Assembly of influenza ribonucleoprotein in vitro using recombinant nucleoprotein

Abstract

The influenza A virus nucleoprotein previously expressed in Escherichia coli after fusion to 32 heterologous amino acids has now been purified and tested for its ability to form complexes with RNA in vitro. By using a simple filter binding assay, we show that ribonucleoprotein (RNP) complexes form readily with single-stranded RNA of viral or nonviral origin but not with double-stranded RNA. The RNP complexes formed were similar to authentic influenza virus RNPs in appearance under the electron microscope, in buoyant density in gradients of cesium chloride, and in sensitivities to pancreatic ribonuclease, to chaotropic reagents, and to high salt. We conclude that nucleoprotein synthesized in E. coli has all the properties required for correct assembly into ribonucleoprotein.