Purification and Ultramicroscopic Observation of the Influenza A Virus Ribonucleoprotein Complex.

Abstract

Influenza A virus (IAV) has an eight-segmented, single-stranded, negative-sense viral genomic RNA (vRNA). Each vRNA strand associates with nucleoproteins and an RNA-dependent RNA polymerase complex to form a viral ribonucleoprotein (vRNP) complex. IAV vRNPs adopt a flexible double-helical configuration that varies in length. Although the transcription and replication of vRNA take place in the context of vRNPs, the precise structural conformation of vRNPs during RNA synthesis remains partially elucidated. To unravel the intricate ultrastructure of the vRNP, it is necessary to purify it while preserving its native functionality. Herein, we introduce a comprehensive protocol for the purification of IAV vRNPs using glycerol gradient ultracentrifugation. Furthermore, we provide a method for the high-speed atomic force microscopy observation of vRNPs during viral RNA synthesis.