Untangling the processes of replication and encapsidation in Picornavirales

为了使病毒能够从一个有机体传播到另一个有机体，它必须保护其遗传物质(基因组)不受恶劣外部环境的影响。对于许多使用核糖核酸(RNA)而不是更常见和更稳定的脱氧核糖核酸(DNA)作为基因组的病毒来说，情况尤其如此。保护是通过由一个或几个外壳蛋白的许多副本组成的蛋白质外壳包围脆弱的基因组来产生病毒颗粒来实现的。当病毒在细胞内繁殖时，病毒基因组和外壳蛋白都会产生。基因组被蛋白质壳包围的过程被称为包裹化，这也发生在细胞内。包膜是高度选择性的，确保只有病毒的基因组，而不是通常存在于宿主细胞中的RNA被整合到病毒颗粒中。尽管包膜在病毒生命周期中至关重要，但对含有RNA的病毒的主要家族之一的包膜机制知之甚少。该家族包含可感染动物或植物的病毒，其中一些成员在医学(脊髓灰质炎病毒、甲型肝炎病毒、普通感冒病毒)、兽医(口蹄疫病毒)和农业(稻米中球状病毒)方面具有重要意义。显然，更好地了解病毒传播的关键过程将对我们抗击这些疾病的能力具有重大意义。这样的理解将有助于开发可作为载体将特定核酸序列输送到细胞的病毒颗粒，以用于各种医疗应用。关于短小核病毒目的包膜作用的信息如此之少的原因之一是，这一过程似乎与复制周期的其他方面密切相关，包括基因组的扩增(复制)和蛋白质合成(翻译)。然而，在John Innes中心(JIC)，我们已经证明，在感染植物的小核病毒目成员--豌豆花叶病毒(CPMV)的情况下，通过在植物中瞬时表达各种病毒成分，有可能解开这些过程。此外，利兹大学(UOL)使用电子显微镜进行的研究表明，可以看到CPMV组装颗粒内的RNA，揭示了基因组如何与外壳蛋白亚基相互作用形成病毒颗粒的细节。在这项提案中，我们希望结合JIC和UOL现有的专业知识来了解CPMV是如何将其基因组具体封装在病毒颗粒中的。我们将调查可以有效包裹的RNA分子是否有大小限制，以及包裹与病毒复制周期的其他方面之间的联系。从这些研究中获得的知识将不仅适用于CPMV，而且适用于小核病毒科的所有成员，并将有助于这些病毒在生物农艺中的应用。

For a virus to be able to spread from one organism to another, it must protect its genetic material (genome) from the harsh external environment. This is particularly true of the many viruses that use ribonucleic acid (RNA) rather than the more common and more stable deoxyribonucleic acid (DNA) for their genomes. Protection is achieved by surrounding the fragile genome by a protein shell made up of many copies of one or a few coat proteins to produce virus particles. Both the viral genome and the coat proteins are produced when a virus multiplies inside a cell. The process by which the genome is surrounded by the protein shell is known as encapsidation and this also takes place within the cell. Encapsidation is highly selective, ensuring that only the genome of the virus, and not the RNA normally present in the host cell, is incorporated into virus particles.Despite its critical importance in the viral life cycle, very little is known about the mechanism of encapsidation in one of the major families of RNA-containing viruses, the Picornavirales. This family contains viruses which can infect either animals or plants, and some members are of great medical (poliovirus, hepatitis A virus, common cold virus), veterinary (foot-and-mouth disease virus) and agricultural (rice tungro spherical virus) importance. Clearly a greater understanding of a process essential for virus spread would be of huge significance to our ability to combat these diseases. Such understanding would help to develop virus particles that can act as vehicles for the delivery of specific nucleic acid sequences to cells for a variety of medical applications. One of the reasons that so little information is available about encapsidation in the Picornavirales is that the process appears to be intimately associated with other aspects of the replication cycle including amplification of the genome (replication) and protein synthesis (translation). However, at the John Innes Centre (JIC), we have shown that it is possible to untangle these processes in the case of the plant-infecting member of the Picornavirales, cowpea mosaic virus (CPMV) using transient expression of the various viral components in plants. Furthermore, studies at the University of Leeds (UoL) using electron microscopy have shown that it is possible to see the RNA within assembled particles of CPMV, revealing details of how the genome interacts with the coat protein subunits to form a virus particle. In this proposal, we wish to combine the expertise available at JIC and UoL to understand how the CPMV specifically encapsidated its genome within viral particles. We will investigate whether there is a size limit on RNA molecules which can be efficiently encapsidated and the linkage between encapsidation and other aspects of the viral replication cycle. The knowledge gained from these studies would be applicable not only to CPMV but to all members of the family Picornavirales and would also aid the application of these viruses in bionanotechnology.