Expanding Reverse Genetics of Positive-Strand RNA Viruses: Optimised Rescue Platforms and Construction of a Novel Fluorescent Reporter Nidovirus

Abstract

Reverse genetics systems are crucial for facilitating the precise manipulation of viruses across a wide spectrum of translational and fundamental research pipelines. Here, we compared Circular polymerase extension reaction (CPER), Gibson assembly, and infectious subgenomic amplicons (ISA) for bacteria-free recovery of a positive sense RNA virus. Through optimisation of CPER, we demonstrated accelerated virus recovery and enhanced viral yields. We further investigated strategies to improve rescue efficiency across diverse positive-sense RNA virus families through incorporation of alternative promoters and non-coding elements. To evaluate the performance of the Aedes aegypti polyubiquitin promoter (AePUb) in tandem with a hammerhead ribozyme (HH Rbz) and a polymerase pause site for virus recovery in insect cells, we constructed a new fluorescent reporter genome using a 20 kb insect-specific mesonivirus. In vitro recovery by CPER of the mesonivirus was achievable in 1 day when using AePUb with HH Rbz, in comparison to a four-day recovery when using the minimal OpIE2-CA promoter. These elements were additionally assessed for rescue of the orthoflaviviruses, Binjari virus (BinJV) and dengue virus 2 (DENV-2), in insect cells (using AePUb); or in mammalian cells (using the CMV promoter) and for launch of DENV2 and SARS-CoV-2. Both BinJV and DENV-2 demonstrated improved rescue with the AePUb promoter and HH Rbz. However, the addition of the HH Rbz and the polymerase pause site to the CMV linker fragment showed no substantial differences to the standard CMV promoter systems for both DENV-2 and SARS-CoV-2, highlighting the context-specific benefits of their implementation. In summary, we demonstrated that a potent constitutive promoter system and a hammerhead ribozyme enhance the efficiency of positive-sense RNA virus rescue using CPER. Importance Reverse genetics systems are often limited by plasmid instability and variable efficiency of promoters across diverse cell lines. Extensive comparative approaches have yielded improvements across a variety of systems, however, there has been a paucity of publications that empirically compare novel advancements to established approaches. Here, we formalised and compared a series of reverse genetics advancements in the form of bacteria-free assembly methods, host promoters, pause sites, and ribozymes. These streamlined approaches expedite the existing methodologies and provide fundamental improvements to the field of synthetic virology. The advancements herein may support applications requiring efficient recovery of low fitness mutants and diverse mutational libraries and barcoded virus populations.