The authors would like to thank Nada Naffakh and Marie-Anne Rameix-Welti (Institut Pasteur) for the rWSN-PB2-Strep virus.

A schematic flowchart of the protocol is shown in Fig. 1 and a table of reagents is presented below.
1. Infection (16 - 24 h)
<ol>
 <li>Seed out HeLa cells in 5 150 mm plastic cell culture dishes in Dulbecco's modified Eagle's medium (DMEM)-high glucose medium, such that they will reach approximately 80% confluency the following day (approximately 5 x 108 cells). It is important that the cells do not reach 100% confluency.</li>
 <li>On the next day, dilute the Strep-tagged influenza virus stock into phosphate-buffered saline (PBS) containing 0.3% bovine serum albumin (BSA) to a multiplicity of infection of 3. </li>
 <li>Remove the growth medium from the cells, wash once with PBS, and add 10 ml PBS containing the virus to the cells. Incubate for 1 h at room temperature.</li>
 <li>Replace the infection medium with DMEM-high glucose, and continue incubation at 37 &deg;C.</li>
</ol>
The length of incubation depends on the phase of infection to be studied (see Discussion).
2. Subcellular Fractionation (3 h)
<ol>
 <li>Wash cells once with cold PBS and then suspend cells in cold PBS with a rubber scraper and transfer to a 50 ml conical tube.</li>
 <li>Pellet cells in a table-top centrifuge for 5 min at 1,000 rpm and then aspirate PBS.</li>
 <li>Resuspend cell pellet in 10 ml cold sucrose buffer (10 mM HEPES pH 7.9, 10 mM KCl, 2 mM Mg Acetate (MgOAc), 3 mM CaCl2, 340 mM sucrose, 1 mM dithiothreitol (DTT), 1% protease inhibitor mix). After suspension, pass cells through a 200 &mu;l pipette tip attached to a 10 ml plastic serum pipette to disrupt cell clumps.</li>
 <li>Incubate 10 min on ice. Gently invert periodically to resuspend cells.</li>
 <li>Add Nonidet P-40 (NP-40) to a final concentration of 0.5% and vortex at high speed for 15 sec. Immediately centrifuge for 10 min at 3,500 x g at 4 &deg;C in a table-top centrifuge.</li>
 <li>Remove supernatant and store at 4 &deg;C. This is the cytoplasmic fraction. The pellet should be smaller and whiter than the original cell pellet. All fractions can be stored for at least 4 h at 4 &deg;C.</li>
 <li>Wash the pellet in 5 ml cold sucrose buffer, re-centrifuge as in step 2.5 and discard the supernatant.</li>
 <li>Resuspend the pellet (containing nuclei) in 1.5 ml cold nucleoplasmic extraction buffer (50 mM HEPES pH 7.9, 150 mM KOAc, 1.5 mM MgCl2, 0.1% NP-40, 1 mM DTT, 1% protease inhibitor mix). </li>
 <li>Transfer to a chilled, all-glass 4 ml Dounce homogenizer with a tight-fitting pestle and homogenize the nuclei with 20 strokes.
 Avoid foaming during homogenization. The resistance should increase after approximately 10 strokes. Efficient homogenization can be confirmed under a phase contrast microscope. 
 </li>
 <li>Transfer the homogenized nuclei to a 1.5 ml microcentrifuge tube and incubate 20 min on a rotating wheel at 4 &deg;C.</li>
 <li>Centrifuge at 16,000 x g in a microcentrifuge at 4 &deg;C for 10 min.</li>
 <li>Remove supernatant and store at 4 &deg;C. This is the nucleoplasmic fraction.</li>
 <li>Resuspend pellet in 1.5 ml digestion buffer (50 mM HEPES pH 7.9, 10 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 1% protease inhibitor mix, 100 U/ml Benzonase (for RNA analysis, substitute with 20 U/ml RNase-free DNase I)) pre-warmed to 37 &deg;C, and incubate 10 min at 37 &deg;C.</li>
 <li>Add 42 &mu;l of 5 M NaCl (for final concentration of 150 mM NaCl) and incubate 20 min further at 4 &deg;C.</li>
 <li>Centrifuge at 16,000 x g in a microcentrifuge at 4 &deg;C for 10 min.</li>
 <li>Remove supernatant and store at 4 &deg;C. This is the ch150 fraction.</li>
 <li>Resuspend pellet in 1.5 ml cold high-salt buffer (50 mM HEPES pH 7.9, 500 mM NaCl, 1.5 mM MgCl2, 0.1% NP-40, 1 mM DTT, 1% protease inhibitor mix) and incubate 20 min on ice.</li>
 <li>Centrifuge at 16,000 x g in a microcentrifuge at 4 &deg;C for 10 min.</li>
 <li>Remove supernatant and store at 4 &deg;C. This is the ch500 fraction.</li>
</ol>
3. Strep-tag Purification (2 h)
<ol>
 <li>Add 300 &mu;l of 5 M NaCl to the cytoplasmic fraction (for final concentration of 150 mM NaCl).</li>
 <li>Aliquot 100 &mu;l of packed Strep-Tactin Sepharose beads per fraction in a 15 ml conical tube.</li>
 <li>Add 10 volumes of Strep wash buffer (20 mM HEPES pH 7.9, 150 mM NaCl (500 mM NaCl for ch500 samples), 1 mM EDTA) to beads, invert the tube to mix, and centrifuge 5 min at 1,000 x g in a tabletop centrifuge.</li>
 <li>Discard the supernatant, repeat step 3.3 twice and then resuspend the bead pellet in an equal volume of Strep wash buffer.</li>
 <li>Add 200 &mu;l of washed Strep-Tactin bead slurry to each fraction (in 1.5 ml microcentrifuge tubes for nuceloplasmic, ch150, and ch500 fractions and a 15 ml conical tube for cytoplasmic fraction).</li>
 <li>Rotate tubes 1h at 4 &deg;C on a rotating wheel.</li>
 <li>Centrifuge tubes 1 min at 1,000 x g at 4 &deg;C. Discard supernatant.</li>
 <li>Add 1 ml cold Strep wash buffer to each tube. Transfer beads from 15 mL tube (cytoplasmic fraction) to 1.5 mL tube. Wash all tubes 1 min at 4 &deg;C, centrifuging after as in step 3.7. Repeat this wash step twice.</li>
 <li>After the last wash step, remove all traces of wash buffer using a flat pipette tip. Add 100 &mu;l elution buffer (Strep wash buffer containing 2.5 mM desthiobiotin) and gently mix. </li>
 <li>Incubate 15 min on ice. Gently mix beads occasionally by tapping the bottom of the tubes.</li>
 <li>Centrifuge tubes 1 min at 1000 x g at 4 &deg;C in microcentrifuge. Collect supernatant containing eluted Strep-tagged vRNPs.</li>
</ol>
4. Representative Results 
The efficiency of the fractionation is best judged by Western blot analysis of the fractionated lysates using antibodies specific for subcellular markers (Fig. 2). Specifically, successful subnuclear fractionation should show little or no cellular RNA polymerase II (Pol II) in the nucleoplasmic, or soluble nuclear fraction4, and most should be extracted with 150 mM NaCl5. A degradation of Pol II is also reproducibly observed in influenza virus-infected cells compared to uninfected cells, consistent with the literature6. 
Purification of vRNPs is best judged by silver or Coomassie staining, as shown for a cytoplasmic eluate in Fig. 3. In our experience, most Strep-PB2 is purified in as part of a fully-formed vRNP, i.e. little soluble polymerase is captured. This is reflected in the high NP:PA/PB1/PB2 ratio observed by silver or Coomassie staining. A lower ratio suggests buffer contamination with RNases, as several ubiquitous RNases (such as RNase A) are known to cleave viral RNA in such a way as to dissociate the polymerase from NP multimers7. Interestingly, treatment with Benzonase alone, while sufficient to digest viral RNA, appears to have no effect on the stochiometric ratios of vRNP proteins. Although we cannot explain this phenomenon, we observed disruption of vRNPs after digestion with other RNases (data not shown), suggesting that Benzonase may leave certain structurally important RNA regions intact. After purification of vRNPs from the cytoplasm and nucleoplasm (performed without nuclease digestion), 8 faint but discrete bands at high molecular weights can be observed by silver staining, which correspond to the predicted molecular weights of the 8 influenza genome segments (see ref. 3). 
The relative amounts of vRNPs purified from each fraction at 9 h post infection are shown in Fig. 4. The distribution of vRNPs varies over the course of infection, with strongest accumulation in the ch150 fraction at early time points, and increased accumulation in the nucleoplasm and cytoplasm at late time points.
<img src="/files/ftp_upload/4028/4028fig1.jpg" alt="Figure 1"/> 
Figure 1. Flow chart of the subcellular fractionation. Adapted from ref. 3.
<img src="/files/ftp_upload/4028/4028fig2.jpg" alt="Figure 2"/> 
Figure 2. Western blot analysis of subcellular fractions from influenza virus-infected cells. 109 HeLa cells were infected with influenza virus strain WSN for 9 h prior to subcellular fractionation. Equal amounts of total protein were loaded in each lane and analyzed using the antibodies shown. RNA Polymerase II (Pol II) was detected using clone 8WG16, which recognizes all forms of the C-terminal domain (the hypophosphorylated band is most prominent). Adapted from ref. 3.
<img src="/files/ftp_upload/4028/4028fig3.jpg" alt="Figure 3"/> 
Figure 3. Silver stain analysis of purified vRNPs. HeLa cells were infected with rWSN (as a negative control) or rWSN-PB2-Strep for 9 h, followed by Strep purification from the cytoplasmic fraction. Asterisk denotes bands which are not visible after RNase digestion. Adapted from ref. 3.
<img src="/files/ftp_upload/4028/4028fig4.jpg" alt="Figure 4"/> 
Figure 4. Silver stain analysis of vRNPs purified from different cellular fractions. 109 HeLa cells were infected with rWSN-PB2-Strep or rWSN for 9 h followed by subcellular fractionation and Strep purification. Equal amounts of eluate from each fraction were loaded. Adapted from ref. 3.

<ol>
	<li>Engelhardt, O. G., Smith, M., Fodor, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+influenza+A+virus+RNA-dependent+RNA+polymerase+with+cellular+RNA+polymerase+II.">Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II.</a> J. Virol. 79, 5812-5812 (2005).</li><li>Garcia-Robles, I., Akarsu, H., Müller, C. W., Ruigrok, R., Baudin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+influenza+virus+proteins+with+nucleosomes.">Interaction of influenza virus proteins with nucleosomes.</a> Virology. 332, 329 (2005).</li><li>Chase, G. P., Rameix-Welti, M. A., Zvirbilene, A., Zvirblis, G., Götz, V., Wolff, T., Naffakh, N., Schwemmle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus+ribonucleoprotein+complexes+gain+preferential+access+to+cellular+export+machinery+through+chromatin+targeting.">Influenza virus ribonucleoprotein complexes gain preferential access to cellular export machinery through chromatin targeting.</a> PLoS Pathogens. 7, e1002187 (2011).</li><li>Kimura, H., Tao, Y., Roeder, R. G., Cook, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+RNA+polymerase+II+and+its+transcription+factors+in+an+HeLa+cell:+little+soluble+holoenzyme+but+significant+amounts+of+polymerases+attached+to+the+nuclear+substructure.">Quantitation of RNA polymerase II and its transcription factors in an HeLa cell: little soluble holoenzyme but significant amounts of polymerases attached to the nuclear substructure.</a> Mol. Cell. Biol. 19, 5383-5383 (1999).</li><li>Henikoff, S., Henikoff, J. G., Sakai, A., Loeb, G. B., Ahmad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+profiling+of+salt+fractions+maps+physical+properties+of+chromatin.">Genome-wide profiling of salt fractions maps physical properties of chromatin.</a> Genome Res. 19, 460 (2009).</li><li>Rodriguez, A., Perez-Gonzalez, A., Nieto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus+infection+causes+specific+degradation+of+the+largest+subunit+of+cellular+RNA+polymerase+II.">Influenza virus infection causes specific degradation of the largest subunit of cellular RNA polymerase II.</a> J. Virol. 81, 5315-5315 (2007).</li><li>Ye, Z., Liu, T., Offringa, D. P., McInnis, J., Levandowski, R. 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No conflicts of interest declared.

While many studies have recently identified individual proteins or cellular networks involved in influenza virus infection8, the functional significance of the majority of these interactions remains unclear. Given the absolute dependence of chromatin-based functions for influenza virus RNA synthesis and the complex biophysical and biochemical nature of the nucleus9, new techniques will be required to elucidate these functions. The subnuclear fractionation we present here, coupled with affinity purification of vRNPs, allows the characterization of crucial viral RNA factories which were previously inaccessible.
Many subcellular fractionation protocols have been previously described in the literature. We observed that most of these traditional methods lead to premature leakage of vRNPs from the nucleus, a phenomenon also described for Pol II10. By using a short incubation of pre-swelled cells with a low concentration of a weak detergent, NP-40, efficient plasma membrane lysis occurred without significant nuclear leakage. Interestingly, performing our subcellular fractionation using A549 and HEK293T cells rather than HeLa cells led to strong nuclear leakage of vRNPs in both cell lines. Lowering the NP-40 concentration to approximately 0.2% reduced this issue; however, due to some variability of these cell lines, optimal conditions should be determined empirically. Another possible altermative is the use of non-human, influenza virus-susceptible cell lines such as MDCK or Vero cells.
Our salt extraction protocol differs from traditional chromatin extraction procedures which often use lower salt concentrations and/or high EDTA concentrations. Our protocol separates chromatin into two specific fractions, a Pol II-high/histone-low fraction and its reciprocal. However, other chromatin extraction methods may also be compatible with the affinity purification of vRNPs. Since most chromatin extraction procedures have focused on DNA-protein interactions as compared to protein-protein interactions, new techniques are required to extract tightly chromatin-bound multi-protein complexes11.
In general, the length of infection should be determined empirically due to variations among virus strains and cell lines. Using the rWSN-PB2-Strep strain at an MOI of 3, we have performed subcellular fractionation at time points varying from 3 to 10 h post infection. In our experience, less than 3 h of infection yields too few purified vRNPs for useful Western blot analysis, while infection longer than 3 h results in strong cytopathic effect and a subsequent mixing of fractions.
The ability to isolate intact, tightly chromatin-bound viral complexes opens up several possibilities. While we utilized a recombinant virus expressing a Strep-tagged PB2, other affinity tags can also be used, as well as traditional immunoprecipitation targeting other viral proteins. Furthermore, while our analysis of vRNPs was limited to the characterization of their protein and RNA components, the purified complexes could also be used to perform functional in vitro synthesis assays. Finally, the separation of vRNPs into coarse functional species through subnuclear fractionation may also allow the closer study of several recently-described post-translational modifications of viral proteins required for efficient viral RNA synthesis12,13.

<table><tbody><tr><td>DMEM-high glucose</td><td>GIBCO, by Life Technologies</td><td>11965-092</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>Protease inhibitor Mix G</td><td>SERVA Electrophoresis</td><td>39101</td><td></td></tr><tr><td>Benzonase Nuclease</td><td>Novagen, EMD Millipore</td><td>71206</td><td>25 U/&amp;mu;l</td></tr><tr><td>DNase I, RNase-free</td><td>Thermo Fisher Scientific, Inc.</td><td>EN0523</td><td>50 U/&amp;mu;l</td></tr><tr><td>Dounce homogenizer</td><td>Wheaton</td><td>432-1271</td><td>Use type &quot;B&quot; pestle</td></tr><tr><td>Strep-Tactin Sepharose</td><td>IBA GmbH</td><td>2-1201-025</td><td>50% suspension column format can also be used</td></tr><tr><td>Desthiobiotin</td><td>IBA GmbH</td><td>2-1000-002</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells at JoVE.com

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

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 ...

affinity-purification-influenza-virus-ribonucleoprotein-complexes

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.0K Views.  Universitätsklinikum Freiburg. 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.

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

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Biochemistry

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

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

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 ...

Biology

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

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 ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

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

Summary

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Chapters in this video

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Generation of Recombinant Influenza Virus from Plasmid DNA

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

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

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes