Rotavirus is a segmented double-stranded RNA (dsRNA) virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses 11 T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a cytomegalovirus support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kb segment 7 dsRNA encoding full-length nonstructural protein 3 (NSP3) fused to UnaG, a 139-amino-acid green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passaging. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 levels in wild-type recombinant SA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wild-type NSP3, formed dimers in vivo Notably, the NSP3-UnaG protein was readily detected in infected cells via live-cell imaging, with intensity levels ∼3-fold greater than those of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deletion or interruption of any of the 12 open reading frames (ORFs) of the virus. Because NSP3 is expressed at higher levels than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be more sensitive tools for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA.IMPORTANCE Previous studies generated recombinant rotaviruses that express FPs by inserting reporter genes into the NSP1 ORF of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses that likely express the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotaviruses encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

中文翻译：

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通过简化的反向遗传学系统生成表达 NSP3-UnaG 融合蛋白的重组轮状病毒。

轮状病毒是一种分段双链 RNA (dsRNA) 病毒，可导致幼儿严重胃肠炎。我们建立了一个高效的简化轮状病毒反向遗传学 (RG) 系统，该系统使用 11 个 T7 质粒，每个质粒表达一个独特的猿猴 SA11 (+) RNA，以及一个用于非洲猪瘟病毒 NP868R 封端酶的巨细胞病毒支持质粒。使用基于 NP868R 的系统，我们生成了重组轮状病毒 (rSA11/NSP3-FL-UnaG)，其具有编码全长非结构蛋白 3 (NSP3) 的基因修饰的 1.5-kb 片段 7 dsRNA，与 UnaG（一种 139 个氨基酸）融合绿色荧光蛋白 (FP)。对 rSA11/NSP3-FL-UnaG 的分析表明，该病毒可以有效复制，并且在 10 轮连续传代中基因稳定。NSP3-UnaG 融合产物在 rSA11/NSP3-FL-UnaG 感染的细胞中表达良好，达到与野生型重组 SA11 感染细胞中 NSP3 水平相似的水平。此外，与功能性野生型 NSP3 一样，NSP3-UnaG 蛋白在体内形成二聚体rSA11/NSP1-FL-UnaG 的 NSP1-UnaG 融合产物。我们的结果表明表达 FP 的重组轮状病毒可以通过操作片段 7 dsRNA 而不会删除或中断病毒的 12 个开放阅读框 (ORF) 中的任何一个。由于 NSP3 在受感染细胞中的表达水平高于 NSP1，因此表达基于 NSP3 的 FP 的轮状病毒可能是研究轮状病毒生物学的比表达基于 NSP1 的 FP 的轮状病毒更敏感的工具。这是包含基因工程片段 7 dsRNA 的重组轮状病毒的第一份报告。重要 以前的研究产生了通过将报告基因插入基因组片段 5 的 NSP1 ORF 中来表达 FP 的重组轮状病毒。不幸的是，NSP1 在受感染细胞中的表达水平很低, 使得表达 FP-fused NSP1 的病毒低于轮状病毒生物学的理想探针。此外，FPs 以破坏 NSP1 的方式插入第 5 段，NSP1 是一种影响病毒生长和发病机制的干扰素拮抗剂。我们已经确定了一种生成表达 FP 的轮状病毒的替代方法，该方法依赖于将报告基因融合到基因组片段 7 的 NSP3 ORF。这是在不中断任何病毒 ORF 的情况下完成的，产生可能表达整套功能性病毒蛋白的重组病毒。鉴于 NSP3 在受感染的细胞中以中等水平产生，编码基于 NSP3 的 FP 的轮状病毒应该是比编码基于 NSP1 的 FP 的轮状病毒更敏感的病毒感染探针。