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Authors: Thao et al.

Link to paper: https://www.nature.com/articles/s41586-020-2294-9_reference.pdf

Journal/ Pre-Print: Nature

Tags: Cell Biology, Molecular Biology

Research Highlights

1. Yeast-based synthetic genomics platform to reconstruct RNA viruses

2. Rapidly engineered chemically synthesized clones of SARS-CoV-2.

3. Replacement of ORF7a with GFP as potential attenuation strategy.

Summary 

This work describes a yeast-based synthetic genomics platform to genetically reconstruct viruses with large RNA genomes encompassing different families and including species such as MHV, rMERS-CoV, SARS-CoV-2, Zika virus and Respiratory Syncytial Virus (RSV). Recombinant virus can be assembled from viral genomic fragments generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA. One step or multistep reassembly by transformation-associated recombination (TAR) cloning using yeast artificial chromosomes (YAC) and S. cerevisiae. A 5’ T7 RNA polymerase promoter allows the generation of synthetic RNA genomes resulting in the recovery of infectious virions upon transfection.

Impact for SARS-CoV2/COVID19 research efforts

Understand the virology and/or cell biology of SARS-CoV2/COVID19

Develop a vaccine for SARS-CoV2/COVID19

Technical advance to allow rapid engineering of RNA viruses, which can follow the evolution of the virus and the pandemic in real time.

Study Type

· In vitro study

Strengths and limitations of the paper

Novelty: TAR cloning in S.cerevisiae has not previously been achieved in RNA viruses with large genomes like coronavirus. Such viruses were previously hard to generate due to their instability in E.coli

Standing in the field: Method components are well documented for similar uses. Use of TAR cloning successfully engineered DNA viruses such as herpes simplex virus 1. Yeast S. cerevisiae previously used as the host for cloning and assembling viruses.

Appropriate statistics: Yes, unpaired student t-tests

Viral model used: MHV strain A59, MERS-CoV, SARS-CoV, SARS-CoV-2, ZIKA, hRSV.

Translatability: None, method for research use not clinical.

Main limitations:

· No replication kinetics for viruses other than SARS-CoV-2 and MHV when testing range of applicability.

· No sequence verification at every stage for SARS-CoV-2

· Replacement of ORF7a with GFP negatively affects virus replication

· No investigation of virus pathogenicity in animal models. Which might be relevant for evaluating potential vaccines based on attenuated recombinant viruses, such as GFP recombinants.