Polycistronic Expression of the Influenza A Virus RNA-Dependent RNA Polymerase by Using the Thosea asigna Virus 2A-Like Self-Processing Sequence.
Polycistronic Expression of the Influenza A Virus RNA-Dependent RNA Polymerase by Using the Thosea asigna Virus 2A-Like Self-Processing Sequence.
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流感的多余体表达A A thosea asigna病毒2a样的自我处理序列，流感A病毒RNA依赖性RNA聚合酶。
DOI：
10.3389/fmicb.2016.00288
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发表时间：
2016
影响因子：
5.2
通讯作者：
Morikawa Y
中科院分区：
文献类型：
--
作者：
Momose F;Morikawa Y
甲型流感病毒的RNA依赖RNA聚合酶(RdRp)由PB2、PB1和PA三个亚基组成，催化病毒RNA基因组的复制和转录。将这些亚基的四个单顺反子表达载体和核蛋白与病毒RNA的表达载体共转染，构建了功能性病毒核糖核蛋白复合体(VRNP)。然而，重组RdRp的比活性通常很低，因为在单细胞水平上，表达水平和三个亚基的比例是不能控制的。为了有效地重建RdRp和vRNP，它们的水平至少需要具有可比性。我们构建了多顺反子表达载体，将这三个亚基的编码序列以不同的顺序连接到拟南芥病毒(Thosea asigna Virus，TaV2A)的类2A自我处理序列中。即使放在最下游，PB1蛋白的表达水平也与单顺反子PB1载体的表达水平相当。相反，PB2和PA的表达水平很低，后者很可能是由于TaV2A衍生的序列附着在该表达系统的氨基和/或羧基末端导致的蛋白酶体降解所致。有趣的是，其中两个结构，其中PB1编码序列位于最上游，在基于荧光素酶的小基因组分析中显示出比单顺反子载体共转染的报告活性高得多的报告活性。当选择性抗生素标记的编码序列进一步位于PB1-PA-PB2开放阅读框的最下游时，很容易建立稳定表达RdRp的细胞，这表明获得抗生素耐药性保证了RdRp上游的表达。在PB2的羧基末端添加亲和标签使我们能够分离重组的vRNP。综上所述，构建的流感病毒RdRp多顺反子表达系统可用于vRNP的功能和结构研究。
The RNA-dependent RNA polymerase (RdRp) of influenza A virus consists of three subunits, PB2, PB1, and PA, and catalyses both viral RNA genome replication and transcription. Cotransfection of four monocistronic expression vectors for these subunits and nucleoprotein with an expression vector for viral RNA reconstitutes functional viral ribonucleoprotein complex (vRNP). However, the specific activity of reconstituted RdRp is usually very low since the expression level and the ratio of the three subunits by transfection are uncontrollable at single-cell levels. For efficient reconstitution of RdRp and vRNP, their levels need to be at least comparable. We constructed polycistronic expression vectors in which the coding sequences of the three subunits were joined with the 2A-like self-processing sequence of Thosea asigna virus (TaV2A) in various orders. The level of PB1 protein, even when it was placed at the most downstream, was comparable with that expressed from the monocistronic PB1 vector. In contrast, the levels of PB2 and PA were very low, the latter of which was most likely due to proteasomal degradation caused by the TaV2A-derived sequences attached to the amino- and/or carboxyl-terminal ends in this expression system. Interestingly, two of the constructs, in which the PB1 coding sequence was placed at the most upstream, showed much higher reporter activity in a luciferase-based mini-genome assay than that observed by cotransfection of the monocistronic vectors. When the coding sequence of selective antibiotic marker was further placed at the most downstream of the PB1-PA-PB2 open reading frame, stable cells expressing RdRp were easily established, indicating that acquisition of antibiotic resistance assured the expression of upstream RdRp. The addition of an affinity tag to the carboxyl-terminal end of PB2 allowed us to isolate reconstituted vRNP. Taken together, the polycistronic expression system for influenza virus RdRp may be available for functional and structural studies on vRNP.