Synthetic herpesvirus genomes with an expanded genetic code

合成生物学家重新设计生物系统，以加速基础和应用研究。在我实验室疱疹病毒研究的基础上，在Dalhousie iGEM合成生物学团队的领导下，我们将使用合成生物学技术来创建一个新的人类疱疹病毒8型(HHV8)突变平台。目前用于HHV8基因组突变的方法效率低，并且复杂的紧凑的病毒基因组结构，许多基因密切相关或重叠，并从两个DNA链转录。在与J·克雷格·文特尔研究所和约翰斯·霍普金斯大学的合作下，我们正在利用酵母中有效的同源重组来组装新的合成HHV8基因组。这一方法将使疱疹病毒基因组在多个位点高效、同时重新编码，并用合成DNA取代基因组的大片段。这一在酵母中组装和突变病毒基因组的新平台将加速必要基因座的定位和基因功能的阐明。只有少数几种HHV8蛋白在病毒感染的背景下得到了彻底的表征。为了克服这一问题，我们将扩展HHV8遗传密码，使具有有用特性的非典型氨基酸(NCAA)能够有效地结合到HHV8蛋白质中。我们的第一个目标将是在酵母中重新编码HHV8基因组，用替代的TAA或TGA停止密码子取代所有29个定位的琥珀(TAG)终止密码子。这将释放标签密码子，以便将NCAA结合到病毒蛋白质中。我们将确认携带这个重新编码的基因组的HHV8病毒表现出正常的基因表达和复制动力学。使用这个修订的基因组模板，我们将创建一个基因组集合，其中标签密码子直接插入到每个带注释的翻译起始点的下游。因此，在缺乏NCAA的正常细胞中，每个重组病毒基本上都是单基因敲除的；添加与正交氨酰-tRNA合成酶/tRNA对匹配的同源NCAA将允许NCAA掺入靶蛋白。这一收集将使我们能够绘制基本基因座并创建这些基因座新的含有NCAA的蛋白质产品，这将有助于结构和功能蛋白质的表征。具体地说，这个系统将允许我们将NCAA与内在荧光结合在一起，这将使我们能够监测病毒蛋白质和衣壳等较大结构的亚细胞定位，从而促进病毒组装和出口的研究。利用合成生物学的原理，我们的NSERC研究计划将创建以前不可用的资源，供广泛共享，用于病毒基因组操作和病毒蛋白功能研究。

Synthetic biologists re-design biological systems to accelerate fundamental and applied research. Building upon a foundation in herpesvirus research in my laboratory and leadership of the Dalhousie iGEM synthetic biology team, we will use synthetic biology techniques to create a new platform for mutagenesis of human herpesvirus-8 (HHV8). Current approaches for HHV8 genome mutagenesis are inefficient, and complicated by compact viral genome architecture, with many genes closely-associated or overlapping, and transcribed from both DNA strands. In collaboration with the J. Craig Venter Institute and Johns Hopkins University we are assembling a new synthetic HHV8 genome by capitalizing on efficient homologous recombination in yeast. This approach will enable efficient, simultaneous re-coding of herpesvirus genomes at multiple loci, as well as substitution of large segments of the genome with synthetic DNA. This new platform for assembling and mutating virus genomes in yeast will accelerate mapping of essential loci and elucidation of gene function. Only a handful of HHV8 proteins have been thoroughly characterized in the context of viral infection. To overcome this, we will expand the HHV8 genetic code to enable efficient site-specific incorporation of non-canonical amino acids (ncAAs) with useful properties into HHV8 proteins. Our first objective will be to re-code the HHV8 genome in yeast, substituting all 29 mapped amber (TAG) stop codons with alternative TAA or TGA stop codons. This will liberate TAG codons for incorporation of ncAAs into viral proteins. We will confirm that HHV8 viruses bearing this re-coded genome display normal gene expression and replication kinetics. Using this revised genome template, we will create a collection of genomes with TAG codons inserted directly downstream of each annotated translation start site. Thus, in normal cells lacking ncAAs, each recombinant virus will essentially be a single-gene knockout; addition of a cognate ncAA matched to the orthogonal aminoacyl-tRNA synthetase/tRNA pair will allow ncAA incorporation into the targeted protein. This collection will allow us to map essential loci and create new ncAA-containing protein products of these loci that will aid structural and functional protein characterization. Specifically, this system will allow us to incorporate ncAAs with intrinsic fluorescence that will allow us to monitor subcellular localization of viral proteins and larger structures like capsids, facilitating studies of viral assembly and egress. Using the principles of synthetic biology, our NSERC research program will create previously unavailable resources to be widely shared for viral genome manipulation and investigation of viral protein function.