Development of Stable Rotavirus Reporter Expression Systems
Development of Stable Rotavirus Reporter Expression Systems
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DOI：
10.1128/jvi.01774-18
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发表时间：
2019-02-01
影响因子：
5.4
通讯作者：
Kobayashi, Takeshi
中科院分区：
文献类型：
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作者：
Kanai, Yuta;Kawagishi, Takahiro;Kobayashi, Takeshi
表达报告基因的工程重组病毒已被开发用于实时监测复制和大规模筛选抗病毒抑制剂。最近，我们报道了使用反向遗传学系统开发了第一个表达NanoLuc （NLuc）荧光素酶的重组报告轮状病毒（RVs）。在这里，我们描述了一种开发稳定的报告rv表达荧光素酶和绿色或红色荧光蛋白的策略。将报告基因插入到NSP1的开放阅读框中，并与由1 ~ 27个氨基酸组成的NSP1肽融合表达。与插入的转基因无关，嵌合nsp1 -报告基因较短的RV报告菌株内外源基因的稳定性高于原RV报告菌株。改良的报告基因RV用于筛选中和性单克隆抗体（mab）。对来自一个单抗克隆（克隆29）的逃逸突变体进行序列分析，发现VP4蛋白441位有一个氨基酸替代（精氨酸到甘氨酸），位于VP5*片段中和表位5-1内。此外，为了表达缺乏NSP1氨基酸1至27的天然报告蛋白，修饰了5‘和3’端序列，以恢复NSP1报告嵌合基因的预测二级RNA结构。这些数据证明了报告型RV在实时监测RV感染方面的效用，并提出了进一步的应用（例如，RV疫苗载体，它可以诱导粘膜免疫对抗肠道病原体）。由于缺乏全面的反向遗传系统，报告型房车的发展一直受到阻碍。最近，我们开发了一种基于质粒的反向遗传系统，可以产生表达NLuc荧光素酶的报告基因rv。原型报告基因RV有一些缺点（即转基因不稳定，并且作为与部分NSP1肽的融合蛋白表达）；然而，改良的报告子RV通过修饰报告子- nsp1嵌合基因的未翻译区克服了这些问题。这种生成稳定报告RV的策略可以扩展到多种转基因，并用于开发RV转导载体。此外，这些数据提高了我们对5‘和3’端序列在基因组复制、组装和包装方面的重要性的理解。
Engineered recombinant viruses expressing reporter genes have been developed for real-time monitoring of replication and for mass screening of antiviral inhibitors. Recently, we reported using a reverse genetics system to develop the first recombinant reporter rotaviruses (RVs) that expressed NanoLuc (NLuc) luciferase. Here, we describe a strategy for developing stable reporter RVs expressing luciferase and green or red fluorescent proteins. The reporter genes were inserted into the open reading frame of NSP1 and expressed as a fusion with an NSP1 peptide consisting of amino acids 1 to 27. The stability of foreign genes within the reporter RV strains harboring a shorter chimeric NSP1-reporter gene was greater than that of those in the original reporter RV strain, independent of the transgene inserted. The improved reporter RV was used to screen for neutralizing monoclonal antibodies (MAbs). Sequence analysis of escape mutants from one MAb clone (clone 29) identified an amino acid substitution (arginine to glycine) at position 441 in the VP4 protein, which resides within neutralizing epitope 5-1 in the VP5* fragment. Furthermore, to express a native reporter protein lacking NSP1 amino acids 1 to 27, the 5'- and 3'-terminal region sequences were modified to restore the predicted secondary RNA structure of the NSP1-reporter chimeric gene. These data demonstrate the utility of reporter RVs for live monitoring of RV infections and also suggest further applications (e.g., RV vaccine vectors, which can induce mucosal immunity against intestinal pathogens).IMPORTANCE Development of reporter RVs has been hampered by the lack of comprehensive reverse genetics systems. Recently, we developed a plasmid-based reverse genetics system that enables generation of reporter RVs expressing NLuc luciferase. The prototype reporter RV had some disadvantages (i.e., the transgene was unstable and was expressed as a fusion protein with a partial NSP1 peptide); however, the improved reporter RV overcomes these problems through modification of the untranslated region of the reporter-NSP1 chimeric gene. This strategy for generating stable reporter RVs could be expanded to diverse transgenes and be used to develop RV transduction vectors. Also, the data improve our understanding of the importance of 5'- and 3'-terminal sequences in terms of genome replication, assembly, and packaging.