Functional Genomics of Insect Baculoviruses

昆虫杆状病毒感染许多农业和森林害虫以及蚊子和黑蝇等有害害虫，使其作为生物防治剂发挥着重要作用。杆状病毒也用作有效的表达载体（例如 Cervarix 疫苗）。其大型双链 DNA 基因组编码约 150 个在病毒复制周期中重要的基因。这些杆状病毒基因中的一些功能是已知的，一些用于初始感染、病毒体蛋白的产生、细胞的排出、细胞和宿主的调节以及病毒传播。人们对其他基因知之甚少。其中包括 me53 基因，它是编码 53 kDa 蛋白质的主要早期基因。 Me53 在感染后不久及之后转录。我们证明它是一种重要的病毒蛋白，因为如果没有它，病毒复制就会受到严重影响。由于它具有与转录因子一致的蛋白质结构域，我们认为它必须在细胞核中发挥作用来调节转录。我们发现 ME53 不仅位于细胞核中（正如转录因子所预期的那样），而且令人惊讶地还位于细胞外周的病灶中，在那里它与病毒包膜蛋白 GP64 共定位。由此我们推测，ME53 可能有助于病毒体在这些 ME53/GP64 病灶处的膜中出芽。但我们仍然有一个问题：ME53 的作用是什么？我们提出了两种假设，一种是关于其在转录调控中的作用，另一种是关于病毒的流出。我们的目标之一是追踪 ME53 删除病毒 (deltaME53) 与野生型病毒相比，病毒复制周期的差异。我们将开始通过电子显微镜进行超微结构比较的搜索（例如观察病毒发生基质以及病毒粒子在细胞核和细胞膜中的组装/运输）。我们还将比较病毒（和细胞）转录谱。为了区分 ME53 的核特异性功能和细胞膜功能，第二个目标是绘制核易位 (NTD) 和膜易位 (MTD) 所需的 ME53 结构域。通过突变这些易位域，我们希望评估 ME53 在不同细胞内位点的作用。例如，突变 NTD 将阻止核易位，并且观察到的任何差异（例如 NTD 突变体的某些病毒甚至宿主基因缺乏调节）必定是由于其核功能，对于 MTD 突变体反之亦然。虽然 ME53 与 GP64 和衣壳蛋白 VP39 相关，但我们怀疑它也与其他病毒（或宿主）蛋白相互作用，例如帮助 ME53 易位到细胞核或质膜。为此，我们将使用遗传、酵母两种杂交筛选和生化“下拉”实验来寻找 ME53 蛋白伴侣（ME53 相互作用组），以从细胞制剂中去除 ME53，但相关蛋白仍然附着。来自这些“下拉”的结合蛋白将通过非常精确的分光光度测量来识别。随后将进行进一步的诱变，以尝试识别 ME53 中对于与不同伙伴结合很重要的域（并突变伙伴本身）。随后将评估无法与某些伙伴结合的 ME53 突变对病毒复制的影响。结合域突变体 ME53 和正常 ME53 之间注意到的任何差异都必须与该特定蛋白质的重要​​性相关。通过这项工作，我们应该更好地了解 ME53 在杆状病毒生命周期中的功能。虽然这些发现推动了基础科学的发展，但我们无法预测这些知识是否有任何实用或商业价值。由于这些病毒被用作生物防治剂，也许这些知识可用于提高其功效并更好地预防加拿大害虫，或提高疫苗生产。

Insect baculoviruses infect many insect pests of agriculture and forests and nuisance pests like mosquitoes and blackflies giving them a major role as biological control agents. Baculoviruses are also used as efficient expression vectors (e.g. Cervarix vaccine). Its large dsDNA genome encodes some 150 genes important in the virus replication cycle. The functions of some of these baculovirus genes are known, some for initial infection, production of virion proteins, egress from the cell, cell and host regulation and virus transmission. Much less is known about other genes. Among these is the me53 gene, a major early gene encoding a 53 kDa protein. Me53 is transcribed shortly after infection and later. We showed it was an important viral protein because in its absence virus replication is severely impacted. Since it has protein domains consistent with transcription factors, we felt it must work in the nucleus to regulate transcription. We found that not only is ME53 located in the nucleus (as expected of a transcription factor) but surprisingly also in foci at the cell periphery where it colocalizes with a viral envelope protein GP64. From this we surmised that ME53 might help virions bud through the membrane at these ME53/GP64 foci. But we are still stuck with the question, what is the role(s) of ME53? We posited two hypotheses, one on its role in transcriptional regulation and one on virus egress. One of our objectives is to follow differences in the virus replication cycle of ME53 deleted viruses (deltaME53) compared to wild type virus. We will start our search by electron microscopy for an ultrastructural comparison (e.g. looking at virogenic stroma and assembly/transit of virions in the nucleus and at the cell membrane). We will also compare virus (and cellular) transcriptional profiles. To differentiate the nuclear-specific function from the cell membrane one of ME53, a second objective is to map domains of ME53 needed for nuclear translocation (NTD) and those for membrane translocation (MTD). By mutating those translocation domains we hope to assess the roles of ME53 at different intracellular sites. For example mutating NTD will prevent nuclear translocation and any differences observed (e.g. lack of regulation of certain viral, or even host, genes for the NTD mutant), must be due to its nuclear function, and vice versa for the MTD mutants. While ME53 associates with GP64 and the capsid protein VP39, we suspect it also interacts with other viral (or host) proteins, for example to help ME53 translocate to the nucleus or plasma membrane. For this we will be looking for ME53 protein partners (the ME53 interactome) using a genetic, yeast two hybrid screen, and biochemical “pull down” experiments to remove ME53 from a cell preparation but with associated proteins still attached. Binding proteins from those “pull downs” will be identified by very precise spectroscophotometric measures. This would be followed by further mutagenesis to try to identify domains in ME53 important for binding to the different partners (and mutate the partners themselves). ME53 mutations unable to bind to certain partners will then be assessed for their affect on virus replication. Any differences noticed between the binding domain mutant ME53 and normal ME53 must relate to the importance of that particular protein.From this work we should better understand the function of ME53 in the baculovirus life cycle. While such discoveries advance fundamental science we cannot predict if there will be any practical or commercial value to this knowledge. As these viruses are used as biological control agents, perhaps this knowledge might be used to improve their efficacy and better protect against Canadian pest insects, or improve vaccine production.