ASSEMBLY OF FOOT-AND-MOUTH-DISEASE VIRUS EMPTY CAPSIDS SYNTHESIZED BY A VACCINIA VIRUS EXPRESSION SYSTEM
ASSEMBLY OF FOOT-AND-MOUTH-DISEASE VIRUS EMPTY CAPSIDS SYNTHESIZED BY A VACCINIA VIRUS EXPRESSION SYSTEM
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DOI：
10.1099/0022-1317-76-12-3089
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发表时间：
1995-12-01
影响因子：
3.8
通讯作者：
BELSHAM, GJ
中科院分区：
文献类型：
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作者：
ABRAMS, CC;KING, AMQ;BELSHAM, GJ
构建了口蹄疫病毒（FMDV）结构蛋白前体P1-2A的cDNA卡带，以及将其裂解为1AB、1C和1D的3C蛋白酶。这些卡带被引入牛痘病毒（VV）转移载体。试图分离表达这些磁带的重组VVs是不成功的。然而，当将P1-2A-3C盒置于噬菌体T7启动子的控制下时，分离出稳定的VV/FMDV重组体。与重组VV vTF7-3（表达T7 RNA聚合酶）共同感染导致正确加工的FMDV衣壳蛋白的产生。蔗糖梯度离心分析表明，形成了与天然空衣壳颗粒（70S）共沉积的物质。电镜显示直径约30毫米的空衣壳样颗粒。利用构象表位特异性单克隆抗体的研究表明，合成颗粒的抗原性与全病毒粒子和天然空衣壳颗粒相似。令人惊讶的是，仅仅修改P1-2A N端肉豆肉酰化一致序列内的单个氨基酸残基就可以分离出重组VV，该重组VV组成性地表达正确加工的蛋白质。然而，从该突变盒中表达的衣壳蛋白未能组装成70S空颗粒。
cDNA cassettes encoding the foot-and-mouth disease virus (FMDV) structural protein precursor (P1-2A) together with the 3C protease, which cleaves this molecule to 1AB, 1C and 1D, were constructed. These cassettes were introduced into vaccinia virus (VV) transfer vectors. Attempts to isolate recombinant VVs constitutively expressing these cassettes were unsuccessful. However, when the P1-2A-3C cassette was placed under the control of the bacteriophage T7 promoter, stable VV/FMDV recombinants were isolated. Co-infection with recombinant VV vTF7-3 (which expresses T7 RNA polymerase) led to the production of correctly processed FMDV capsid proteins. Analysis by sucrose gradient centrifugation showed that material which co-sedimented with natural empty capsid particles (70S) was formed. Electron microscopy revealed empty capsid-like particles with diameters of about 30 mm. Studies using monoclonal antibodies specific for conformational epitopes indicated that the antigenicity of the synthetic particles was similar to whole virions and natural empty capsid particles. Surprisingly, merely the modification of a single amino acid residue within the myristoylation consensus sequence at the N terminus of P1-2A allowed the isolation of a recombinant VV which constitutively expressed the correctly processed proteins. However, the capsid proteins expressed from this mutant cassette failed to assemble into 70S empty particles.