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通过病毒衣壳蛋白对单链 RNA 分子的相对包装效率进行体外定量。

In vitro quantification of the relative packaging efficiencies of single-stranded RNA molecules by viral capsid protein.

机构信息

Department of Chemistry and Biochemistry, University of California, Los Angeles, California, USA.

出版信息

J Virol. 2012 Nov;86(22):12271-82. doi: 10.1128/JVI.01695-12. Epub 2012 Sep 5.

Abstract

While most T=3 single-stranded RNA (ssRNA) viruses package in vivo about 3,000 nucleotides (nt), in vitro experiments have demonstrated that a broad range of RNA lengths can be packaged. Under the right solution conditions, for example, cowpea chlorotic mottle virus (CCMV) capsid protein (CP) has been shown to package RNA molecules whose lengths range from 100 to 10,000 nt. Furthermore, in each case it can package the RNA completely, as long as the mass ratio of CP to nucleic acid in the assembly mixture is 6:1 or higher. Yet the packaging efficiencies of the RNAs can differ widely, as we demonstrate by measurements in which two RNAs compete head-to-head for a limited amount of CP. We show that the relative efficiency depends nonmonotonically on the RNA length, with 3,200 nt being optimum for packaging by the T=3 capsids preferred by CCMV CP. When two RNAs of the same length-and hence the same charge-compete for CP, differences in packaging efficiency are necessarily due to differences in their secondary structures and/or three-dimensional (3D) sizes. For example, the heterologous RNA1 of brome mosaic virus (BMV) is packaged three times more efficiently by CCMV CP than is RNA1 of CCMV, even though the two RNAs have virtually identical lengths. Finally, we show that in an assembly mixture at neutral pH, CP binds reversibly to the RNA and there is a reversible equilibrium between all the various RNA/CP complexes. At acidic pH, excess protein unbinds from RNA/CP complexes and nucleocapsids form irreversibly.

摘要

虽然大多数 T=3 单链 RNA(ssRNA)病毒在体内包装约 3000 个核苷酸(nt),但体外实验表明可以包装广泛的 RNA 长度。在适当的溶液条件下,例如,豇豆花叶病毒(CCMV)衣壳蛋白(CP)已被证明可以包装长度从 100 到 10000nt 的 RNA 分子。此外,在每种情况下,只要组装混合物中 CP 与核酸的质量比为 6:1 或更高,它就可以完全包装 RNA。然而,正如我们通过在有限量的 CP 中竞争的两种 RNA 头对头测量所证明的那样,RNA 的包装效率可以有很大差异。我们表明,相对效率与 RNA 长度呈非单调关系,对于 CCMV CP 偏好的 T=3 衣壳而言,3200nt 是最佳包装长度。当两种相同长度的 RNA(因此具有相同的电荷)竞争 CP 时,包装效率的差异必然是由于它们的二级结构和/或三维(3D)大小的差异。例如,雀麦花叶病毒(BMV)的异源 RNA1 被 CCMV CP 包装的效率比 CCMV 的 RNA1 高三倍,尽管这两种 RNA 的长度几乎相同。最后,我们表明在中性 pH 的组装混合物中,CP 可逆地与 RNA 结合,并且在所有各种 RNA/CP 复合物之间存在可逆平衡。在酸性 pH 下,过量的蛋白质从 RNA/CP 复合物中解吸,核衣壳不可逆形成。

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