreased the viruliferous rate of insect more than 60%. And the RNAi effect steadily lasted for more than 12 d, which was nearly a circulative transmission period. qRT-PCR assay showed that knockdown of Pns11 caused more than 80% reduction in the relative expression levels of P8 and Pns11 genes. It was suggested that RNAi targeting on Pns11 gene inhibited the viral accumulation in body of N. cincticeps, and decreased the number of viruliferous insect. Immuno-fluorescence assay displayed that at 6 d after microinjection, RDV and Pns11 were detected in the epithelia cells of filter chamber and midgut of N. cincticeps in control, while blocked in 1~2 epithelia cells of filter chamber in treatment of Pns11 knockdown. At 12 d after microinjection, RDV and Pns11 were detected in the whole of intestines and salivary gland in control, while still blocked in 2~3 epithelia cells of filter chamber in the treatment of Pns11 knockdown. These observations indicated that knockdown of Pns11 inhibited the spread of RDV in insect vector via blocking viral replication. Finally, capability of viral transmission by insect was tested. The result showed that when Pns11 was knocked down, insect vectors were unable to transmit RDV in 14 d after microinjection. These results confirmed that Pns11 was a viral replication factor, which was necessary for RDV infection and multiplication. It also verified Pns11 was an effective target to block viral replication and transmission. This study theoretically bases for virus disease control via blocking RDV multiplication and circulation in insect vector.
[1]Belhouchet M, Mohd Jaafar F, Firth AE, et al.Detection of a Fourth Orbivirus Non-Structural Protein [J].PLoS One, 2011, 6(10):e25697-
[2]Cao X, Zhou P, Zhang X, et al.Identification of an RNA silencing suppressor from a plant double-stranded RNA virus[J].Journal of Virology, 2005, 79(20):13018-13027
[3]Chen H, Chen Q, Omura T, et al.Sequential infection of Rice dwarf virus in the internal organs of its insect vector after ingestion of virus[J].Virus Research, 2011, 160:389-394
[4]Chen H, Zheng L, Mao Q, et al.Development of continuous cell culture of brown planthopper to trace the early infection process of oryzaviruses in insect vector cells[J].Journal of Virology, 2014, 88(8):4265-4274
[5]Chen Q, Chen H, Mao Q, et al.Tubular structure induced by a plant virus facilitates viral spread in its vector insect[J].PLoS Pathogens, 2012, 8:e1003032-
[6]Chen Q, Wang H, Ren T, et al.Interaction between nonstructural protein Pns10 of rice dwarf virus and cytoplasmic actin of leafhoppers is correlated with insect vector specificity [J].Journal of General Virology, 2015, 96(4):933-938
[7]Contin R, Arnoldi F, Campagna M, et al.Rotavirus NSP5 orchestrates recruitment of viroplasmic proteins[J].Journal of General Virology, 2010, 91:1782-1793
[8]Criglar JM, Hu L, Crawford SE, Hyser JM, et al.A novel form of rotavirus NSP2 and phosphorylation-dependent NSP2-NSP5 interactions are associated with viroplasm assembly[J].Journal of Virology, 2014, 88(2):786-798
[9]Eichwald C, Arnoldi F, Laimbacher AS, et al.Rotavirus viroplasm fusion and perinuclear localization are dynamic processes requiring stabilized microtubules[J].PLoS One, 2012, 7(10):e47947-
[10]Hagiwara K, Higashi T, Miyazaki N, et al.The amino-terminal region of major capsid protein P3 is essential for self-assembly of single-shelled core-like particles of Rice dwarf virus [J].Journal of Virology, 2004, 78(6):3145-3148
[11]Hunter W, Ellis J, vanEngelsdorp D, et al. Large-Scale Field Application of RNAi Technology Reducing Israeli Acute Paralysis Virus Disease in Honey Bees (Apis mellifera, Hymenoptera: Apidae)[J].PLoS Pathogens, 2010, 6(12):e1001160-
[12]Jia D, Chen H, Zheng A, et al.Development of an insect vector cell culture and RNA interference system to investigate the functional role of Fijivirus replication protein[J].Journal of Virology, 2012, 86:5800-5807
[13]Kimura I.A study of Rice dwarf virus in vector cell monolayers by fluorescent antibody focus counting[J].Journal of General Virology, 1986, 67(10):2119-2124
[14]Li Y, Bao YM, Wei CH, et al.Rice dwarf phytoreovirus segment S6-encoded nonstructural protein has a cell-to-cell movement function[J].Journal of Virology, 2004, 78(10):5382-5389
[15]Mao Q, Zheng S, Han Q, et al.New model for the genesis and maturation of viroplasms induced by fijiviruses in insect vector cells[J].Journal of Virology, 2013, 87:6819-6828
[16]Mao ZJ, Li Y, Xu H, et al.The 42K protein of Rice dwarf virus is a post-translational cleavage product of the 46K outer capsid protein [J].Archives of Virology, 1998, 143(9):1831-1938
[17]Omura T, Yan J, Zhong B, et al.The P2 protein of Rice dwarf phytoreovirus is required for adsorption of the virus to cells of the insect vector[J].Journal of Virology, 1998, 72:9370-9373
[18]Patton JT, Silvestri LS, Tortorici MA, et al.Rotavirus genome replication and morphogenesis: role of the viroplasm[J].Current Topics in Microbiology and Immunology, 2006, 309:169-187
[19]Price DRG, Gatehouse JA.RNAi-mediated crop protection against insects[J].Trends in Biotechnology, 2008, 26:393-400
[20]Ren B, Guo Y, Gao F, et al.Multiple functions of Rice dwarf phytoreovirus Pns10 in suppressing systemic RNA silencing[J].Journal of Virology, 2010, 84(24):12914-12923
[21]Shimizu T, Yoshii M, Wei T, et al.Silencing by RNAi of the gene for Pns12, a viroplasm matrix protein of Rice dwarf virus, results in strong resistance of transgenic rice plants to the virus[J].Plant Biotechnology Journal, 2009, 7(1):24-32
[22]Suzuki N, Kusano T, Matsuura Y, et al.Novel NTP binding property of rice dwarf phytoreovirus minor core protein P5[J].Virology, 1996, 19(2):471-474
[23]Suzuki N, Tanimura M, Watanabe Y, et al.Molecular analysis of rice dwarf phytoreovirus segment S1: Interviriral homology of the putative RNA-dependent RNA polymerase between plant- and animal-infecting reoviruses[J].Virology, 1992, 190(1):240-247
[24]Touris-Otero F, Martínez-Costas J, Vakharia VN, et al.Avian reovirus nonstructural protein μNS forms viroplasm-like inclusions and recruits protein σNS to these structures[J].Virology, 2004, 319(1):94-106
[25]Ueda S, Uyeda I.The rice dwarf phytoreovirus structural protein P7 possesses non-specific nucleotide acids binding activity in vitro[J]., 1997, :-
[26]Uyeda I, Kudo H, Yamada N, et al.Nucleotide sequence of Rice dwarf virus genome segment 4[J].Journal of General Virology, 1990, 71(10):2217-2222
[27]Wei T, Chen H, Ichiki-Uehara T, et al.Entry of Rice dwarf virus into cultured cells of its insect vector involves clathrin-mediated endocytosis[J].Journal of Virology, 2007, 81:7811-7815
[28]Wei T, Kikuchi A, Moriyasu Y, et al.The spread of Rice dwarf virus among cells of its insect vector exploits virus-induced tubular structures[J].Journal of Virology, 2006a, 80(17):8593-8602
[29]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[J].Archives of Virology, 2006b, 151:1701-1712
[30]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].Journal of General Virology, 2006c, 87(2):429-438
[31]Xu H, Li Y, Mao Z, et al.Rice dwarf phytoreovirus segment S11 encodes a nucleic acid binding protein[J].Virology, 1998, 240(2):267-272
[32]Yu N，Christiaens O, Liu J, et al.Delivery of dsRNA for RNAi in insects: an overview and future directions[J].Insect Science, 2013, 20:4-14
[33]Zhang C, Liu Y, Liu L, et al.Rice black streaked dwarf virus P9-1, an alpha-helical protein, self-interacts and forms viroplasms in vivo[J].Journal of General Virology, 2008, 89:1770-1776
[34]Zheng L, Chen H, Liu H, et al.Assembly of viroplasms by viral nonstructural protein Pns9 is essential for persistent infection of rice gall dwarf virus in its insect vector[J].Virus Research, 2015, 196(22):162-169
[35]Zhong BX, Shen YW, Omura T.RNA-binding domain of the key structural protein P7 for the Rice dwarf virus particle assembly[J].Acta Biochimica et Biophysica Sinica, 2005, 37(1):55-60
[36]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[J].Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(49):19547-19552
[37]陈倩，张玲华，黄海宁，等.水稻矮缩病毒非结构蛋白 Pns6 是病毒的复制因子[J]., 2015, :-
[38]尹哲, 吉栩, 吴云锋, 等.水稻矮缩病毒外壳蛋白P9具有体内转录激活活性[J].中国农业科技导报, 2007, 9(3):61-65
[39]郑立敏, 刘华敏, 陈红燕, 等.干扰水稻瘤矮病毒(RGDV)非结构蛋白(Pns12)的表达抑制病毒在介体昆虫培养细胞内的复制[J].农业生物技术学报, 2014, 22(11):1321-1328
