A yeast-based reverse genetics system to generate HCoV-OC43 reporter viruses encoding an eighth subgenomic RNA
Bibliographic record
Abstract
ABSTRACT Coronaviruses have large, positive-sense single-stranded RNA genomes that challenge conventional strategies for mutagenesis. Yeast genetics has been used to manipulate large viral genomes, including those of herpesviruses and coronaviruses. This method, known as transformation-associated recombination (TAR), involves assembling complete viral genomes from dsDNA copies of viral genome fragments via homologous recombination in Saccharomyces cerevisiae . Here, we report our development of a TAR assembly and mutagenesis system for the endemic, seasonal human coronavirus (HCoV) strain OC43. HCoV-OC43 generally causes mild respiratory symptoms and is classified as a biosafety level 2 agent, making it useful for studying fundamental aspects of coronavirus biology and for comparative studies of more highly pathogenic betacoronaviruses. Following cDNA synthesis from HCoV-OC43 viral RNA, we generated five plasmids encompassing ~7.2 kb portions of the ORF1ab gene, the NS2 to M segment, or the N gene and structured to facilitate reporter gene insertions in the M -to- N intergenic region. Using these plasmids, we completed independent assemblies of yeast centromeric plasmids encoding ORF1ab , NS2a to N , as well as full-length HCoV-OC43 plasmids. A wild-type virus (OC43 YA ), as well as mClover3-H2B (OC43-mClo YA ), mRuby3-H2B (OC43-mRuby YA ), and mCardinal (OC43-mCard YA ) reporter viruses, were rescued. The OC43-mClo YA reporter virus replicated comparably to an OC43 reference strain and produced the mClover3-H2B protein from a novel subgenomic RNA through insertion of an eighth body transcription regulatory sequence, preventing the need to delete or mutate viral genes. This updated HCoV-OC43 reverse genetics system will contribute to a better understanding of betacoronavirus host-pathogen interactions and can accelerate studies of novel antivirals. IMPORTANCE Coronaviruses are ubiquitous pathogens that infect humans resulting in both mild and severe respiratory infections. Human coronavirus strain OC43 (HCoV-OC43) is one of many viruses responsible for common colds and is a useful model of more severe coronavirus infections. In this study, we describe an updated HCoV-OC43 mutagenesis system that uses yeast to capture six DNA fragments of the viral RNA genome and assemble them into full-length genomes in yeast/bacterial plasmids. The design of this system allowed for the rapid assembly and rescue of functional HCoV-OC43 viruses, including fluorescent reporter viruses with expanded genetic capacity. This updated reverse genetics system will enhance our ability to monitor viral replication, through building new reporter viruses, while also enhancing the study of betacoronavirus biology through the generation of mutant HCoV-OC43 viruses.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".