Vaccinia virus DNA binding proteins and their role in virion morphogenesis

在天花被根除、天花不再是一个医学问题的35年后，痘病毒仍然是一个令人着迷的生物学难题。痘病毒的原型，牛痘病毒（VAC），在一个约200 kbp的双链DNA基因组中编码bbb200个基因，病毒粒子的合成需要许多精心安排的生化步骤。在这个过程中，病毒必须复制其基因组并将其包装成成熟的病毒颗粒，同时试图避免触发或主动抑制先天免疫系统，否则会阻止感染。VAC编码许多DNA结合蛋白，其中许多是催化转录和DNA复制所需的酶。有趣的是，文献和生物信息学鉴定了至少7个以上的病毒基因，编码DNA结合蛋白，其中许多是必不可少的，其中一些的功能仍然不清楚。我们将利用我们目前获得的NSERC奖开发的RNA干扰方法，结合先进的显微镜、病毒反向遗传学和其他技术，进一步探索这些特征不明显的蛋白质所提供的功能。我们预测它们支持病毒复制、基因组包装和/或ddna触发的宿主反应的病毒衰减。作为第一步，我们已经建立了一组siRNA，通过逆转录酶PCR判断，选择性地抑制VAC基因表达。空斑试验已被用于测量对病毒生长的影响。到目前为止，我们已经确定了siRNA靶向I3L、I6L或E8R，可以明显降低人类细胞中的基因表达和病毒复制。sirna对E5R和J1R也有类似的渗透作用，但没有那么强，我们正在筛选其他sirna来改进方法。使用超分辨率荧光显微镜，我们还观察到一种新的病毒DNA结构，可能代表早期包装中间体。从电子显微镜来看，成熟的病毒颗粒包裹着由DNA包裹着蛋白质核心组成的管状结构。在病毒粒子组装和包装之前，我们对管状病毒DNA结构的观察是第一次在成熟颗粒之外发现这种结构。设计了一种方法来可视化这些假定的包装中间体，我们将确定这些结构的组成，并测试哪些VAC DNA结合蛋白在其组装或加工中发挥作用。最后，我们对这样一个事实很感兴趣，即在24小时的复制周期内，受感染的细胞产生的病毒DNA几乎与细胞核中发现的病毒DNA一样多，并且尽管存在越来越多的细胞DNA传感器（例如cGAS， DAI, Pol-III， STING和TLR9）。众所周知，VAC可以阻断某些病原体相关分子模式的检测（例如dsRNA和5 ' -三磷酸盖帽RNA），还可以抑制下游信号传导，否则会诱导炎症、细胞凋亡和/或干扰素。我们假设一种或多种vacc编码的DNA结合蛋白也可能作为细胞质DNA传感器的抑制剂发挥尚未确定的作用。特别令人感兴趣的是细胞的DNA损伤感知系统，因为病毒复制产生的DNA中间体应该触发损伤反应。这可以阻断G2-M的细胞周期，这对病毒复制的支持很差。事实上，我们已经观察到，尽管存在片段化的dsDNA，但VAC感染不会引发DNA损伤反应，正如TUNNEL试验所表明的那样。这些初步数据为更好地定义这些VAC dna结合蛋白的生物学研究提供了基础。这些研究还将在高级病毒学、分子生物学和最先进的显微成像方面提供高质量的早期职业培训。

Poxviruses remain a fascinating biological puzzle, 35 years after variola was eradicated and smallpox ceased to be a medical problem. The prototypic poxvirus, vaccinia virus (VAC), encodes >200 genes within a ~200 kbp duplex DNA genome, and virion synthesis requires many carefully orchestrated biochemical steps. During this process the virus must replicate its genome and package it into mature virus particles, whilst trying to avoid triggering, or actively inhibiting, the innate immune system that would otherwise block infection. VAC encodes numerous DNA binding proteins, of which many are enzymes required to catalyze transcription and DNA replication. Interestingly, the literature and bioinformatics identify at least seven more virus genes, encoding DNA binding proteins, many of which are essential and where the function(s) of some are still unclear. We will use the RNA interference methods developed with our current NSERC award, in conjunction with advanced microscopy, virus-reverse genetics, and other technologies to further explore the functions served by these poorly characterized proteins. We predict that they support viral replication, genome packaging, and/or viral attenuation of a dsDNA-triggered host response. As a first step we have established a panel of siRNA’s that selectively inhibit VAC gene expression as judged by reverse transcriptase PCR. Plaque assays have been used to measure effects on virus growth. To date we have established siRNA’s targeting I3L, I6L, or E8R that clearly decrease both gene expression and virus replication in human cells. Similar, but not so strongly penetrant effects have been seen with siRNAs against E5R, and J1R and we are screening additional siRNAs to improve the methods. Using super resolution fluorescence microscopy, we have also observed a novel viral DNA structure that may represent an early packaging intermediate. From electron microscopy it has been suggested that mature viral particles enclose tubular structures composed of DNA wrapped around a protein core. Our observation of tubular viral DNA structures, prior to virion assembly and packaging, is the first discovery of such structures outside of the mature particle. Having devised a way to visualize these putative packaging intermediates, we will determine the composition of these structures and test which VAC DNA binding proteins play a role in their assembly or processing. Lastly, we have been intrigued by the fact that over a 24 hr replication cycle, an infected cell makes almost as much viral DNA as is found in the nucleus and does so despite the presence of a growing list of cellular DNA sensors (e.g. cGAS, DAI, Pol-III, STING, and TLR9). VAC is known to block detection of certain pathogen-associated molecular patterns (e.g. dsRNA and 5’-triphosphate capped RNA) and also inhibits the downstream signaling that would otherwise induce inflammation, apoptosis, and/or interferons. We hypothesize that one or more of the VAC-encoded DNA-binding proteins may also serve a still unidentified role as inhibitors of cytoplasmic DNA sensors. Of particular interest is the cell’s DNA damage sensing system, since virus replication produces DNA intermediates that should trigger a damage response. This can block the cell cycle at G2-M, which is poorly supportive of virus replication. In fact, we have observed that VAC infection does not trigger a DNA damage response despite the presence of fragmented dsDNA, as indicated by TUNNEL assay. These preliminary data provide a basis for a program of studies designed to better define the biology of these VAC DNA-binding proteins. These studies will also provide high-quality early-career training in advanced virology, molecular biology, and state-of-the-art microscopic imaging.