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.
Paramyxoviruses (PVs) cause a heavy disease burden; reverse genetics is an invaluable tool for making attenuated paramyxoviral viral vaccines, and indispensable for basic studies, but is extremely inefficient. We have increased the efficiency of rescuing recombinant PVs by 1,000 fold. This transformative improvement enables us to perform functional studies on a genome wide scale to gain a better understanding of PVs.
