Expression of Separate Heterologous Proteins from the Rotavirus NSP3 Genome Segment Using a Translational 2A Stop-Restart Element
Expression of Separate Heterologous Proteins from the Rotavirus NSP3 Genome Segment Using a Translational 2A Stop-Restart Element
复制标题
使用翻译 2A 停止-重启元件表达来自轮状病毒 NSP3 基因组片段的单独异源蛋白
DOI：
10.1128/jvi.00959-20
复制
发表时间：
2020
影响因子：
5.4
通讯作者：
J. Patton
中科院分区：
文献类型：
--
作者：
Asha A. Philip;J. Patton
轮状病毒是婴幼儿严重胃肠炎的主要原因。最近，开发了一种高效的反向遗传学系统，该系统允许轮状病毒分段双链RNA基因组的遗传操作。使用反向遗传学系统，我们表明，它是可能的，以修改轮状病毒基因组片段（第7段），使病毒获得的能力，表达一个单独的异源蛋白除了完整的病毒蛋白质。通过这种方法，我们已经产生了野生型样轮状病毒，表达各种荧光报告蛋白，包括UnaG（绿色），mRuby（远红色），mKate（红色），和TagBFP（蓝色）。这类病毒株将在通过活细胞成像技术探索轮状病毒生物学和发病机制方面具有价值。值得注意的是，我们的工作表明，轮状病毒基因组是非常灵活的，能够容纳大量的异源RNA序列，提高了使用病毒作为疫苗表达载体的可能性。轮状病毒dsRNA基因组全长18.5kbp，分片段，表达6种结构蛋白和6种非结构蛋白。我们研究了使用最近开发的基于质粒的轮状病毒反向遗传学（RG）系统来产生重组病毒的可能性，所述重组病毒除了表达12种病毒蛋白之外还表达单独的异源蛋白。为了解决这个问题，我们用编码融合到荧光报告蛋白的NSP 3的ORF（即，UnaG、mRuby、mKate或TagBFP）。在融合连接处插入的是捷申病毒翻译2A停止-重新启动元件，其设计用于指导NSP 3和荧光蛋白的单独表达。用修饰的pT 7/NSP 3载体制备的重组轮状病毒生长良好，并且通常遗传稳定，并且它们表达NSP 3和通过活细胞成像可检测到的单独的荧光蛋白。由重组病毒产生的NSP 3是功能性的，诱导细胞poly（A）结合蛋白的核积累。对NSP 3 ORF的进一步修饰表明，有可能产生编码除NSP 3之外的2种异源蛋白（mRuby和UnaG）的重组病毒。我们的结果表明，通过节段7的修饰，轮状病毒基因组的大小可以增加到至少19.8 kbp，并可用于生产重组轮状病毒表达的病毒蛋白和多种异源蛋白的完整的补充。表达荧光蛋白的重组轮状病毒的产生对于通过活细胞成像研究轮状病毒的复制和致病性将是有价值的，并且表明轮状病毒将被证明是有用的表达载体。重要性轮状病毒是婴幼儿严重胃肠炎的主要原因。最近，开发了一种高效的反向遗传学系统，该系统允许轮状病毒分段双链RNA基因组的遗传操作。使用反向遗传学系统，我们表明，它是可能的，以修改轮状病毒基因组片段（第7段），使病毒获得的能力，表达一个单独的异源蛋白除了完整的病毒蛋白质。通过这种方法，我们已经产生了野生型样轮状病毒，表达各种荧光报告蛋白，包括UnaG（绿色），mRuby（远红色），mKate（红色），和TagBFP（蓝色）。这类病毒株将在通过活细胞成像技术探索轮状病毒生物学和发病机制方面具有价值。值得注意的是，我们的工作表明，轮状病毒基因组是非常灵活的，能够容纳大量的异源RNA序列，提高了使用病毒作为疫苗表达载体的可能性。
Rotaviruses are a major cause of severe gastroenteritis in infants and young children. Recently, a highly efficient reverse genetics system was developed that allows genetic manipulation of the rotavirus segmented double-stranded RNA genome. Using the reverse genetics system, we show that it is possible to modify one of the rotavirus genome segments (segment 7) such that virus gains the capacity to express a separate heterologous protein in addition to the full complement of viral proteins. Through this approach, we have generated wild-type-like rotaviruses that express various fluorescent reporter proteins, including UnaG (green), mRuby (far red), mKate (red), and TagBFP (blue). Such strains will be of value in probing rotavirus biology and pathogenesis by live cell imagining techniques. Notably, our work indicates that the rotavirus genome is remarkably flexible and able to accommodate significant amounts of heterologous RNA sequence, raising the possibility of using the virus as a vaccine expression vector. ABSTRACT The segmented 18.5-kbp dsRNA genome of rotavirus expresses 6 structural and 6 nonstructural proteins. We investigated the possibility of using the recently developed plasmid-based rotavirus reverse genetics (RG) system to generate recombinant viruses that express a separate heterologous protein in addition to the 12 viral proteins. To address this, we replaced the NSP3 open reading frame (ORF) of the segment 7 (pT7/NSP3) transcription vector used in the RG system with an ORF encoding NSP3 fused to a fluorescent reporter protein (i.e., UnaG, mRuby, mKate, or TagBFP). Inserted at the fusion junction was a teschovirus translational 2A stop-restart element designed to direct the separate expression of NSP3 and the fluorescent protein. Recombinant rotaviruses made with the modified pT7/NSP3 vectors were well growing and generally genetically stable, and they expressed NSP3 and a separate fluorescent protein detectable by live cell imaging. NSP3 made by the recombinant viruses was functional, inducing nuclear accumulation of cellular poly(A)-binding protein. Further modification of the NSP3 ORF showed that it was possible to generate recombinant viruses encoding 2 heterologous proteins (mRuby and UnaG) in addition to NSP3. Our results demonstrate that, through modification of segment 7, the rotavirus genome can be increased in size to at least 19.8 kbp and can be used to produce recombinant rotaviruses expressing a full complement of viral proteins and multiple heterologous proteins. The generation of recombinant rotaviruses expressing fluorescent proteins will be valuable for the study of rotavirus replication and pathogenesis by live cell imagining and suggest that rotaviruses will prove useful as expression vectors. IMPORTANCE Rotaviruses are a major cause of severe gastroenteritis in infants and young children. Recently, a highly efficient reverse genetics system was developed that allows genetic manipulation of the rotavirus segmented double-stranded RNA genome. Using the reverse genetics system, we show that it is possible to modify one of the rotavirus genome segments (segment 7) such that virus gains the capacity to express a separate heterologous protein in addition to the full complement of viral proteins. Through this approach, we have generated wild-type-like rotaviruses that express various fluorescent reporter proteins, including UnaG (green), mRuby (far red), mKate (red), and TagBFP (blue). Such strains will be of value in probing rotavirus biology and pathogenesis by live cell imagining techniques. Notably, our work indicates that the rotavirus genome is remarkably flexible and able to accommodate significant amounts of heterologous RNA sequence, raising the possibility of using the virus as a vaccine expression vector.
荧光和光活性蛋白的生长和发光工具箱。
影响因子：
13.8
作者：
Rodriguez EA;Campbell RE;Lin JY;Lin MZ;Miyawaki A;Palmer AE;Shu X;Zhang J;Tsien RY
通讯作者：
Tsien RY