RNA replication-independent, DNA linearization-dependent expression of reporter genes from a SARS-CoV-2...

Area of Science:

• Virology
• Molecular Biology
• Genetic Engineering

Background:

• RNA replicons are viral genetic constructs used for gene expression and replication.
• Current methods involve in vitro RNA transcription and electroporation, which can be inefficient.
• Generating replicons within cells after DNA transfection is a desirable alternative.

Purpose of the Study:

• To investigate the feasibility of expressing genes from a DNA-based SARS-CoV-2 replicon after transfection into mammalian cells.
• To elucidate the mechanism of gene expression from the DNA replicon, particularly its dependence on T7 RNA polymerase and viral replication.

Main Methods:

• A bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon was constructed under a T7 promoter.
• HEK293T cells expressing T7 RNA polymerase (T7 RNAP) were transfected with the BAC DNA.
• Reporter gene expression (GFP, luciferase) was analyzed, along with effects of DNA linearization, T7 RNAP, remdesivir, and SARS-CoV-2 N protein.

Main Results:

• Reproducible, albeit low, expression of reporter proteins was observed from the transfected BAC DNA.
• Expression required linearization of the BAC DNA and occurred independently of T7 RNAP and viral RNA self-replication.
• Remdesivir treatment did not affect gene expression, further indicating a lack of self-replication.
• SARS-CoV-2 N protein enhanced expression from RNA replicons but not from the DNA replicon BAC.

Conclusions:

• Transfection of a large DNA encoding a coronaviral replicon can lead to reproducible gene expression via an unknown mechanism.
• This DNA-based expression pathway bypasses the need for T7 RNA polymerase and RNA self-replication.
• Findings suggest a novel method for DNA-based RNA replicon applications, offering valuable insights for future research.