Influenza A virus segments five and six can harbor artificial introns allowing expanded coding capacity.
Influenza A virus segments five and six can harbor artificial introns allowing expanded coding capacity.
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甲型流感病毒片段5和6可以含有人工内含子，从而扩大编码能力。
DOI：
10.1371/journal.ppat.1009951
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发表时间：
2021-09
期刊：
影响因子：
6.7
通讯作者：
Heaton NS
中科院分区：
文献类型：
--
作者：
Froggatt HM;Burke KN;Chaparian RR;Miranda HA;Zhu X;Chambers BS;Heaton NS
甲型流感病毒编码其基因组跨越八个负义RNA区段。六个最大的片段产生通常不剪接的mRNA转录物;然而，两个最小的片段被主动剪接以产生必需的病毒蛋白NEP和M2。因此，病毒利用RNA剪接有效地扩大了病毒编码能力，而不增加基因组区段的数量。作为理解为什么剪接不能更广泛地用于基因组片段的第一步，我们设计并插入了一个人工内含子到正常的非剪接NA片段中。这种插入是耐受的，尽管病毒mRNA被不完全剪接，但我们仅观察到对病毒适应性的微小影响。为了利用未剪接的病毒RNA，我们编码了与病毒ORF同框的报告荧光素酶基因，使得当内含子未被去除时，将产生报告蛋白。这种方法，我们也表明，可以应用于NP编码段和在不同的病毒遗传背景，导致高水平的报告蛋白的表达与病毒复制的动力学或在实验感染的动物引起疾病的能力的影响最小。这些数据共同表明，流感病毒基因组比以前认识到的更耐受剪接，并且可以利用这些知识来开发具有生物技术应用效用的病毒遗传平台。与大多数宿主mRNA不同，一些病毒mRNA编码多个离散的功能蛋白。A型流感病毒用来增加八个RNA基因组片段中两个片段的蛋白质产物的一种方法是剪接。剪接需要宿主机器去除病毒mRNA的一部分，即内含子，以产生不同的mRNA产物。虽然只有某些流感病毒片段自然剪接，我们感兴趣的是是否额外的片段可以剪接产生多种蛋白质。我们将携带报告基因的人工内含子插入到H1N1甲型流感病毒的非剪接基因组片段中，发现这种修饰被病毒很好地耐受。我们进一步证明，一个无关的H3 N2甲型流感病毒可以类似地支持剪接和表达报告蛋白从人工内含子。这些发现对我们理解病毒如何利用有限的基因组扩展其编码能力具有重要意义。此外，在拼接的内含子序列中编码报告蛋白也代表了一种产生报告病毒的新方法，需要对病毒RNA进行有限的操作。
Influenza A viruses encode their genomes across eight, negative sense RNA segments. The six largest segments produce mRNA transcripts that do not generally splice; however, the two smallest segments are actively spliced to produce the essential viral proteins NEP and M2. Thus, viral utilization of RNA splicing effectively expands the viral coding capacity without increasing the number of genomic segments. As a first step towards understanding why splicing is not more broadly utilized across genomic segments, we designed and inserted an artificial intron into the normally nonsplicing NA segment. This insertion was tolerated and, although viral mRNAs were incompletely spliced, we observed only minor effects on viral fitness. To take advantage of the unspliced viral RNAs, we encoded a reporter luciferase gene in frame with the viral ORF such that when the intron was not removed the reporter protein would be produced. This approach, which we also show can be applied to the NP encoding segment and in different viral genetic backgrounds, led to high levels of reporter protein expression with minimal effects on the kinetics of viral replication or the ability to cause disease in experimentally infected animals. These data together show that the influenza viral genome is more tolerant of splicing than previously appreciated and this knowledge can be leveraged to develop viral genetic platforms with utility for biotechnology applications. Unlike most host mRNAs, some viral mRNAs encode multiple discrete, functional proteins. One method influenza A viruses use to increase the protein products from two of their eight RNA genome segments is splicing. Splicing requires host machinery to remove part of the viral mRNA, the intron, to generate a different mRNA product. Although only certain influenza viral segments naturally splice, we were interested in whether additional segments could splice to produce multiple proteins. We inserted artificial introns harboring reporter genes into otherwise nonsplicing genomic segments of an H1N1 influenza A virus and found that this modification was well tolerated by the virus. We further demonstrated that an unrelated H3N2 influenza A virus could similarly support splicing and express a reporter protein from an artificial intron. These findings have implications for our understanding of how viruses expand their coding capacity with a limited genome. Additionally, encoding reporter proteins in spliced intronic sequences also represents a new method of generating reporter viruses requiring limited manipulation of the viral RNA.
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影响因子：
5.8
作者：
Gultyaev, Alexander P.;Heus, Hans A.;Olsthoorn, Rene C. L.
通讯作者：
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DOI：
10.1099/vir.0.042499-0
发表时间：
2012-07
期刊：
The Journal of general virology
影响因子：
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作者：
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通讯作者：
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影响因子：
16.6
作者：
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