Polyprotein processing in Southampton virus: Identification of 3C-like protease cleavage sites by in vitro mutagenesis
Polyprotein processing in Southampton virus: Identification of 3C-like protease cleavage sites by in vitro mutagenesis
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DOI：
10.1128/jvi.70.4.2605-2610.1996
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发表时间：
1996-04-01
影响因子：
5.4
通讯作者：
Lambden, PR
中科院分区：
文献类型：
--
作者：
Liu, BL;Clarke, IN;Lambden, PR
从一组重叠PCR扩增子构建了小的圆形结构病毒南安普顿病毒（SV）的基因组克隆。序列分析证实不存在突变和PCR产物的准确连接。将SV cDNA克隆到载体中，用于体外产生RNA并随后通过兔网织红细胞裂解物进行翻译。对应于病毒多聚蛋白的N-末端和C-末端区域的两种多肽在大肠杆菌中表达，并用于产生用于检测翻译产物的鼠抗血清。三个主要的翻译产物的113，48，和41 kDa的被确定在一个耦合的转录-翻译系统。大的113-kDa的蛋白质与抗血清反应，提出了对C-末端区域的多蛋白，并代表了前体的病毒RNA聚合酶。体外检测到的48-kDa蛋白与针对多聚蛋白N末端的抗血清特异性反应，表明在SV中在基因组5'末端的三个串联的符合读框的AUG密码子处开始翻译。编码病毒多蛋白的大型开放阅读框的一系列嵌套3'缺失用于定义病毒蛋白酶的翻译起始位点和基因组位置。结果与以下模型一致，其中病毒基因组的翻译起始于核苷酸位置5处开始的三个框内AUG密码子之一，并且其中活性病毒蛋白酶在位于NhEI（核苷酸3052）和SphI（核苷酸4056）之间的区域翻译后产生，导致大前体蛋白的快速切割。通过定点诱变推定的活性位点半胱氨酸（Cys-1238）来消除病毒3C样蛋白酶活性，导致产生约200 kDa的大蛋白，其与N-末端和C-末端抗血清反应。两个潜在的多聚蛋白裂解位点含有优选的小核糖核酸病毒QG识别位点被确定在推定的2C样解旋酶区域的多聚蛋白的两侧。在这些位置的蛋白水解将产生相对分子质量与在兔网织红细胞系统中检测到的产物相同的产物。定点诱变用于引入单个碱基变化，其导致氨基酸399和762处的谷氨酰胺残基被脯氨酸残基取代。这些突变完全消除了多蛋白在这些位置的裂解，并产生了分子量与Q-399或Q-762处单次裂解的预测大小相匹配的替代产物。这些数据表明，小的、圆形结构的病毒南安普顿病毒产生3C样蛋白酶，该蛋白酶在399和762位具有两个主要切割位点。在这些位置的蛋白水解裂解释放推定的病毒2C样解旋酶。
A genomic clone of the small, round-structured virus Southampton virus (SV) was constructed from a set of overlapping PCR amplicons. Sequence analysis confirmed the absence of mutations and accurate ligation of the PCR products. The SV cDNA was cloned into a vector for in vitro production of RNA and subsequent translation by rabbit reticulocyte lysate. Two polypeptides corresponding to the N-terminal and C-terminal regions of the viral polyprotein were expressed in Escherichia call and used to produce murine antisera for detection of translation products. Three major translation products of 113, 48, and 41 kDa were identified in a coupled transcription-translation system. The large 113-kDa protein reacted with antisera raised against the C-terminal region of the polyprotein and represents a precursor of the viral RNA polymerase. The 48-kDa protein detected in vitro reacted specifically with antisera raised against the polyprotein N terminus, showing that translation was initiated in SV at the three tandem in-frame AUG codons at the 5' end of the genome. A series of nested 3' deletions of the large open reading frame encoding the viral polyprotein was used to define the translation initiation site and genomic location of the viral protease. The results are consistent with a model in which translation of the viral genome is initiated at one of the three in-frame AUG codons starting at nucleotide position 5 and in which active viral protease is produced following translation of a region located between NhEI (nucleotide 3052) and SphI (nucleotide 4056), resulting in rapid cleavage of a large precursor protein. Abolition of the viral 3C-like protease activity by site-directed mutagenesis of the putative active-site cysteine (Cys-1238) resulted in production of a large protein of approximately 200 kDa which reacted with both N-terminal and C-terminal antisera. Two potential polyprotein cleavage sites containing the preferred picornaviral QG recognition site were identified on either side of the putative 2C-like helicase region of the polyprotein. Proteolysis at these positions would give rise to products with relative molecular masses identical to those of the products detected in the rabbit reticulocyte system. Site-directed mutagenesis was used to introduce a single base change which resulted in the substitution of glutamine residues with proline residues at amino acids 399 and 762. These mutations completely abolished cleavage of the polyprotein at these positions and gave rise to alternative products with molecular masses which matched the predicted sizes for a single cleavage at either Q-399 or Q-762. These data indicate that the small, round-structured virus Southampton virus produces a 3C-like protease which has two primary cleavage sites at positions 399 and 762. Proteolytic cleavage at these positions releases the putative viral 2C-like helicase.