Expression and Cellular Localization of Dendrolimus puntatus Cytoplasmic Polyhedrosis Virus NSP1 in Insects

doi:10.13560/j.cnki.biotech.bull.1985.2016.11.024

Abstract

Abstract: Genome segment 5 of Dendrolimus puntatus cytoplasmic polyhedrosis virus（DpCPV）was predicted to encode a protein of 881 amino acids with a molecular mass of 101 kD non-structural protein（NSP1）. In order to study the function of the DpCPV NSP1 protein,PCR primers were designed according to the S8 segment genome sequence. The antigen of DpCPV 1 genome S5-1 piece's area（1-600 bp）was in prokaryotic expression,and the polyclonal antibodies against the expressed proteins were raised in rabbits. The diluted virus was used to infect Autographa californica,midgut anatomical samples were taken in each day after infection,and the synthesis curve of NSP1 protein vs time was measured by Western blot. The results of Western blot indicated that the synthesized protein expressed by S5 segment was observed in the first day of infection;in addition to full length protein（101 kD）,20 kD and 80 kD protein bands also were detected,indicating that the expression of NSP1 was initiated in early stage and cleaving of NSP1 protein occurred. Moreover,for the first time,the DpCPV 1 S5 fragment was expressed in insect cells and subcellular localization of insects was observed. The results revealed that the NSP1 protein was present in the cytoplasm of the cells.

Key words: Dendrolimus puntatus cytoplasmic polyhedrosis virus, NSP1, polyclonal antibody preparation, eukaryotic expression, cellular localization

JIN Liang, XU Cui-ping, WANG Jin-chang, GUAN Li-mei, ZHANG Wen-chao, HUANG Chao, GAO Xue-mei, WANG Hong-xiu. Expression and Cellular Localization of Dendrolimus puntatus Cytoplasmic Polyhedrosis Virus NSP1 in Insects[J]. Biotechnology Bulletin, 2016, 32(11): 208-214.

References

[1] King AMQ, Adams MJ. International committee on taxonomy of viruses[C]// Virus Taxonomy：Classification and Nomenclature of Viruses：Ninth Report of the International Committee on Taxonomy of Viruses, Elsevier, 2012.
[2] 农业部农药检定所. 农药登记公告汇编[M]. 北京：中国农业大学出版社, 2008.
[3] 赵同海, 陈昌洁, 徐静, 张青文. 松毛虫质型多角体病毒的宿主域与交叉感染[J]. 昆虫学报, 2004, 47（1）：117-123.
[4] Graham RI, Rao S, Possee RD, et al. Detection and characterisation of three novel species of reovirus（Reoviridae）, isolated from geographically separate populations of the winter moth Operophtera brumata（Lepidoptera：Geometridae）on Orkney[J]. J Invertebr Pathol, 2006, 91（2）：79-87.
[5] Cheng L, Sun JC, Zhang K, et al. Atomic model of a cypovirus built from cryo-EM structure provides insight into the mechanism of mRNA capping[J]. PNAS, 2011, 108：1373-1378.
[6] Yang CW, Gang J, Liu HR, et al. Cryo-EM structure of a transcribing cypovirus[J]. PNAS, 2012, 109（16）：6118-6123.
[7] Yang J, Cheng ZY, Zhang SL, et al. A cypovirus VP5 displays the RNA chaperone-like activity that destabilizes RNA helices and accelerates strand annealing[J]. Nucleic Acids Res, 2014, 42（4）：2538-2554.
[8] 文力, 张珈敏. 马尾松毛虫质型多角体病毒NS5蛋白基因的cDNA克隆及序列分析[J]. 中国病毒学, 2003, 18（1）：49-53.
[9] 张万菊, 赵淑玲, 张小霞, 等. 马尾松毛虫质多角体病毒NS5蛋白的表达和功能初步分析[J]. 中国病毒学, 2006, 21（4）：401-404.
[10] 汪洋, 张珈敏, 李杨, 等. 马尾松毛虫CPV基因组第7片段的cDNA克隆及序列分析[J]. 武汉大学学报：理学版, 2004, 50（2）：216-222.
[11] 段兵, 赵淑玲, 张海元, 等. 文山松毛虫质型多角体病毒S8片段cDNA克隆与原核表达[J]. 中国病毒学, 2004, 19（6）：627-631.
[12] 胡建芳, 张珈敏, 杨娟, 等. 单引物法扩增马尾松毛虫CPV基因组第8片段及其序列分析[J]. 中国病毒学, 2003, 18（1）：39-43.
[13] Zhao SL, Liang CY, Zhang WJ, et al. Characterization of the RNA-binding domain in the Dendrolimus punctatus cytoplasmic polyhedrosis virus nonstructural protein p44[J]. Virus Research, 2005, 114（1）：80-88.
[14] Hagiwara K, Kobayashi J, Tomita M, et al. Nucleotide sequence of genome segment 5 from Bombyx mori cypovirus 1[J]. Archives of Virology, 2001, 146：181-187.
[15] Cheng CG, Shao YP, Su L, et al. Interactions among Dendrolimus punctatus cypovirus proteins and identification of the genomic segment encoding its A-spike[J]. Journal of General Virology, 2014, 95：1532-1538.
[16] 刘润忠, 谢天恩, 彭辉银. 文山松毛虫质型多角体病毒形态结构及理化性质的研究[J]. 中国病毒学, 1992, 7（1）：69-79.
[17] 肖宇宙, 孙修炼, 汤显春, 等. 甜菜夜蛾增值马尾松毛虫质型多角体病毒研究[J]. 应用与环境生物学报, 2010, 16（1）：84-90.
[18] 马永平, 孟小林, 徐进平. 文山松毛虫质型多角体病毒在Sf21细胞中离体增值实验[J]. 中国病毒学, 2002, 17（2）：137-141.
[19] Jin L, Dai CW, Qin TC, et al. Molecular characterization of protein p50 of Dendrolimus punctatus cytoplasmic polyhedrosis virus[J]. Journal of Basic Microbiology, 2013, 53（1）：37-44.