摘要
登革病毒(dengue virus)是人类登革热(DF)、登革出血热／登革休克综合征(DHF／DSS)的病原体。自上世纪80年代以来，登革热和登革出血热在世界范围内的暴发流行日益频繁。尤其是在热带及亚热带地区，登革病毒感染导致的发病率及病死率急剧上升。登革热亦是我国一种严重的蚊媒传播性病毒病，我国南方及东南沿海各省区均为登革热流行区。
登革病毒感染严重威胁全球近半数人的健康，然而时至今日仍然没有有效的治疗药物和疫苗。因而探索抗登革病毒感染的新策略成为当今登革研究的热点。衣壳蛋白靶向病毒灭活(capsid targeted viral inactivation；CTVI)是近年来新兴的基于蛋白水平的抗病毒策略，与目前流行的流行的基于核苷酸水平的策略(如RNAi，反义核酸，核酶等)具有特异、高效、不易产生突变株等优点。将CTVI策略应用于抗登革病毒感染充满了吸引力。
登革病毒衣壳蛋白位于位于病毒颗粒的内部，并不诱导产生中和抗体，因而对其研究较少，同时其加工过程中无需糖基化，因而我们首先在大肠杆菌中对登革2型病毒衣壳蛋白(D2C)及其前体(D2C_I)进行了表达。结果表明表达的D2C及D2C_I均能被抗登革2型病毒衣壳蛋白单克隆抗体特异识别，相对分子量大小分别为11和13KD，与DNAstar软件包分析一致。利用重组表达的衣壳蛋白我们对其体外自组装活性进行分析，结果表明无论是成熟的衣壳蛋白还是其前体均不能自组装形成衣壳样颗粒。利用ProteinPredict软件包对登革2型病毒43株衣壳蛋白进行二级结构预测，结果发现：存在一个高度疏水区，Blast分析显示该区域高度保守；存在4个α螺旋和2个正电荷富集区。同时，利用大肠杆菌GI724／pLEX表达系统对葡萄球菌核酸酶(staphylococcal nuclease；SN)进行了表达。结果表明重组表达的SN相对分子量大小约为17kD，表达的目的蛋白占菌体蛋白总量37％。将重
摘要
组表达的SN蛋白免疫日本大耳白兔，制备了抗SN多克隆抗体，
Westem Blot分析显示制备的多克隆抗体具有良好的特异性，为后续研
究奠定了物质基础。
为了研究登革病毒衣壳蛋白靶向核酸酶作为抗病毒因子的可行
J险，我们首先构建了登革2型病毒衣壳蛋白靶向葡萄球菌核酸酶融合
蛋白DZC一SN，原核表达的DZC一SN相对分子量大小约为3OkD，具有
良好的抗原性，可被抗DZC抗体特异识别;同时具有良好的核酸酶活
性，能够对DNA进行切割。这些结果表明与DZC融合表达的SN能够
正确折叠并具有良好的生物学活性，为进一步探讨登革病毒衣壳蛋白
靶向抗病毒作用的研究奠定了基础。
在此基础上，我们构建了表达DZC一SN的真核表达载体，转染哺
乳动物细胞系BHKZI后，经压力筛选获得了稳定表达DZC一SN的细胞
系，说明DZC一SN对哺乳动物细胞不具毒性，与MTT分析结果一致。
真核表达DZC一SN能够为抗DZC及 SN的特异抗体识别，分子量大小
与原核表达基本一致(3 okD)，DNA消化试验表明DZc一sN具有良好的
核酸酶活性。将稳定表达DZC一sN的细胞系命名为pc/DZc一sN，同时
获得了对照细胞系pc/DZC一SN*。然后以我国登革2型病毒分离株DZ一43
分别感染该两种细胞系及正常BHKZI细胞，收集细胞培养上清中产生
的子代病毒，分别研究其感染性及对乳鼠的致病性，是为预防模型。
对于业以感染登革病毒的细胞，通过阳离子转染试剂将重组质粒导入
细胞，收集子代病毒并分析其感染性，是为治疗模型。结果表明DZC一SN
呈现出良好的抗病毒效果:在治疗模型中导致子代病毒感染性降低
12~60倍，在预防模型中感染性降低倍数高达1护一104。而核酸酶活性
缺失突变体对照DZC一SN*仅观察到轻微的抗病毒效果(感染性降低
1.5一巧倍)，说明子代病毒感染性的降低主要是由于靶向核酸酶
DZC一SN的核酸酶活性所致。为分析DZC一SN抗病毒作用的确切机制，
我们收集各细胞系中产生的子代病毒颗粒进行Westem Blot分析，结
果表明自BHK/DZC一sN及BHK/DZc一SN*细胞中产生的病毒颗粒中均
可检测到SN及SN*的存在，说明DZC一SN在病毒组装过程中被共同装
，l匕
摘要
配入子代病毒颗粒内部。DNA消化试验表明，装配入子代病毒颗粒内
部的sN具有良好的核酸酶活性，能够降解病毒基因组。进一步我们采
用RT一PCR的方法分析了子代病毒完整基因组含量，结果表明
BHK/DZC一SN细胞中产生的子代病毒颗粒中完整基因组含量与正常
BHKZI细胞相比降低倍数在1护以上，与感染性降低倍数基本吻合。
这说明DZC一SN的抗病毒效果并非降低子代病毒颗粒的数量，而是通
过降解病毒基因组从而导致病毒感染性丧失。
总之，我们首次在细胞水平上证实了CTVI策略应用于抗登革病
毒感染的可行性，随着体内研究及相关技术的不断发展，CTVI必将成
为抗人类登革病毒感染的有力武器。
Dengue virus, a member of the flavivirus genus of enveloped RNA virus, is one of the most significant viral pathogens and now presents in over 100 countries. Annually, the four serotypes of dengue virus collectively cause 100 million or more cases of infection. Dengue infection is usually characterized by fever and joint pains, but more serious syndromes, dengue hemorrhagic fever and dengue shock syndrome(combined mortality, up to 5%), sometimes occur following infection. Although intense efforts have been made to control those diseases, dengue is still endemic and epidemic and threatens the health of over 2.5 billion people in urban and rural areas of the tropics and subtropics. Dengue infection has now been a major global public health problem.
Currently, no vaccine or effective antiviral treatment exists for the prevention or treatment of infections with dengue virus. This situation has spurred urgently the interest in search for new strategy against dengue. One potential approach, termed intracellular inhibition or immunization, was designed to render cells resistant to viral pathogens by introducing genes encoding antiviral macromolecules into a cell culture or an individual. Inhibition of dengue viral multiplication by such a strategy had been established with different antiviral genes, most of which were nucleic-acid-based. Capsid targeted viral inactivation (CTVI), first proposed by Natsoulis and Boeke, represented a conceptually powerful protein-based antiviral approach. In this strategy the viral capsid protein was designed as carrier to target a deleterious enzyme, e.g., a nuclease, a proteinase and even a single chain antibody bind to a native viral protein, specifically into progeny virions during assembly to prevent the production of in
fectious viral particles and subsequent spread of de novo infection. CTVI had been
thoroughly investigated in the experimental treatments for several important virus, such as murine leukemia virus, hepatitis B virus and human immunodeficiency virus, showing a promising prospect as an antiviral strategy.
In this paper, we firstly cloned and expressed the dengue 2 virus capsid protein, termed with D2C, and staphylococcal nuclease(SN) genes respectively with E.coli GI724/pLEX system from Invitrogen. The recombinant expressed D2C was about 11kDa and could be recognized by monoclonal antibody 8H8 raised against dengue 2 virus A15 strains capsid protein. Unexpectedly, there were no evidence showing the ability of self-assembly into capsid-like particles in vitro of recombinant D2C protein. The bacterial expressed SN was immuned rabbits to prepare polyantibody against SN for further detection of eukaryotic expression, and the prepared antisera were specfic to recognize recombinant SN and SN*.
To explore the feasibility of CTVI as an antiviral strategy against dengue infection, first of all we fused SN gene to D2C gene at the carboxyl terminus, to construct the antiviral effector D2C-SN. This orientation was chosen so that the nuclease could be inserted within the particles where it would have access to the viral RNA. The E.coli. expressed D2C-SN was about 30kDa and could be recognized by Mab 8H8. What's more, the recombinant expressed D2C-SN had proper nuclease activity to degrade DNA or RNA with appropriate Ca2+ concentrations. Those data supported the further research.
Based on the previous work, we inverted the constructed D2C-SN and D2C-SN* gene fragments with EcoR I and Xho I sites into the eukaryotic expression vector pcDNA6 to obtain the desired recombinant palsmids pc/D2C-SN and pc/D2C-SN*. Firstly, we examined the temporary expression of D2C-SN in mammalian BHK21 cells. After transfection by electroporation, RT-PCR? Western Blot and indirect immunofluorescence were used to detect the expression of D2C-SN. The results indicated that D2C-SN could be expressed and detected with specific antibody against D2C and SN. And the results of MTT assay showed that D2C-SN were no cytotoxic to the host cell. Secondly, we set up prophylactic and therapeutic models to demonstrat
引文
[1] Rice CM. 1996. Flaviviridae: the viruses and their replication, p931-959. In Fields BN, Knipe DM and Howley PM (ed.). Fields Virology.
[2] Rigau-Perez JG, Clark GG, Gubler DJ et al. Dengue and dengue haemorrhagic fever. Lancet. 1998, 352: 971-977.
[3] 杨佩英，秦鄂德主编。1999，登革热和登革出血热。第一版，人民军医出版社。
[4] Bielefeldt-Ohmann H. Pathogenesis of dengue virus diseases: missing pieces in the jigsaw. Trends Microbiol, 1997, 5:409-413.
[5] Leyssen P, DeClercq E, Neyts J. Perspectives for the treatment of infections with Flaviviridae. Clin Microbiol Rev. 2000,13: 67-82.
[6] Baeuerle PA, Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell. 1988, 53:211-217.
[7] Natsoulis G, Boeke JD. New antiviral strategy using capsid-nuclease fusion proteins. Nature. 1991, 352: 632-635.
[8] VanBrocklin M, Ferris AL, Hughes SH et al. Expression of a murine leukemia virus Gag-Escherichia coli RNase HI fusion polyprotein significantly inhibits vires spread. J Virol. 1997, 71: 3312-3318.
[9] VanBrocklin M, Federspiel MJ. Capsid-targeted viral inactivation can eliminate the production of infectious murine leukemia virus in vitro. Virology. 2000, 267:111-123.
[10] Natsoulis G, Seshaiah P, Federspiel MJ et al. Targeting of a nuclease to murine leukemia virus capsids inhibits viral multiplication. Proc Natl Acad Sci USA. 1995, 92: 364-368.
[11] Beterams G, Nassal M. Significant interference with hepatitis B virus replication by a core-nuclease fusion protein. J Biol Chem. 2001, 276: 8875-8883.
[12] Wu XY, Liu HM, Xiao HL et al. Inhibition of human and simian immunodeficiency virus -protease function by targeting Vpx-protease -mutant fusion protein into viral particles. J Virol. 1996, 70: 3378-3384.
[13] Liu J, Li YH, Xue CF et al. Targeted ribonuclease can inhibit replication of hepatitis B virus. World J Gastroenterol. 2003, 9: 295-299.
[14] Kobinger GP, Borsetti A, Nie ZL et al. Virion-targeted viral inactivation of human immunodeficiency virus type 1 by using Vpr fusion proteins. J Virol. 1998, 72: 5441-5448.
[15] Okui N, Kitamura Y, Kobayashi N, et al. Viron-targeted viral inactivation: new therapy against viral infection. Mol Urol. 2001, 5: 59-66.
[16] Giordano V, Jin DY, Rekosh D et al. Intravirion targeting of a functional anti-human immunodeficiency virus ribozyme directed to pol. Virology. 2000, 267:174-84.
[17] Kuhn RJ, Zhang W, Rossmann MG et al. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell. 2002, 108:717-725.
[18] Schumann G, Hermankova M, Cannon K et al. Therapeutic effect of a Gag-nuclease fusion protein against retroviral infection in vivo. J Virol. 2001, 75: 7030-7041.
[19] 于曼，秦鄂德，陈水平等。登革2型PrM基因的重组病毒对不同型别登革病毒复制的阻断作用。中国生物化学与分子生物学报。2002，18：568-572。
[20] Jones CT, Ma LX, Burgner JW et al. Flavivirus capsid is a dimeric alpha-helical protein. J Virol. 2003, 77: 7143-7149.
[21] Khromykh AA, Westaway EG. RNA binding properties of core protein of the flavivirus Kunjin. Arch Virol. 1996, 141: 685-699.
[22] Boeke JD, Hahn B. Destroying retrovirus from within. Trends Microbiol. 1996, 4:421-426.
[23] Tucker PW, Hazen EE, Cotton FA. Staphylococcal nuclease reviewed: a prototypic study in contemporary enzymology Mol Cell Biochem. 1979, 23: 67-86.
[24] 秦成峰，于曼，陈水平等。登革病毒衣壳蛋白在大肠杆菌中的表达及其自组装活性的研究。生物技术通讯，2003，14：444-446。
[25] Pupo-Antunez M, Rodriguez H, Vazquez S et al. Monoclonal antibodies raised to the dengue-2 virus which recognize viral structural protein. Hybridoma, 1997, 16: 347-353.
[26] 汪家政，范明主编。2001，蛋白质技术手册。第一版，科学出版社
[27] Serwer P, Khan SA, Griess GA. Non-denaturing gel electrophoresis of biological
nanoparticles: viruses. J Chromatogr A. 1995,698:251-261.
[28] Lachica RV, Genigeorgis C, Hoeprich PD. Metachromatic agar-diffusion methods for detecting staphyloccal nuclease activity. Applied Microbiol. 1971, 21: 585-587.
[29] 胡志君，杨敬，赵卫等。我国2株登革2型病毒基因组的全序列分析。中国病毒学。2002，17：17-21。
[30] Gubler DJ, Kuno G. Dengue and dengue hemorrhagic fever. London: CAB international, 1997,175-199.
[31] Wang SH, Syu WJ, Huang KJ et al. Intracellular localization and determination of a nuclear localization signal of the core protein of dengue virus. J Gen Virol. 2002, 83:3093-3102.
[32] Ma LX, Jones CT, Groesch TD et al. Solution structure of dengue virus capsid protein reveals another fold. Proc Natl Acad Sci USA. 2004, 101: 3414-3419.
[33] Markoff L, falgout B, Chang A. A conserved internal hydrophobic domain mediates the stable membrane integration of the dengue virus capsid protein. Virology. 1997, 233:105-117.
[34] Konishi E, Fuji A. Dengue type 2 virus subviral extracellular particles produced by a stably transfected mammalian cell line and their evaluation for a subunit vaccine. Vaccine. 2002, 20: 1058-67.
[35] Lorenz IC, Kartenbeck J, Mezzacasa A et al. Intracellualr assembly and secretion of recombinat subviral particles from tick-borne encephalitis virus. J Virol. 2003, 77: 4370-4382.
[36] Konishi E, Fujii A, Mason PW. Generation and characterization of a mammalian cell line continuously expressing Japanese encephalitis virus subviral particles. J Virol. 2001, 75:2204-12.
[37] Cuatrecasas P, Fuchs S, Anfinsen CB. Catalytic properties and specificity of the extracellular nuclease of staphylococcus aureus. J Biol Chem. 1967, 242:1541-1547.
[38] 司徒镇强，吴军正主编。1996，细胞培养。第一版。世界图书出版公司。
[39] Feigel, Polly and Ipsen Johannes. 1970. Bancroft's Introduction to Biostatistics. 2nd edition. Harper and Row, New York.
[40] 赵卫，曹虹，张文炳等。登革病毒甲基纤维素微量蚀斑技术的建立。第一军医大学学报。
2001，12：28-29。
[41] 胡志君。D2-43病毒株全序列测定及NS2基因的免疫及保护研究。博士学位论文。2000。
[42] 赵卫。我国登革2型病毒毒力相关位点研究。博士学位论文。2001。
[43] Jin D, Liu J, Xue CF et al. Anti-HBV effect of TAT-HBV targeted ribbonuclease. World J Gastroenterol. 2003, 9: 1525-1528.
[44] 秦成峰，胡志君，陈水平等。登革病毒衣壳蛋白与葡萄球菌核酸酶融合蛋白在大肠杆菌中的共表达。军事医学科学院院刊。2004，28：104-107。
[45] 胡志君，杨敬，赵卫等。登革2型病毒43株NS1基因的克隆及在真核细胞中的表达。军事医学科学院院刊。2001，25：5-9。
[46] Beterams G, Bottcher B, Nassal M. Packaging of up to 240 subunits of a 17kDa nuclease into the interior of recombiant hepatitis B virus capsids. FEBS Lett, 2000, 481: 169-176.
[47] Mellors JW, Rinaldo CR, Gupta P et al. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science. 1996, 272:1167-1170.
[48] Kummerer BM, Rice CM. Mutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious particles. J Virol. 2002, 76: 4773-4784.
[49] Schumann G, Cannon K, Ma WP et al. Antiretroviral effect of a gag-RNase HI fusion gene, Gene Ther. 1997, 4: 593-599.