A platform for RNA virus cloning
A study in Nature reports on the suitability of the yeast Saccharomyces cerevisiae as a platform for the assembly and maintenance of diverse RNA virus genomes, including SARS-CoV-2, that enables their genetic manipulation and functional characterization.During outbreaks, virus isolates are used for the development of diagnostics, in vivo models, antiviral therapeutics and vaccines. Viral genomes can be cloned from chemically synthesized DNA if the availability of virus isolates is limited, but established approaches using Escherichia coli are often inadequate to accommodate the large genomes of RNA viruses such as coronaviruses (family Coronaviridae).
Credit: Gavin Hellier/AlamyThao et al. applied transformation-associated recombination (TAR) cloning to the mouse hepatitis virus (MHV) containing a GFP gene, which has an established reverse-genetics platform. Overlapping DNA fragments covering the MHV-GFP genome and a TAR vector were transformed into yeast, upon which the DNA fragments assemble by homologous recombination, generating a yeast artificial chromosome (YAC) comprising the full-length viral cDNA. Notably,>90% of screened clones showed correct assembly of the YAC, indicating high efficiency of assembly. Infectious virus was successfully recovered from two individual clones by isolating and linearizing the YACs for in vitro transcription to generate viral RNA, which was then transfected into the BHK-MHV-N hamster cell line, jointly with an mRNA encoding the MHV nucleocapsid protein, to produce and amplify the virus. Recovered virus exhibited the same replication kinetics as parental MHV-GFP.The team set out to establish whether the synthetic genomics platform could be applied to MERS-CoV, using a low-copy bacterial artificial chromosome (BAC) to clone the virus from eight overlapping, PCR-amplified DNA fragments. The approach was also applied to a mutated MERS-CoV clone, which had a GFP gene inserted. YAC clone assemblies and virus rescues from cloned DNA were successful, ascertaining the applicability of this platform to a broader range of viruses, including genetically modified virus genomes.Further experiments determined that viral genomes could be stably maintained and that this platform was applicable to other difficult to clone viruses, such as the ZIKA virus (family Flaviviridae) and human respiratory syncytial virus (family Paramyxoviridae), including directly from a clinical sample without prior knowledge of the virus genotype.Strikingly, within 1 week of receiving synthetic DNA fragments of SARS-CoV-2 based on genome sequences released in January 2020, cloning and rescue of recombinant SARS-CoV-2 and SARS-CoV-2-GFP was achieved.“synthetic genomics platform for the rapid generation and functional characterization of evolving RNA viruses”
Taken together, this study showcases the utility of a synthetic genomics platform for the rapid generation and functional characterization of evolving RNA viruses during an outbreak from different starting materials, including virus isolates, cloned DNA, synthetic DNA or clinical samples.
Author informationAffiliationsNature Reviews GeneticsLinda KochAuthorsLinda KochYou can also search for this author in Corresponding authorCorrespondence to
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