A world of virus structures: understanding how non-icosahedral capsids are built

病毒可能是地球上最成功的病原体。它们无处不在，它们感染每一种其他类型的有机体，包括植物、动物(包括人类)、真菌、所有类型的细菌，甚至其他病毒。无论我们在哪里寻找他们，他们都已经被找到了。因此，它们具有巨大的社会重要性，直接影响到我们的生活，因为它们影响到人和动物的健康、农业，从而影响到真正全球范围的粮食安全。所有的病毒都面临着一个共同的挑战，它们必须将自己的基因组信息(DNA或RNA)打包在一个叫做衣壳的保护性容器中，这个容器可以保护基因组不受外部环境的影响，并将其完好无损地传递给新的细胞，开始新一轮的感染。制造这些衣壳的一种方法是用一种单一类型的蛋白质构建高度对称的容器。然而，要做到这一点，单一类型的蛋白质必须采用多种形状来构建合适大小的容器--很像制作足球所需的六边形和五边形。构建合适的衣壳，并完美地构建它，是病毒复制周期的一个基本部分，但我们对这种“构象转换”是如何发生的理解非常差。它可能发生的一种方式是让蛋白质与基因组DNA或RNA中的特定序列结合；这种结合将驱动构象变化。然而，人们对这一过程知之甚少。在某种程度上，这是因为对于绝大多数病毒来说，衣壳具有上述非常高的对称性，这意味着当我们解决它们的结构时，对称性平均会洗掉蛋白质(每个位置相同)与DNA或RNA(不是这样，因为它必须具有编码病毒基因的唯一序列)之间任何特定相互作用的细节。因此，当我们试图研究组装所涉及的分子机制时，许多病毒所依赖的高度对称性是非常没有帮助的。在这项提议中，我们想要利用我们实验室中两项令人振奋的最新发现，这将使我们能够克服这一障碍，并首次发现允许这些病毒高效自我组装的神秘规则。我们一直在研究两个不同的病毒家族，这两个家族都是全球粮食和纺织作物的重要病原体，因此是发达国家和发展中国家粮食安全和农业经济的主要威胁；双生病毒和UmbraVirus。在每一种情况下，病毒都进化出一种(不同的)新颖创新，这意味着衣壳具有一种不再像通常情况下那样对称的“非标准”结构。值得注意的是，在我们用5倍对称性(而不是二十面体病毒的60倍对称性)解决的每个病毒的初步结构中，我们现在可以看到DNA(对于双生病毒)和RNA(对于UmbraVirus)与病毒外壳蛋白结合的细节。这项拨款申请将使我们能够解决这些非标准病毒衣壳的高分辨率结构，而根本不需要任何对称平均。结合生化和生物信息学实验，我们将揭示基因组结合的细节，这如何改变蛋白质构象，以及这些特征在病毒基因组中的位置。这将(A)提供令人着迷的新的基本生物学见解，这些见解对理解病毒如何工作非常重要；(B)提供一种机械性的理解，可能导致防止病毒发挥作用的新方法；以及(C)明确病毒组装的规则，使我们能够改变病毒组装的方式，例如，为生物技术应用制造不同大小的衣壳。

Viruses are probably the most successful pathogens on earth. They are everywhere, and they infect every other type of organism, including plants, animals (including humans), fungi, bacteria of all types, and even other viruses. Wherever we have looked for them, they have been found. As a result, they are of huge societal importance, impacting directly on our lives because of their effects on human and animal health, agriculture, and thus on food security on a truly global scale. All viruses face a common challenge, in that they must package their genomic information (either DNA or RNA) within a protective container called a capsid, that shields the genome from the external environment, and delivers it intact to a new cell to start a new round of infection. One way in which these capsids are made is to build a highly symmetric container from a single type of protein. However, to do this the single type of protein has to adopt multiple shapes to build a container of the right size - much like the hexagons and pentagons that are needed to make a football. Building the right capsid, and building it perfectly, is a fundamental part of the viral replication cycle, but our understanding of how this 'conformational switching' happens is very poor. One way that it could occur is for the protein to bind to defined sequences within the genomic DNA or RNA; this binding would drive the conformational change. However, this process is poorly understood. In part, this is because for the vast majority of viruses the capsids have the very high symmetry described above, which means that when we solve their structures, symmetry averaging washes out details of any specific interactions between the protein (which is the same in each position) and the DNA or RNA (which is not, because it has to have a unique sequence that encodes the virus' genes). The high symmetry many viruses rely on is therefore tremendously unhelpful when we try to study the molecular mechanisms involved in assembly. In this proposal we want to exploit two hugely exciting recent discoveries in our laboratories, that will allow us to overcome this barrier and discover, for the first time, the cryptic rules that allow these viruses to efficiently self-assemble. We have been working on two different families of virus that are each important pathogens of food and textile crops globally, and thus are major threats to food security and agricultural economies across the developed and developing world; Geminiviruses and Umbraviruses. In each, the virus has evolved a (different) novel innovation that means the capsid has a "non-standard" structure which is no longer quite as symmetric as is normally the case. Remarkably, in the preliminary structure of each which we have solved with 5-fold symmetry (rather than the 60-fold symmetry for an icosahedral virus), we can now see details of DNA (for Geminiviruses) and RNA (for Umbraviruses) bound to the viral coat proteins. This grant application will allow us to solve high resolution structures of these non-standard virus capsids without any symmetry averaging at all. Together with biochemical and bioinformatics experiments, we will uncover the details of genome binding, how this changes protein conformation, and where these features lie within the viral genome. This will (a) provide fascinating new fundamental biological insights that are important in understanding how viruses work, (b) provide a mechanistic understanding that could lead to new ways to prevent them working, and (c) make clear the rules for virus assembly that could allow us to change the way viruses assemble, to make capsids of, for example, different sizes for biotechnology applications.