Efficient Plasmid-Based Rescue of T7 RNA Polymerase-Driven Calicivirus Reverse Genetics Systems in Mammalian Cells...

Area of Science:

• Virology
• Molecular Biology

Background:

• Caliciviruses, including norovirus, are significant human and animal pathogens.
• Viral reverse genetics is crucial for understanding virus biology and developing vaccines.
• Existing calicivirus reverse genetics systems have limitations, such as reliance on in vitro transcription or helper viruses.

Purpose of the Study:

• To develop a novel and efficient system for murine norovirus (MNV) reverse genetics.
• To improve viral replication and protein expression in rescued MNV.
• To provide a robust platform for assessing viral mutants without helper viruses.

Main Methods:

• Integration of vaccinia capping enzymes (D1R and D12L) encoded on plasmids into an MNV reverse genetics system.
• Co-expression of T7 RNA polymerase, D1R, and D12L in cell culture.
• Utilizing standard BSR-T7 cells and transgenic BSR-T7 cells expressing the murine CD300LF receptor (BSR-T7^CD300LF).

Main Results:

• The novel system, incorporating D1R, D12L, and T7 RNA polymerase, significantly increased rescued MNV titers and viral protein abundance.
• Viral titers in BSR-T7^CD300LF cells expressing the MNV receptor were 100-1000-fold higher compared to standard BSR-T7 cells.
• The system demonstrated robustness and increased throughput for assessing viral mutants.

Conclusions:

• The developed reverse genetics system provides a more efficient and robust method for MNV research.
• This system facilitates higher throughput analysis of viral mutants compared to traditional methods.
• It eliminates the need for helper viruses, simplifying the reverse genetics process for caliciviruses.