首页> 外文期刊>Virus Research: An International Journal of Molecular and Cellular Virology >Mutations in the alpha-helical region of the amino terminus of the Maize rayado fino virus capsid protein and CP:RNA ratios affect virus-like particle encapsidation of RNAs
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Mutations in the alpha-helical region of the amino terminus of the Maize rayado fino virus capsid protein and CP:RNA ratios affect virus-like particle encapsidation of RNAs

机译:玉米rayado fino病毒衣壳蛋白氨基末端的α-螺旋区中的突变和CP:RNA的比率影响RNA的病毒样颗粒衣壳化

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Viral-based nanoplatforms rely on balancing the delicate array of virus properties to optimally achieve encapsidation of foreign materials with various potential objectives. We investigated the use of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) as a multifunctional nanoplatform and their potential application as protein cages. MRFV-VLPs are composed of two serologically related, carboxy co-terminal coat proteins (CP1 and CP2) which are capable of self-assembling in Nicotiana benthamiana plants into 30 nm particles with T=3 symmetry. The N-terminus of CP1 was targeted for genetic modification to exploit the driving forces for VLP assembly, packaging and retention of RNA in vivo and in vitro. The N-terminus of MRFV-CP1 contains a peptide sequence of 37 amino acids which has been predicted to have an alpha-helical structure, is rich in hydrophobic amino acids, facilitates CP-RNA interactions, and is not required for self-assembly. Amino acid substitutions were introduced in the 37 amino acid N-terminus by site-directed mutagenesis and the mutant VLPs produced in plants by a Potato virus X (PVX)-based vector were tested for particle stability and RNA encapsidation. All mutant CPs resulted in production of VLPs which encapsidated non-viral RNAs, including PVX genomic and subgenomic (sg) RNAs, 18S rRNA and cellular and viral mRNAs. In addition, MRFV-VLPs encapsidated GFP mRNA when was expressed in plant cells from the pGD vector. These results suggest that RNA packaging in MRFV-VLPs is predominantly driven by electrostatic interactions between the N-terminal 37 amino acid extension of CP1 and RNA, and that the overall species concentration of RNA in the cellular pool may determine the abundance and species of the RNAs packaged into the VLPs. Furthermore, RNA encapsidation is not required for VLPs stability, VLPs formed from MRFV-CP1 were stable at temperatures up to 70 degrees C, and can be disassembled into CP monomers, which can then reassemble in vitro into complete VLPs either in the absence or presence of RNAs. (C) 2014 Published by Elsevier B.V.
机译:基于病毒的纳米平台依靠平衡各种微弱的病毒特性来最佳地实现具有各种潜在目标的外来物质的衣壳化。我们调查了玉米雷亚达菲诺病毒(MRFV)-病毒样颗粒(VLP)作为多功能纳米平台的用途及其作为蛋白质笼的潜在应用。 MRFV-VLP由两个与血清学相关的羧基共末端外壳蛋白(CP1和CP2)组成,它们能够在本氏烟草植物中自组装成T = 3对称的30 nm颗粒。 CP1的N端用于基因修饰,以利用VLP在体内和体外组装,包装和保留RNA的驱动力。 MRFV-CP1的N端包含37个氨基酸的肽序列,该序列已被预测具有α-螺旋结构,富含疏水性氨基酸,促进CP-RNA相互作用,并且不需要自组装。通过定点诱变将氨基酸取代引入37个氨基酸的N端,并测试基于马铃薯病毒X(PVX)的载体在植物中产生的突变VLP的颗粒稳定性和RNA衣壳化。所有突变的CP均导致VLP产生,所述VLP包裹了非病毒RNA,包括PVX基因组和亚基因组(sg)RNA,18S rRNA以及细胞和病毒mRNA。另外,当在来自pGD载体的植物细胞中表达时,MRFV-VLPs使GFP mRNA衣壳化。这些结果表明,MRFV-VLPs中的RNA包装主要由CP1的N端37个氨基酸延伸与RNA之间的静电相互作用驱动,并且细胞池中RNA的总体物种浓度可能决定了该物种的丰度和物种。包装到VLP中的RNA。此外,RNA衣壳化对于VLP的稳定性不是必需的,由MRFV-CP1形成的VLP在高达70摄氏度的温度下是稳定的,可以分解为CP单体,然后可以在不存在或存在的情况下在体外重组为完整的VLP。 RNA。 (C)2014由Elsevier B.V.发布

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