The
influenza A viral genome consists of eight negative-sense, single stranded
RNA molecules, individually packed with multiple copies of the
influenza A
nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The
influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of
influenza vRNPs and the replication of the
influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the
influenza A virion with
detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the
influenza A virion on a 33-70% discontinuous
glycerol gradient by velocity sedimentation. The fractions obtained from the
glycerol gradient are then analyzed on via SDS-PAGE after staining with
Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The
glycerol gradient purification is a modification of that from Kemler et al. (1994)(1), and the negative staining has been performed by Wu et al. (2007).(2).