Rescue of Recombinant Arenaviruses in Virus-Susceptible Mammalian Cells

Begin with transfection complexes carrying plasmids for arenavirus protein expression and plasmids encoding antigenomic RNA under host-specific promoters.

Add virus-susceptible mammalian cells and incubate to allow the complexes to attach to the cell surface.

Plate the mixture into culture wells and incubate to facilitate cell attachment and plasmid uptake.

The plasmids enter the nucleus, where host RNA polymerase II transcribes the protein-encoding plasmids into mRNAs.

Simultaneously, host RNA polymerase I transcribes antigenomic RNA-encoding plasmids into antigenomic RNA templates for genome replication.

Using the mRNAs, the host cell synthesizes nucleoprotein and polymerase.

Nucleoprotein coats the templates, enabling the viral polymerase to synthesize viral genomic RNAs, which are coated by nucleoprotein to form viral ribonucleoprotein complexes.

Replace the transfection medium with a nutrient-rich medium and incubate to support viral genome replication and protein expression.

These proteins assemble with viral ribonucleoprotein complexes at the host cell membrane, where budding releases recombinant arenaviruses.

First, prepare 250 microliters of Opti-MEM media and depending on the viru