A VIRAL CLEAVAGE SITE CASSETTE - IDENTIFICATION OF AMINO-ACID SEQUENCES REQUIRED FOR TOBACCO ETCH VIRUS POLYPROTEIN PROCESSING
A VIRAL CLEAVAGE SITE CASSETTE - IDENTIFICATION OF AMINO-ACID SEQUENCES REQUIRED FOR TOBACCO ETCH VIRUS POLYPROTEIN PROCESSING
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DOI：
10.1073/pnas.85.10.3391
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发表时间：
1988-05-01
影响因子：
11.1
通讯作者：
DOUGHERTY, WG
中科院分区：
文献类型：
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作者：
CARRINGTON, JC;DOUGHERTY, WG
烟草蚀纹病毒（TEV）的成熟病毒编码蛋白是由一个大的前体蛋白水解加工产生的。负责大多数这些裂解的蛋白酶是病毒编码的49-kDa蛋白。TEV多聚蛋白中所有已知或预测的切割位点的侧翼都是保守序列基序Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser或Gly，Gln-Ser或Gly二肽之间存在易断裂键。通过使用无细胞系统来操作和表达克隆的cDNA序列，含有TEV多蛋白的推定蛋白水解切割位点的25个氨基酸的区段已被引入TEV衣壳蛋白序列中。该重组蛋白在引入的切割位点被49-kDa蛋白酶切割，从而证明了功能性切割位点的可移植性。通过构建含有部分或全部该基序的工程蛋白质来测试保守氨基酸序列在确定底物活性中的作用。一个蛋白，窝藏保守的7-氨基酸片段的插入被切割的49 kDa TEV蛋白酶。通过微序列分析显示，合成前体的切割准确地发生在预期的Gln-Ser二肽之间。含有仅产生Gln-Ser或仅产生保守序列的丝氨酸部分的插入的蛋白质对49-kDa蛋白酶不敏感。
Mature viral-encoded proteins of tobacco etch virus (TEV) arise by proteolytic processing of a large precursor. The proteinase responsible for most of these cleavages is a viral-encoded 49-kDa protein. All known or predicted cleavage sites inthe TEV polyprotein are flanked by the conserved sequences motif Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly, with the scissile bond located between the Gln-Ser or Glydipeptide. By using cell-free systems to manipulate and express cloned cDNA sequences, a 25-amino acid segment containing a putative proteolytic cleavage site of the TEV polyproteins has been introduced into the TEV capsid protein sequence. This recombinant protein is cleaved by the 49-kDa proteinase at the introduced cleavage site, thus demonstrating portability of a functional cleavage site. The role of the conserved amino acid sequence in determining substrate activity was tested by construction of engineered proteins that contained part or all of this motif. A protein that harbored an insertion of the conserved 7-amino acid segment was cleaved by the 49-kDa TEV proteinase. Cleavage of the synthetic precursor was shown to occur accurately between the expected Gln-Ser dipeptide by microsequence analysis. Proteins containing insertions that generated only the Gln-Ser, or only the serine moiety of the conserved sequence, were insensitive to the 49-kDa proteinase.