Remodeling of Viral Ribonucleoprotein Activity Through Recombinant Polymerase and Host Protein

Begin with influenza virus-derived viral ribonucleoprotein or vRNP complexes.

The vRNP complex contains an RNA polymerase bound to the viral RNA genome, essential for viral RNA synthesis.

Add increasing amounts of a recombinant viral polymerase, along with a recombinant host protein, to mimic host-mediated regulation of vRNP activity.

Introduce capped RNA as a primer to initiate transcription.

Add nucleotide and polymerase cofactors to promote vRNP activity, and RNase inhibitors to inactivate the RNase enzyme. Then, incubate.

At low concentrations of recombinant polymerase and host protein, the capped RNA binds to the vRNP, initiating transcription of viral RNA to produce messenger RNA.

At higher concentrations, the recombinant host protein facilitates attachment of the recombinant viral polymerase to vRNPs, promoting polymerase dimer formation.

This dimer induces conformational changes that alter polymerase positioning.

This switches vRNP activity from transcription to replication, initiating cRNA synthesis, confirming recombinant polymerase and host protein-dependent remodeling of vRNPs.