Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

Summary

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

Abstract

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

Protocol

Part 1: Disruption of the Influenza A Virion

1. Add 750 µl of MNT buffer (20 mM MES, 150 mM NaCl, 30 mM Tris, pH 7.5) into one Beckman polycarbonate centrifuge tube (11 mm x 34 mm) designed to fit into a TLA-120.2 rotor for use in a Beckman Optima Max-E ultracentrifuge.
2. Add 500 µl of the influenza A virus (H3N2 X-31 A/AICHI/68 strain; 2 mg/ml) into that tube. Mix the virus with the MNT buffer by pipetting up and down several times.
3. Centrifuge for 10 minutes at 109,000 x g, 4 °C, in a Beckman Optima Max-E centrifuge using a TLA-120.2 rotor.
4. Remove the supernatant, and resuspend the pellet in 500 µl disruption buffer (100 mM KCl, ....

Discussion

The purification of vRNPs is based on the procedure described by Kemler et al. (1994).¹ We and others have also used this protocol to isolate vRNPs to study their nuclear import.^2,4,5

We recommend the use of RNase-free tips and tubes when manipulating vRNPs because the viral genome is composed of RNA, and therefore degrades easily in the presence of RNA. In addition, all buffers should be made in water that is RNase free. The protocol described here was performe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
H3N2 X-31 A/AICHI/68 influenza A	Charles River Laboratories	490715
Tris	Sigma-Aldrich	T1503
MES	Sigma-Aldrich	M-8250
Glycerol	Fisher Scientific	G33-1
Octylglucoside	Sigma-Aldrich	O-8001
Lysolecithin	Sigma-Aldrich	L-4129
Dithiothreitol	Sigma-Aldrich	D-9779
Coomassie brilliant blue G-250	Kodak	1367796
diethyl pyrocarbonate (DEPC)-treated water	Invitrogen	750024
Uranyl Acetate	Ted Pella, Inc.	19481
Ammonium Molybdate	Fisher Scientific	A-674
Optima MAX-E Ultracentrifuge	Beckman Coulter Inc.	434491
MLS-50 Rotor Package, Swinging Bucket	Beckman Coulter Inc.	367280
TLA-120.2 Rotor Assembly, Fixed-Angle, Titanium	Beckman Coulter Inc.	362046
Eppendorf Thermomixer	Lauda Brinkmann	022670000