中蜂囊状幼虫病病毒_CSBV_核酸的结构研究_英文_

中蜂囊状幼虫病病毒_CSBV_核酸的结构研究_英文_ 电 子 显 微 学 报 Vol221 ,No13 第 21 卷 第 3 期 200226 2002 年 6 月Journal of Chinese Electron Microscopy Society () Article ID : 100026281 20020320331204 Study on structure of the Chinese Sacbrood Virus’nucleic acid 3ZHANG Qin2fen , YANG Yi2feng , L IANG Yu2yao , LU Xin2ying , ZHANG J ing2qiang ( )State Key Lab for Biocontrol , Zhongshan University , Guangdong Guangzhou 510275 , China () ( ) Abstract : The primary and three2dimensional 32Dstructure of Chinese Sacbrood Virus CSBVnucleic acid were determined ( ) and analyzed by applying the cryo2electronic microscopy cryo EM, computer reconstruction methods and the molecular biology techniques. The possible relations between the primary and the 32D structures were also discussed. Key words : Chinese Sacbrood Bee Virus ; nucleic acid ;primary structure ;three2dimensional structure CLC number : Q52214 ;Q71 Document code : A nucleic acid 1 Introduction The CSBV sample for cryo2EM study was prepared 6 ( ) according to the methods of Chiu W 1986. About 2 The techniques of X2ray crystallography and cryo EM μ have previously been used in investigating the structure of , 5 l aliquot of the purified virus was applied to a copper EM grid coated with a holey carbon support film. the cap sid proteins of viruses , such as the Blue Tongue 1 ,2 ( The grid was blotted nearly dry , cooled rapidly about Virus and Hepatitis B Virus. These methods can also 3 ) - 10degrees Centigrade per secondby plunging the be used to study nucleic acids structure since they are of similar importance to that of the cap sid proteins. grid into liquid nitrogen , and virus particles were ( ) embedded in a layer of vitrified ice . The holder was then Chinese sacbrood bee virus CSBVbelongs to the small RNA virus family , the Picornaviridae . The genome rapidly and carefully inserted into the J EM24000 TEM. of the virus is + ssRNA and there are four structural Images were recorded at 60 ,000 magnification , 400 keV 3 2 proteins at its cap sid. It can infect both larva and adult with an electron dose of ,12èΠ! . Images of each area honey bees and therefore seriously impacts Chinese bee were recorded twice as follows. The first image was close2 3 ,4 ( ) farming. In this paper , the three2dimensional 32Dto2focus and the second one was far2from2focus which was and the primary structure of Chinese Sacbrood Virus 1 um further away from the defocus value of the first one . ( ) CSBVnucleic acid were determined and analyzed by Both images were recorded on Kodak2SO2163 films , ( ) applying cryo2electronic microscopy cryo EM, computer which were developed in full strength D219 for 12min and reconstruction methods and molecular biology techniques. fixed for 20 min at 20 ?. Micrographs with the appropiate The possible relations between the primary and the 32D defocus , minimal astigmatism or movement and a suitable structures were also discussed. particle concentration were selected and digitized using () ZEISS SCAI microdensitometer with a step pixelsize of 2 Materials and methods μμ7m and further averaged to 14m per pixel , leading to a 2133 ! Πpixel step size on the object . Data processing 211 Virus , pla smid and bacteria was carried out on SGI O2 workstation. The digitized Bee larvae naturally infected with CSBV were harvested from the bee farms in Guangdong province and images were displayed and boxed out individual particle 5 images with a size of 200 pixel ×200 pixel , and floated purified according to the methods of Feng J ianxun. PGEM2Teasy was purchased from Promega Corporation , by using an X2window based on the program EMTOOL . ( ) USA. E. coli DH5a was kindly donated by the Ocean The image resolution , contrast transfer function CTF and the defocus values of the micrographs were eventually Functional Genomes Lab , Zhongshan University. determined based on the incoherently averaged Fourier 212 Determination of the 32D structure of virus Received date : 2002203215 () Foundation item : National Natural Science Foundation of China . No : 30070169. () Biogra phy : Zhang Qinfen 1966 - , female , Jiangsu , Lecturer . () 3 Author f or correspondence : Zhang Jingqiang 1939 - , male , Guangdong , Professor . 7 ( ) transforms of the boxed2out particles. Five focal pairs the nucleic acid like a membrane Fig. 1. The nucleic of micrographs from different specimen areas that had the acid was located in the middle of the virus and arranged best quality were selected based on the results of the 2fold axes of the loosely in ribbon shape . Each of 3 () preprocessing. Using the build template procedure , three nucleic acid NAwere like a“W”and had two points to contact the VP4 . Each“W”began with a“arrowhead” particles with the lowest self2common2lines analysis phase and had a dot at the base . It could also be observed that residuals were selected and their centers and orientations the NA of CSBV on 22fold and 52fold axes was arranged in (θψω ) ,, , x, ywere refined. These particles were 0 0 symmetry from figure 2 and 3 . There were two parts of the then used as templates to eliminate the incorrect nucleic acid with swelled symmetry along the 22fold axes orientation estimation and evaluate the handedness of that contacted with the VP4 through the ribbon shaped other particles. The orientation and center parameters nucleic acid. The NA along 52fold axes was also of ribbon estimated above were further refined by minimizing the shape and arranged in a“V”shape . All of these showed cross common lines phase residual . Those particles with clearly that the NA of the CSBV was arranged in 52322 phase residuals < 55?were selected and their centers and symmetry format . orientations were refined simultaneously and iteratively using GlobalRefine . 3D reconstruction was obtained by Fourier inversion using the Fourier2Bessel synthesis method. 213 Primary structure determination of CSBV nucleic acid According to the sequence of Sacbrood Bee Virus 8 () SBV, the primers were designed and the sequence2 primary structure was determined on an AB I PRISM377 DNA Sequencer after RT2PCR , ligation , transfer and Fig. 1 Three2fold central slice 32Dstructure of the virus genome 9 () at 215nm resolution 2 pixel thick. cloning according to the methods of Zhang Qinfen. ( ) The capsid ?of CSBV was composed VP1 ,VP2 , VP3 and VP4 214 Analysis of the nucleic acid sequence ( ) ( ) β which like a membrane encircled the nucleic acid ? ?. A software package named“YYF”was designed to () Each of 32fold axes of the nucleic acid NAshaped as“W”and analyze the nucleic acid sequence using the comparability ( had two points to contact the VP4 ) . Each“W”began with a of different segments and the number of the nucleotides as ( ) ( ) “arrowhead”?and had a dot at the base ?. ( ) parameters. The repeated sequences comparability = 1 ( ) and the resembled sequences comparability = 0175 312 Primary structure of CSBV nucleic acid could be found using YYF. Sequences of other The 8740 nucleotide sequence of CSBV nucleic acid picrornaviruses for which the 32D structure has been 10 ,11 was obtained except the 3’and 5’ends through RT2PCR , determined, such as Foot and Mouth Disease Virus molecular cloning methods and it has been accepted by ( ) ( ) ( FMDVGenebank : X00429, Poliovirus GeneBank : () the GenBank accepted number AF469603. It contained ) NC2002058 and BmCPV Genebank : AF433559 , ( ) a large open reading frame ORF commencing at AF433560 were also analyzed by using YYF. nucleotide 138 . There was a second in2frame AUG at 3 Results 163 , but AUG 138 may be the translation initiation site since it occurred in a context AUUAUGG identical to that 32D structure of the CSBV’s nucleic acid 3 . 1 () of many invertebrate initiating codons ANNAUGG. The A 215nm resolution 32D structure of CSBV genome ORF contained 8574 nucleotides and ended with UAG obtained by using cryo2EM and computer was stop codon at nucleotide 871128713 . reconstruction methods. A three2fold central slice with 2 Analysis of the obtained nucleotide sequences of pixel thickness demonstrated that the cap sid of CSBV was CSBV revealed that it contained a lot of repeated composed of VP1 , VP2 , VP3 and VP4 which encircled 第 3 期 ZHANG Qin2fen et al . : Study on structure of the Chinese Sacbrood Virus’nucleic acid 333 an obvious feature of the primary structure of CSBV nucleic acid. 4 Discussion The spatial structure of virus NA could be affected by many factors , such as the structures of the cap sid proteins and the relations between the NA and the cap sid proteins. Although most NA of icosahedral viruses are not 12 symmetrically arranged, a few , such as the Bean Pod 13 () Mottle Virus BPMV, are. Fig. 2 Two2fold central slice 32D structure of the virus genome In BPMV , the NA was ordered along the 32fold. at 215nm resolution. The analysis of the sequence of BPMV NA revealed fifteen 52nucleotide sequences which repeated 14,19 times and 14 a 18bp fragment with a gap repeated 15 times. Although there was no parallelism between the sequence and spatial structure , the authors suggested that the repeated sequence may play an important role for the 14 ordered spatial structure. In CSBV , there were more than 60 repeated 52 nucleotides sequences with more than 20 copies , and some with more than 35 copies. There were also some sequences that were very similar to each other . Related to the spatial structure of CSBV NA showed icosahedral Fig. 3 Five2flod central slice 32D structure of the virus genome symmetry. In some other viruses that also belong to at 215nm resolution. picornaviridae , such as FMDV and the Poliovirus , the nucleic acid were arranged irregularly in their high sequences. By using the software package YYF , sixty 52 resolution spatial structures. Corresponding to the 32D nucleotide segments which had more than 20 copies were structure of their nucleic acids , their NA sequence also found , such as TGATA , TTGGA and TAAGG . Some of had few reiterated sequences. There were only 6 and 7 them had more than 35copies , such as ATTTT. Some reiterated 52nucleotide sequences which had 20 copies in resembled segments with different nucleotides , such as 10 ,11 FMDV and Poliovirus respectively. The number of 8bp or 12bp ,and had more than 10 copies , for example : repeated sequences in CSBV was 10 times more than those 69 TGAAGATT , 5732 TGAAGATG , in FMDV and Poliovirus. 83 TGAAGTTT , 6022 AGAAGATT , In BmCPV , whose 32D structure was determined by 848 AGAAGATT , 6074 TCAAGATT , using the same cryo2EM methods as used in this study , 934 TGAAGTTT , 6134 TGAAGATT , the spatial structure of NA was arranged as an irregularly 2429 TGAAGATG , 7103 GGAAGATT , 15 coiled shape. There were no reiterated 52nucleotide 3458 7565 AGAAGATT , TGGAGATT , sequence which had more than 20 copies in all of its 4549 AGAAGATT , 8276 TGATGATT , nucleic acid segments. TGAAGATG , 4997 8687 TGACGATT. According to the obtained results and these analysis , This resembled sequence was repeated 16 times and there it could be suggested that the repeated sequences may were only 2 different nucleotides between each two play some roles in the ordered 32D structure of CSBV There were more than 40 82nucleotide segments. nucleic acid. () sequences which had 16 copies comparability = 0175 . Acknolo wledgements : We thank Dr . Z H Zhou , All of these results showed that repeated sequences were ( ) ( 维结 构 J , 中 国 科 学 C 辑 Zhang Jingqiang , Feng () University of Texas 2 Houston Medical Schooland Dr . Jianxun , Liang Yuyao , et al . Three2dimensional structure of () Donghua Chen Baylor College of Medicine , Houstonfor ( the Chinese Sacbrood Bee VirusJ . 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Analysis of the nucleotide sequence of Bean Pod Mottle Virus middle 6 Chiu W. Electron microscopy of frozen hydrated biological component RNAJ . Virology , 1991 , 183 :405 2409. 2257. specimenJ . Annu Rev Biophys Chem , 1986 , 15 :237 15 Zhang H , Zhang J , Yu X ,et al . Visualization of protein 2RNA 7 Zhou Z H , Hrdt S , Wang B , et al . CTF determination of ice 2 interaction in cytoplasmic polyhedrosis virus J . J Viro , embedded single particle using a graphics interface J . J of () 1999 , 73 2:162421629. Structural Biology , 1996 , 116 :2162222. 8 张景强 ,冯建勋 ,梁玉尧 ,等 . 中蜂囊状幼虫病病毒的三 ( ) 中蜂囊状幼虫病病毒 CSBV核酸的结构研究 3 英 ,张景强 张勤奋 ,杨艺峰 ,梁玉尧 ,卢 ()广州中山大学生物防治国家重点实验室 ,结构生物学研究室 ,广东 广州 510275 ( ) 摘 要 : 本文分别应用冷冻电镜与计算机三维重构技术和分子生物学技术对中蜂囊状幼虫病病毒 CSBV的核酸的三维结构 和核酸一级结构进行测定和分析 ,并对两者之间关系进行探索 。 关键词 : 中蜂囊状幼虫病病毒 ;核酸 ;一级结构 ;三维结构 3 通讯作者