Influence of RNA on icosahedral virus particle structure

病毒是非常成功的病原体，可以感染所有类型的生物，包括植物、动物(包括人类)、真菌和所有类型的细菌。它们本质上是细胞内的寄生虫，将它们的遗传物质引入宿主细胞，并颠覆正常的细胞功能，以制造更多的自身副本。它们通常以病毒颗粒的形式存在于环境中，其中病毒核酸，无论是DNA还是RNA，被一个由病毒基因组编码的一种或多种蛋白质亚单位的多个副本组成的壳所包围。在某些情况下(包膜病毒)，它们还被宿主起源的膜所包围。蛋白质壳的目的是保护微妙的遗传信息不受细胞外恶劣环境的影响，并使病毒能够成功传播到其他宿主。这通常涉及到媒介的吸收和传播，例如昆虫或真菌，而核酸必须能够在这个过程中存活下来。一旦进入易受感染的宿主体内，病毒就会经历一个包括几个阶段的“复制周期”，从而制造更多的自身副本：揭开颗粒的外壳以释放病毒基因组，病毒基因的表达，病毒核酸的复制，以及将新合成的核酸封装成颗粒，这些颗粒随后被释放以感染进一步的宿主。这都是一个精心设计的过程，病毒基因组的复制和封装通常密切相关。与宿主不同，将病毒核酸特定地包裹到具有感染性的颗粒中是病毒复制周期中至关重要的一步。这一过程必须导致病毒颗粒的形成，从而有效地保护不稳定的遗传物质。这导致了对RNA病毒中已定义的RNA序列元件或“包装信号”的识别。这样的包装信号被设想为标记病毒RNA，使得它以一种通过外壳蛋白亚基从细胞分子的混合物中被特异性识别的方式有效地被条形码识别，从而赋予封装选择性。然而，我们最近的工作表明，包装的选择性由感染细胞内RNA分子的复制决定，外壳蛋白的合成与RNA复制紧密耦合。在这样的“复制工厂”中，病毒的RNA基因组并不与其他非复制的RNA竞争，这表明“条形码”可能不是必须的选择性包装。相反，它们的存在可能是为了确保将RNA掺入颗粒中的过程有效地进行，以产生完全具有传染性的病毒粒子。我们还获得了复制RNA的长度可能控制所得颗粒的结构的证据。为了确定潜在包装信号在复制RNA中的作用，我们将使用基于植物病毒马铃薯X(PVX)的载体PEF，该载体同时产生复制RNA和植物内的外壳蛋白。这与病毒感染期间在体内发生的情况非常相似。我们最近已经证明，从PEF复制的RNA可以被不同的、无关的病毒的外壳蛋白所包裹。因此，PEF是检测包装信号在病毒复制中的作用的理想系统。将检测包装信号的消除、复制或突变对颗粒组装和形态的影响，以更全面地了解病毒组装的机制。这些研究不仅将有助于更好地了解病毒复制周期的关键阶段，而且还将使特定的RNA能够被有意地包装在定义的结构的衣壳中，用于生物纳米技术和创建新的RNA递送系统。

Viruses are extremely successful pathogens that infect organisms of every type, including plants, animals (including humans), fungi, and bacteria of all types. They are essentially intracellular parasites that introduce their genetic material into host cells and subvert the normal cellular functions to make more copies of themselves. They are normally present in the environment in the form of virus particles in which the viral nucleic acid, either DNA or RNA, is surrounded by a shell made up of multiple copies of one or more type of protein subunit encoded by the virus genome. In some cases (enveloped viruses) they are further surrounded by a membrane of host origin. The purpose of the protein shell is to protect the delicate genetic information from the, often harsh, environment outside the cell and to enable the virus to successfully spread to other hosts. This often involves uptake and transmission by vectors, such as insects or fungi, and the nucleic acid must be able to survive this process. Once inside a susceptible host, the virus makes more copies of itself by undergoing a "replication cycle" that includes several stages: uncoating of the particles to release the viral genome, expression of viral genes, replication of the viral nucleic acid and the encapsidation of the newly synthesised nucleic acid into particles that are then released to infect further hosts. This is all a carefully choreographed process, with replication and encapsidation of the viral genome usually closely linked. The specific encapsidation of viral, as opposed to host, nucleic acid into infectious particles is a vital step in the replication cycle of viruses. The process must result in the formation of virus particles that protect the labile genetic material effectively. This has led to the identification of defined RNA sequence elements or "packaging signals" in RNA viruses. Such packaging signals have been envisaged as labelling the viral RNA so that it is effectively "barcoded" in such a way that it is specifically recognised from a mixture of cellular molecules by the coat protein subunits, thereby conferring encapsidation selectivity. However, our recent work has shown that selectivity of packaging is determined by replication of an RNA molecule within infected cells, with synthesis of the coat protein being tightly coupled to RNA replication. In such "replication factories" the viral RNA genome is not in competition with other, non-replicating RNAs, suggesting that "barcoding" may not be required selective packaging. Rather, they may be present to ensure that the incorporation of RNA into particles proceeds efficiently to produce fully infectious virions. We have also obtained evidence that the length of replicating RNA may control the architecture of the resulting particles.To determine the role of potential packaging signals in the context of a replicating RNA, we will use a vector, pEff, based on the plant virus, potato virus X (PVX), that simultaneously produces replicating RNA and the coat proteins within plants. This closely mimics the situation that occurs in vivo during a viral infection. We have recently shown that the replicating RNA from pEff can be encapsidated by the coat protein from different, unrelated viruses. Thus, pEff is an ideal system for examining the role of packaging signals in the context of viral replication. The effect of elimination, duplication or mutation of the packaging signals on the assembly and morphology of the particles will be examined to obtain a more complete understanding of the mechanism of virus assembly. These studies will not only lead to a greater understanding of a critical stage of the viral replication cycle but will also enable specific RNAs to be deliberately packaged in capsids of defined architecture for use in bionanotechnology and the creation of novel RNA delivery systems.