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 fusion of the viral and the late endosomal membrane⁹. Once the IAV capsid (here also referred to as viral "core") has escaped from late endosomes (LEs), it is uncoated in the cytosol followed by transport of the viral ribonucleoproteins (vRNPs) into the nucleus — the site of virus replication and transcription^10-13. Prior to acid activation of HA the virus experiences a gradual decrease in pH in the endocytic system, which primes the core for its subsequent disassembly^14-16. In this "priming" step the M2 ion channels in the viral membrane mediate the influx of protons and K^+10,14,16. The change in ionic concentration in the virus interior disturbs the interactions build up by the viral matrix protein M1 and the eight vRNP bundle, and facilitates IAV uncoating in the cytoplasm following membrane fusion^10,14-17.

Direct and quantitative analysis of the priming step has been hampered by the fact that endosomes are difficult to access experimentally. The entry process is, moreover, highly non-synchronous. In addition, end-point assays, such as qRT-PCR of released viral RNA or infectivity measurements, do not provide a detailed picture about the biochemical state of the viral capsid at any given step of entry. While perturbation of endosomes by siRNA or drug treatment has significantly contributed to the understanding of IAV entry^18-20, fine-tuning is difficult and prone to unspecific side effects in the tightly controlled endosome maturation program.

To avoid these problems, we have adapted a previously developed in vitro protocol based on the use of velocity gradient centrifugation¹⁷. In contrast to other attempts ^21,22,23,24, which were mostly based on combinations of proteolytic cleavage and detergent treatment followed by EM analysis, this approach enabled an easily quantifiable result. Centrifugation through different gradient layers allows the sedimenting particles to be exposed to and react with changing conditions in a controlled manner. In the presented protocol, IAV derived from clarified allantoic fluid or purified viral particles are sedimented into a two-layer glycerol gradient in which the bottom layer contains the non-ionic detergent NP-40 (Figure 1). As the virus enters the second, detergent-containing layer, the viral lipid envelope and envelope glycoproteins are gently solubilized and left behind. The core, composed of the eight vRNP bundle and surrounded by a matrix layer, sediments as a stable structure into the pellet fraction. Viral core proteins, such as M1 and vRNP-associated nucleoprotein (NP), can be identified in the pellet by SDS-PAGE and Coomassie staining. In particular, taking advantage of commercially available gradient gels and staining with the highly sensitive colloidal Coomassie²⁵, enables high precision and detection of even small amounts of viral core-associated proteins.

This sets the basis for testing whether different conditions such as pH, salt concentration, and putative uncoating factors have effects on the sedimentation behavior of core components and core stability. To this aim, only the lower, detergent-containing layer in the glycerol gradient is modified by introducing the factor or condition of interest. The technique has been particularly valuable in investigating the effect of different pH values and salt concentrations on the integrity of the IAV core¹⁶. Intermediate steps of IAV uncoating could be monitored including dissociation of the matrix layer at mildly acidic pH (<6.5) followed by vRNP dissociation at pH 5.5 and lower^16,17 (Figure 1). The latter step was further enhanced by the presence of high K⁺ concentration in the glycerol layer, reflecting a late endocytic environment¹⁶. Thus, as the virus and the core sedimented through the gradient they experienced a changing milieu that mimics conditions in endosomes. The outcome was a stepwise disassembly of the viral core in vitro, complementing results derived from cell biology assays.

The method presented here has enabled fast and highly reproducible analysis of IAV (X31 and A/WSN/33) and IBV (B/Lee/40) uncoating triggered by acidic pH and increasing K^+16,17 as well as disassembly of paramyxovirus cores upon alkaline pH exposure¹⁷. It is conceivable that the approach can be adapted to other enveloped viruses to gain insights into the biochemical properties of the viral capsid structure and capsid disassembly during cell entry.

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 dissolving 7.88 g Tris Hydrochloride in 80 ml dH₂O each and adjust the pH to 7.4 and 6.4, respectively, by titrating with concentrated HCI. Bring the buffer to a final volume of 100 ml with dH₂O.
4. Prepare 50% glycerol solution in dH₂O and stir well until the glycerol is homogenously mixed. Filter the solution through a Steritop filter unit (0.22 µm pore size) into a glass bottle.
5. Prepare 10% NP-40 solution and 1 M NaCl in dH₂O.
6. Prepare 25x concentrated protease inhibitor by dissolving two tablets in 4 ml ddH₂O.

2. Preparation of Glycerol Gradients

1. Prepare 5 ml detergent buffer master mix (300 mM NaCl, 2% NP-40, 2x concentrated protease inhibitor) solution for each pH condition to test (pH 7.4, 6.4, 5.8, 5.4, and 5.0). For this purpose, mix 9 ml of 1 M NaCl, 6 ml of 10% NP-40, 2.4 ml of the 25x protease inhibitor stock, and 9 ml ddH₂0.
2. For each pH condition pipet 4.4 ml of the master mix into a 50 ml conical tube.
3. For pH values above 5.8 add 0.6 ml of the respective pH-adjusted 500 mM Tris stock solution to the tube. For pH 5.8 and lower add 0.6 ml of the respective pH-adjusted 500 mM MES stock solution.
4. Make 5 ml of buffer solution containing 300 mM NaCl, 2x protease inhibitor, 60 mM Tris adjusted to pH 7.4 and ddH₂O. This will serve as the detergent-free control gradient buffer.
5. If necessary, fine-adjust the pH of the solutions to pH 7.4, 6.4, 5.8, 5.4, and 5.0 by adding concentrated HCI or NaOH solutions, respectively.
6. Add 5 ml of the 50% glycerol stock to 5 ml of the detergent-containing and detergent-free buffer mixtures resulting in six different 25% glycerol solutions. Verify the pH values by using pH indicator strips.
   NOTE: In case the measured pH differs significantly from the desired value, the respective solutions should be prepared again.
7. Prepare 15% glycerol solution by mixing 50% glycerol stock with dH₂O in a 3:7 ratio.

3. Ultracentrifugation of IAV

1. Add 3 ml 15% glycerol solution into ultra-clear centrifugation tubes (13.2 ml, 14 mm x 89 mm) by using a 5 ml syringe and a needle (21 G, 9 cm long). Do not leave drops at the inner wall of the tube as this might disturb the integrity of the gradient. Repeat this for a total of six centrifugation tubes, one for each of the five pH conditions to test and one for the control sample.
2. Carefully place 3.4 ml 25% glycerol solution under the 15% glycerol layer by using a 5 ml syringe and a long needle. Take care to not mix the two layers. Repeat this for all six conditions to test with the respective pH-adjusted glycerol solutions.
   NOTE: Work in class II biosafety cabinet for the following steps.
3. Gently overlay the glycerol gradients with 30 µl clarified allantoic fluid containing IAV (X31, H3N2) diluted in 1 ml MNT buffer (corresponds to around 20-30 µg of total viral protein) for each gradient.
4. Balance opposing tubes and place them into a SW41 ultracentrifugation rotor. Centrifuge for 150 min, at 55,000 x g, and 12 °C.
5. After the centrifugation, carefully remove the supernatant, i.e., both glycerol layers by using a clean Pasteur pipette and an aspirator. Resuspend the pellet in 40 µl (1x) non-reducing LDS sample buffer. It is important to pipet up and down several times to dissolve the pellet completely. Transfer the sample into a 1.5 ml microcentrifuge tube.

4. SDS-PAGE of Pellet Fractions and Coomassie Staining

1. Heat all samples at 95 °C for 10 min. At this point the samples could be stored at -20 °C until they are analyzed by SDS-PAGE.
2. Load 20 µl of the dissolved pellets onto a pre-cast gradient (4-12%) Bis-Tris mini gel and run for 1 hr at 200 V in 1x MOPS SDS running buffer.
3. Make fixation solution with 40% methanol and 10% glacial acetic acid in ddH₂O.
4. Incubate the gel in fixation solution for 1 hr and stain overnight in a 15 cm cell culture dish with a sufficient volume of colloidal Coomassie solution while gently shaking at room temperature.
   NOTE: It is important to close the dish in order to avoid evaporation of the staining solution.
5. Destain the gel in ddH₂O. Replace the ddH₂O every 15-20 min until the gel background becomes clear. Store the gel in ddH₂O at 4 °C until it is scanned for band quantification.
6. Scan the gel at high resolution and use commercially available or custom-made software for quantification of protein band intensities. Subtract the background signal from a region close to the respective bands and normalize these values to the detergent-free control samples (at pH 7.4).

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