Specific Packaging of Custom RNA Molecules into Cowpea Mosaic Virus-like Particles.
Specific Packaging of Custom RNA Molecules into Cowpea Mosaic Virus-like Particles.
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DOI：
10.1007/978-1-0716-2241-4_7
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发表时间：
2022-01-01
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影响因子：
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通讯作者：
Lomonossoff, George P
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作者：
Peyret, Hadrien;Lomonossoff, George P
最近在植物病毒豇豆花叶病毒（CPMV）中基因组复制和包装动力学方面的发现已经导致开发了一种用于将所选RNA分子特异性包装到CPMV的病毒样颗粒（VLP）中的新方法。由于现代基因合成和分子克隆方法，对应于感兴趣的RNA序列的DNA序列可以被克隆到合适的表达质粒中，用于在植物中瞬时表达。我们在这里描述了一种方法，用于确保该RNA序列将在植物细胞中通过复制依赖性RNA包装被包装在CPMV的VLP内。这需要与CPMV外壳蛋白前体一起共表达CPMV复制机制。这些组分在本氏烟草（Nicotiana benthamiana）植物的叶中共表达，并且这种共表达导致产生大量含有所选RNA序列的VLP。这些VLP易于从植物组织中提取和纯化，并且在冷藏条件下稳定数月。然后，这些VLP可用于各种不同的应用，例如RT-qPCR中的RNA递送或对照试剂。
Recent discoveries in the dynamics of genome replication and packaging in the plant virus Cowpea mosaic virus (CPMV) has led to the development of a novel method for specifically packaging an RNA molecule of choice into virus-like particles (VLPs) of CPMV. Thanks to modern gene synthesis and molecular cloning methods, the DNA sequence corresponding to an RNA sequence of interest can be cloned into a suitable expression plasmid for transient expression in plants. We describe here a method for ensuring that this RNA sequence will be packaged within VLPs of CPMV in plant cells by replication-dependent RNA packaging. This requires co-expression of the CPMV replication machinery alongside the CPMV coat protein precursor. These components are co-expressed in the leaves of the Nicotiana benthamiana plant and this co-expression results in the production of large quantities of VLPs that contain the RNA sequence of choice. These VLPs are easy to extract and purify from the plant tissue, and are stable for months in refrigerated conditions. These VLPs can then be used for a variety of different applications, such as RNA delivery or control reagents in RT-qPCR.