Functional interrogation of paramyxovirus genomes with efficient reverse genetics

描述（由申请人提供）：完全从克隆cDNA中克隆感染性病毒的反向遗传学或从头生成是挽救许多引起严重发病率和死亡率的副粘病毒（PV）的重组减毒活疫苗株的宝贵工具，也是PV生物学基础研究不可缺少的工具。尽管取得了这些进展，大多数副粘病毒（pv）的反向遗传系统在过去15年中仍然是出了名的低效。在106-107个转染细胞中，营救效率为~1。这是一个限制障碍。副粘病毒的反向遗传需要病毒支持质粒、t7启动子驱动的病毒抗基因组和丰富的T7RNAP的精确组合，T7RNAP通常由表达T7RNAP的重组痘苗（rVV-T7RNAP）提供，或者在某些情况下，由稳定表达T7RNAP的细胞系提供。这两种策略都不适合制作用于人体试验的重组减毒活病毒候选疫苗，这需要符合GMP的细胞系、条件和试剂。为了克服这些问题，我们定制设计了一个密码子优化的T7聚合酶（T7opt）基因，该基因可以使用单步转染方法从克隆的cdna中完全拯救rpv。该方法用于拯救所有五个主要副粘病毒属的代表性病毒。使用T7opt可以在没有rVV-T7RNAP的情况下有效地挽救pv。在转录的抗基因组中添加锤头核酶（Hh-Rbz）序列，救援效率进一步提高，在某些情况下可达3-4 log（103个转染细胞中约1个）。我们设计了Hh-Rbz的活性和位置，以确保通过预先设计的转录起始3Gs的自催化切割来遵守“六规则”，这些3Gs来自全T7启动子。我们的T7opt-HhRbz方法更快、更安全、更有效，并且可以轻松适应GMP合规条件。我们的目标是利用我们高效的T7opt-HhRbz系统提供的救援效率的革命性改进，以功能性地询问和探索PV生物学中以前无法进入的表型和遗传景观。在这样做的过程中，我们还希望扩大衰减选择和分子工具的范围，以供更广泛的研究人员开发减毒副粘病毒活疫苗。为实现既定目标，我们提出了以下具体目标：目标1。探索其他策略，进一步提高我们的T7opt- HhRbz系统的救援效率和稳健性，以确保我们的反向遗传系统的广泛适用性。进一步的改进将促进减毒疫苗的开发，并在AIM 2中实现更高通量的实验分析，这是使用插入突变对选定的副粘病毒基因组进行功能性查询。我们的策略利用了六法则对我们有利，而基于T7opt- HhRbz的高效救援使这一目标可行。我们将用一组生物学相关的选择压力来询问插入诱变文库的表型景观。

DESCRIPTION (provided by applicant): Reverse genetics or de novo generation of infectious virus entirely from cloned cDNA is an invaluable tool for rescuing recombinant live-attenuated vaccine strains for many paramyxoviruses (PVs) that cause significant morbidity and mortality, and is indispensable for basic studies on PV biology. Despite these advances, reverse genetics systems for most paramyxoviruses (PVs) have remained notoriously inefficient for the past 15 years. Rescue efficiency, when documented, is ~1 in 106-107 transfected cells. This is a limiting barrier. Reverse genetics for paramyxoviruses require an exacting combination of viral support plasmids, a T7-promoter driven viral antigenome, and an abundant supply of T7RNAP, usually provided by recombinant vaccinia expressing T7RNAP (rVV-T7RNAP), or in some cases, by cell lines stably expressing T7RNAP. Neither strategy is compatible with making recombinant live-attenuated virus vaccine candidates for human trials, which requires GMP compliant cell lines, conditions and reagents. To overcome these many issues, we have custom- designed a codon-optimized T7 polymerase (T7opt) gene that enables the rescue of rPVs entirely from cloned cDNAs using a single-step transfection methodology. This method was used to rescue representative viruses from all five major Paramyxovirinae genera. The use of T7opt makes efficient rescue of PVs possible without rVV-T7RNAP. Rescue efficiency was further enhanced, up to 3-4 logs in some cases (~1 in 103 transfected cells), with the addition of a hammerhead ribozyme (Hh-Rbz) sequence in the transcribed antigenome. We engineered the activity and placement of this Hh-Rbz to ensure adherence to the "rule of six" by pre-designed autocatalytic cleavage of the transcript-initiating 3Gs derived from the full T7 promoter. Our T7opt-HhRbz methodology is faster, safer, more efficient, and allows the possibility of easy adaptation to GMP compliant conditions. Our objective is to leverage the transformative improvement in rescue efficiencies provided by our highly efficient T7opt-HhRbz system to functionally interrogate and explore a previously inaccessible phenotypic and genetic landscape in PV biology. In so doing, we also hope to broaden the range of attenuation options and molecular tools available to the broader field of researchers working on developing live-attenuated paramyxovirus vaccines. We proposed the following Specific Aims to fulfill our stated objective: AIM 1. Explore additional strategies to further enhance the rescue efficiency and robustness of our T7opt- HhRbz system in order to ensure the broadest applicability of our reverse genetics system. Additional improvements will facilitate attenuated vaccine development, and enable higher throughput analysis of the experiments in AIM 2, which is to functionally interrogate select Paramyxovirinae genomes using insertional mutagenesis. Our strategy utilizes the rule of six to our advantage while our high efficiency T7opt- HhRbz based rescue makes this aim feasible. We will interrogate the phenotypic landscape of our insertional mutagenesis library with a set of biologically relevant selective pressures.