In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Summary

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Abstract

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

Introduction

Influenza A virus (IAV) is an enveloped virus and belongs to the family of Orthomyxoviridae. Its genes are encoded on a segmented, negative-sense and single-stranded RNA genome. In humans, IAV causes respiratory infections, which occur in seasonal epidemic outbreaks and bears the potential for global pandemics¹. Upon binding to sialic acid residues on the host cell surface², IAV is internalized by clathrin-dependent endocytosis and clathrin-independent pathways^3-8. The acidic milieu (pH < 5.5) in the endocytic vacuoles triggers a major conformational change in the IAV spike glycoprotein hemagglutinin (HA), which results in....

Protocol

1. Preparation of Buffers and Stock Solutions

1. Prepare MNT buffer (20 mM MES, 30 mM Tris and 100 mM NaCl) by dissolving 470 mg Tris Hydrochloride, 390 mg MES hydrate and 580 mg NaCl in 80 ml ddH₂O. Adjust the buffer to pH 7.4 and bring it to a final volume of 100 ml with ddH₂O.
2. Prepare three identical solutions of 500 mM MES buffer by dissolving 9.76 g MES hydrate in 80 ml dH₂O each. Adjust the buffers to pH 5.8, 5.4, and 5.0, respectively, by titrating with concentrated NaOH. Bring each buffer to a final volume of 100 ml with dH₂O.
3. Prepare two identical solutions of 500 mM Tris buffer by disso....

Results

As already discussed, priming in endosomes is required to render the IAV core uncoating competent. Protonation of the core weakens interaction of M1 and vRNPs (composed of the viral RNA, NP, and the polymerase complex PB1/PB2/PA). This process is initiated when incoming virus is exposed to a pH of 6.5 (or lower) in early endosomes (EEs) and continues until the virus fuses at around pH 5.0 in LEs.

In order to mimic the decrease o.......

Discussion

Viral capsids are metastable macromolecular complexes. While assembly of virions requires the encapsidation and condensation of the virus genome, initiation of the next round of infection depends on disassembly of this compact capsid structure. Viruses have evolved to exploit various cellular mechanisms to control the coating-uncoating cycle, including cellular receptors, chaperones, proteolytic enzymes, physical forces provided by motor proteins or helicases as well as pH and ionic switches^26,27. Here, we des.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
cOmplete™, EDTA-free protease inhibitor tablets	Sigma-Aldrich	11873580001	The stock solution can be stored at 2 to 8 °C for 1 to 2 weeks
Glacial acetic acid	Merck Millipore	100063
Glycerol anhydrous BioChemica	AppliChem	A1123
Hydrochloric acid	Merck Millipore	100317
Long injection needle (21 G, 9 cm, bevel or blunt-end)
MES hydrate	Sigma-Aldrich	M8250
Methanol	Merck Millipore	106009
NP-40	Sigma-Aldrich	I8896	Now commercially available as IGEPAL® CA-630
NuPAGE 4-12% Bis-Tris mini gels, 10 wells, 1.0 mm	Life Technologies	NP0321
NuPAGE LDS sample buffer (4x)	Life Technologies	NP0008
NuPAGE MOPS SDS running buffer (20x)	Life Technologies	NP0001
pH indicator strips, pH 4.0-7.0	Merck Millipore	109542
QC Colloidal Coomassie Stain	BIO RAD	1610803
Sodium chloride	Merck Millipore	106406
Sodiumhydroxide	Merck Millipore	106498
Steritop filter unit	Merck Millipore	SCGPT05RE
SW41 Ti, ultracentrifuge rotor set	Beckman Coulter	331336
Thinwall, Ultra-clear centrifuge tubes, 13.2 ml, 14 mm x 89 mm	Beckman Coulter	344059
Tris hydrochloride	AppliChem	A1087
X31 Influenza A virus (H3N2), egg-grown, clarified allantoic fluid	Virapur		Freshly thawed on 4 °C