Efficient plasmid-based rescue of T7 RNA polymerase-driven calicivirus reverse genetics systems in mammalian cells using vaccinia virus RNA capping enzymes

Abstract

The caliciviruses include important human and animal pathogens such as norovirus, sapovirus and feline calicivirus. Viral reverse genetics is performed to understand the fundamental biology of these viruses, as well as a potential route to generate live-attenuated vaccines. Calicivirus reverse genetics systems have typically relied on either on the production of in vitro-transcribed RNA or plasmid-based rescue either from a mammalian promoter, or through supplementing with helper enzymes through means of a helper virus. Here, we present a novel system integrating vaccinia capping enzymes D1R and D12L encoded on plasmids as part of a system for Murine Norovirus (MNV) reverse genetics. Addition of D1R, D12L and T7 RNA polymerase-expressing plasmids increases the viral titres of rescued MNV in both BSR-T7 cells and transgenic BSR-T7CD300LF cells, and viral polyprotein abundance. When the murine norovirus receptor is expressed in BSR-T7CD300LFcells, viral titres increased 100-1000-fold compared over standard BSR-T7 cells. This system offers a robust, high-throughput means of assessing viral mutants.