A yeast-based reverse genetics system to generate HCoV-OC43 reporter viruses encoding an eighth sgRNA
Bibliographic record
Abstract
ABSTRACT Coronaviruses have large, positive-sense single-stranded RNA genomes that challenge conventional strategies for mutagenesis. Here, we report the development of a new reverse genetics system for the endemic human coronavirus (HCoV) OC43 that utilizes transformation-associated recombination (TAR) to assemble complete viral genomes from dsDNA genome fragments via homologous recombination in Saccharomyces cerevisiae . Following cDNA synthesis from HCoV-OC43 viral RNA, we used TAR to capture fragments of the HCoV-OC43 genome to store as sequence-validated dsDNA parts. We performed combinatorial assembly in yeast to obtain an intact dsDNA copy of the HCoV-OC43 genome sufficient to launch viral replication upon introduction into human cells, yielding the yeast assembled OC43 YA virus. We also expanded the OC43 YA genome by inserting an eighth body transcription regulatory sequence (B-TRS) and an mClover3-H2B reporter gene between the M and N genes, designed to allow the reporter protein to be translated from its own subgenomic mRNA. We thoroughly evaluated OC43 YA and the OC43-mClo YA reporter virus, and demonstrated comparable viral gene expression, fitness in cell culture, and susceptibility to antivirals, compared to their natural progenitor. In summary, this new HCoV-OC43 reverse genetics system provides a modular platform for mutagenesis and combinatorial assembly of HCoV-OC43 genomes, and demonstrates the feasibility of expanding the genome while avoiding disruption of native coding sequences. IMPORTANCE Human coronavirus OC43 (HCoV-OC43) is an endemic human coronavirus that typically causes relatively mild respiratory illnesses and displays seasonal patterns of infection. We developed a new system to assemble DNA copies of HCoV-OC43 genomes and generate recombinant viruses for research purposes. This system uses yeast, first to capture segments of DNA encompassing the entire RNA-based viral genome, and then to stitch them together into complete DNA genome copies that can be amplified in bacteria and introduced into human cells to initiate an infectious cycle, ultimately yielding recombinant viruses with comparable properties to their natural progenitors. We also devised a strategy to expand the viral genome, adding a gene for a reporter protein encoded by an additional eighth subgenomic mRNA. This yeast-based genome assembly system provides a modular platform for rapid mutagenesis and combinatorial assembly of HCoV-OC43 genomes and demonstrates the feasibility of expanding the genome.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 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 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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 source (direct Gemma or distilled Codex), 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".