This work was supported by grants from the Canada Foundation for Innovation (CFI), the Canadian Institute of Health Research (CIHR), and the Natural Sciences and Engineering Research Council of Canada (NSERC).

Part 1: Disruption of the Influenza A Virion
<ol>
<li>Add 750 &micro;l 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.</li>
<li>Add 500 &micro;l 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.</li>
<li>Centrifuge for 10 minutes at 109,000 x g, 4 &deg;C, in a Beckman Optima Max-E centrifuge using a TLA-120.2 rotor.</li>
<li>Remove the supernatant, and resuspend the pellet in 500 &micro;l disruption buffer (100 mM KCl, 5 mM MgCl2, 5% (w/v) glycerol, 50 mM octylglucoside, 10 mg/ml lysolecithin, 1.5 mM dithiothreitol, 100 mM MES, pH 5.5).</li>
<li>Vortex vigorously, and then shake at 31 &deg;C for 20 minutes in an eppendorf thermomixer.</li>
</ol>
Part 2: Glycerol Gradient Sediment Velocity Centrifugation
<ol>
<li>Prepare a glycerol gradient by placing the following amounts of glycerol into a Beckman ultraclear centrifuge tube (13 mm x 51 mm): 1 ml 70% (v/v) glycerol, 0.75 ml 50% glycerol, 0.375 ml 40% glycerol, and 1.8 ml 33% glycerol. Glycerol solutions are made by mixing pure glycerol with NM buffer (150 mM NaCl, 50 mM MES, pH 5.5). Also prepare a balance tube containing both glycerol and buffer.</li>
<li>Vortex the disrupted viral sample again, and load it onto the glycerol gradient.</li>
<li>Centrifuge the gradient for 3.75 hours at 217,000 x g, 4 &deg;C, in a Beckman MLS-50 swinging-bucket rotor.</li>
<li>After the centrifugation is complete, manually collect 250 &micro;l aliquots of the gradient starting from the top of the tube. Keep fractions on ice or at 4 &deg;C.</li>
</ol>
Part 3: Analysis of the Glycerol Gradient Fractions by SDS Polyacrylamide Gel Electrophoresis
<ol>
<li>Remove 20 &micro;l from each fraction to a fresh tube.</li>
<li>Add 5 &micro;l of 5x SDS-PAGE sample buffer.</li>
<li>Heat to 95 &deg;C for 5 minutes.</li>
<li>Spin down the samples briefly and load them onto a 10% polyacrylamide gel, and include molecular weight markers in one of the wells.</li>
<li>Run the samples on the gel until the bromphenol blue loading dye reaches close to the bottom of the gel.</li>
<li>Stain the gel with Coomassie blue.</li>
</ol>
Part 4: Concentration of the vRNP Fractions
<ol>
<li>Choose the glycerol fractions containing mainly NP, combine them, and distribute them into two Beckman polycarbonate centrifuge tubes (11 mm x 34 mm).</li>
<li>Fill each tube with diethyl pyrocarbonate (DEPC)-treated Ultrapure water, and pipet up and down several times to mix.</li>
<li>Centrifuge for 4.5 hours at 157,000 x g, 4&deg;C, in a Beckman TLA-120.2 rotor.</li>
<li>Remove the supernatant, and resuspend the pellet in 50 &micro;l of DEPC-treated water.</li>
<li>If desired, the A280 of the concentrated vRNPs can be measured. As NP is the major component present in the pooled fractions, an estimate of the molarity of NP can be calculated using its extinction coefficient of 55,350 M-1 cm-1 (as determined via ProtParam3).</li>
<li>Aliquot the purified vRNPs, and store them frozen at -80 &deg;C for later use.</li>
</ol>
Part 5: Negative Staining of vRNPs
<ol>
<li>Dilute 1 &micro;l of purified vRNP into 9 &micro;l of freshly-made and filtered-twice MNT buffer (20 mM MES, 150 mM NaCl, 30 mM Tris, pH 7.5). Other buffers are also OK to dilute the vRNPs and to wash the specimen grid (step 5.5), but do not use PBS if uranyl acetate is used for negative staining.</li>
<li>Glow-discharge a specimen (copper TEM) grid previously coated with a parlodion and carbon film for 30 seconds.</li>
<li>Hold the freshly glow-discharged specimen grid with tweezers, and apply a 5-&micro;l drop of diluted vRNP onto the specimen grid.</li>
<li>Leave the drop of specimen on the grid for 8 minutes.</li>
<li>While waiting, place a small strip of Parafilm on the bench, and dispense 2 drops containing 10 &micro;l each of MNT buffer (to wash the grid), and a 80-&micro;l drop of the freshly made staining solution (1% ammonium molybdate or 1% uranyl acetate) onto the Parafilm.</li>
<li>After the 8-minutes of adsorption, wick off part of the sample solution from the specimen grid with a piece of filter paper (cut into triangles). Do not let the specimen grid to dry.</li>
<li>Wash specimen grid in 2 drops containing 10 &micro;l each of MNT buffer for a total time of 1 minute. This is done by carefully lowering the specimen grid on the drop, and then wicking off part of the sample solution from the specimen grid with a piece of filter paper (without letting the specimen grid dry).</li>
<li>Immediately after wicking off the last drop of buffer from specimen grid, completely submerge the specimen grid within the stain droplet and wait 1 minute.</li>
<li>Completely wick off the stain solution from the specimen grid with a piece of filter paper.</li>
<li>Allow the specimen grid to air-dry for several minutes before observation under a transmission electron microscope.</li>
</ol>
Part 6: Representative Results:
<img src="/files/ftp_upload/old/1105_2009_1_22_1/1105_Figure2.png" alt="Figure 2" width="600" height="215" />
Figure 1
As influenza A NP (~56 kDa) is the major protein found in viral ribonucleoprotein complexes, the NP band generally is the strongest band in the fractions containing the vRNPs (Figure 1). In addition to NP, each influenza vRNP also contains a copy of a trimeric RNA polymerase (molecular weights of 82, 86 and 86.5 kDa) complex. These may or may not be visible by Coomassie blue staining because their abundance is low compared to that of NP. The polymerases, however, can be detected if, instead of a Commassie blue stain, a silver stain of the gel is performed. The influenza matrix protein M1 (~ 28 kDa) should be minimally present in the fractions where the NP protein peak fractions are present.
<img src="/files/ftp_upload/old/1105_2009_1_22_2/1105_3.png" alt="Figure 3" width="600" height="360" />
Figure 2
Negatively-stained vRNPs as visualized under a transmission electron microscopy should yield vRNPs that resemble rod-shaped particles with variable length that are approximately 30 nm to 120 nm in length (Figure 2a). The oligomeric NP is organized as a chain of NP molecules that is further folded into a double helical repeat structure, so loops on either ends of these rod-shaped particles can sometimes be seen (Figure 2b).

<ol>
	<li>Kemler, I., Whittaker, G., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+microinjected+influenza+virus+ribonucleoproteins.">Nuclear import of microinjected influenza virus ribonucleoproteins.</a> Virology. 202, 1028-1033 (1994).</li><li>Wu, W. W. H., Weaver, L. L., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+the+nuclear+localization+sequences+on+influenza+a+ribonucleoprotein+complexes.">Ultrastructural analysis of the nuclear localization sequences on influenza a ribonucleoprotein complexes.</a> J Mol Biol. 374, 910-916 (2007).</li><li>Gasteiger, E., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Proteomics Protocols Handbook. , 571-607 (2005).</li><li>Wu, W. W. H., Sun, Y. H. B., Pant&#233;, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+influenza+A+viral+ribonucleoprotein+complexes+is+mediated+by+two+nuclear+localization+sequences+on+viral+nucleoprotein.">Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoprotein.</a> Virol J. 4, 49-49 (2007).</li><li>Babcock, H. P., Chen, C., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+single-particle+tracking+to+study+nuclear+trafficking+of+viral+genes.">Using single-particle tracking to study nuclear trafficking of viral genes.</a> Biophys J. 87, 2749-2758 (2004).</li></ol>

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...

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

purification and visualization of influenza a viral ribonucleoprotein complexes

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

Watch this Scientific Journal Video about Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes at JoVE.com

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

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.

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

purification-visualization-influenza-viral-ribonucleoprotein

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.9K Views.  University of British Columbia - UBC. 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.

Video: Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Immunology and Infection

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

Environment

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

Biochemistry

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

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.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

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.

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

Summary

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Chapters in this video

High-throughput Detection Method for Influenza Virus

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization