Proteolytic processing of a human astrovirus nonstructural protein.
Proteolytic processing of a human astrovirus nonstructural protein.
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人星状病毒非结构蛋白的蛋白水解加工。
DOI：
10.1099/0022-1317-83-1-25
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发表时间：
2002
期刊：
影响因子：
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通讯作者：
Matsui,SuzanneM
中科院分区：
文献类型：
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作者：
Kiang,David;Matsui,SuzanneM
为了分析人星状病毒血清1型（HAstV-1）开放阅读框（ORF）-1a编码的3C样丝氨酸蛋白酶的活性，体外转录并鉴定了ORF-1a。通过免疫沉淀法用针对重组C-末端ORF-1a片段的特异性抗体鉴定的翻译产物包括全长101 kDa（p101）蛋白和38 kDa条带（p38）。此外，当FLAG表位插入ORF-1a产物的N末端时，64 kDa蛋白质（p64）被抗FLAG抗体免疫沉淀。预测形成HAstV-1 3C样丝氨酸蛋白酶的催化三联体的氨基酸（Ser-551，Asp-489，His-461）的突变导致不可检测水平的p38，支持HAstV-1 3C样丝氨酸蛋白酶的参与以及这些氨基酸在p101加工成p38和p64中的重要性。与野生型相比，不涉及预测的3C样丝氨酸蛋白酶基序的p101的高达420个氨基酸的N-末端缺失没有改变p38的水平。C-末端缺失分析将p38定位于ORF-1a的C末端。预测裂解位点的P1残基突变（在已知的人和绵羊星状病毒序列中是保守的）导致无法检测到p38，支持Gln-567↓Thr-568二肽的裂解。这些结果表明，p101在依赖于病毒3C样丝氨酸蛋白酶的过程中在Gln-567↓Thr-568处被切割成N端p64片段和C端p38产物。
To analyse the activity of the putative 3C-like serine protease encoded in open reading frame (ORF)-1a of human astrovirus serotype 1 (HAstV-1), ORF-1a was transcribed and translatedin vitro. Translation products, identified by immunoprecipitation with specific antibodies against recombinant C-terminal ORF-1a fragments, include the full-length 101 kDa (p101) protein and a 38 kDa band (p38). In addition, a 64 kDa protein (p64) was immunoprecipitated by an anti-FLAG antibody when a FLAG epitope was inserted at the N terminus of the ORF-1a product. Mutation of the amino acids predicted to form the catalytic triad of the HAstV-1 3C-like serine protease (Ser-551, Asp-489, His-461) resulted in undetectable levels of p38, supporting the involvement of the HAstV-1 3C-like serine protease and the importance of these amino acids in the processing of p101 into p38 and p64. N-terminal deletions of up to 420 aa of p101 that did not involve the predicted 3C-like serine protease motif did not alter levels of p38 compared to wild-type. C-terminal deletion analysis localized p38 to the C terminus of ORF-1a. Mutation of the P1 residue of the predicted cleavage site, which is conserved among known human and sheep astrovirus sequences, resulted in no detectable p38, supporting cleavage at the Gln-567↓Thr-568 dipeptide. These results suggest that p101 is cleaved into an N-terminal p64 fragment and a C-terminal p38 product at Gln-567↓Thr-568 in a process that is dependent on the viral 3C-like serine protease.