Generation of a Recombinant Virus Using a Protein Kinase R-Based System

Take cells that express protein kinase R (PKR), an antiviral protein. PKR detects virus-derived RNA and becomes activated, triggering signaling that restricts viral replication.

Add a mutant virus that lacks the PKR antagonist and cannot replicate in PKR-competent cells.

Incubate to allow viral entry and release of the viral DNA genome into the cytoplasm, where viral transcription produces single-stranded and double-stranded viral RNAs.

Replace the medium.

Add transfection complexes that contain a vector encoding a gene of interest and a PKR antagonist. Incubate to allow vector uptake.

The vector integrates via homologous sequences into the viral genome and expresses the PKR antagonist.

The antagonist sequesters virus-derived RNA and prevents PKR activation, allowing viral replication and virion production.

Harvest the cells. Repeatedly freeze–thaw the cells, then sonicate to lyse them and release virions.

The released virions include recombinant viruses that express the PKR antagonist, which can be further selected in PKR-competent cells.

For recombinant virus generation, infect a confluent monolayer of suitable cells with the virus to be recombined at a multiplicity of infection of one in