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. A. <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+influenza+virus+matrix+protein+with+ribonucleoproteins.">Association of influenza virus matrix protein with ribonucleoproteins.</a> J. Virol. 73, 7467-7467 (1999).</li><li>Watanabe, T., Watanabe, S., Kawaoka, T., Y, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+networks+involved+in+the+influenza+virus+life+cycle.">Cellular networks involved in the influenza virus life cycle.</a> Cell Host Microbe. 7, 427 (2010).</li><li>Engelhardt, O. 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P., Baetz, K., Figeys, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+proteins+and+DNA--proteomic+role+in+the+field+of+chromatin+research.">Of proteins and DNA--proteomic role in the field of chromatin research.</a> Mol. Biosyst. 6, 30 (2010).</li><li>Wu, C. Y., Jeng, K. S., Lai, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SUMOylation+of+matrix+protein+M1+modulates+the+assembly+and+morphogenesis+of+influenza+A+virus.">The SUMOylation of matrix protein M1 modulates the assembly and morphogenesis of influenza A virus.</a> J. Virol. 85, 6618 (2011).</li><li>Liao, T. L., Wu, C. Y., Su, W. C., Jeng, K. S., Lai, M. 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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 purif...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>DMEM-high glucose</td>
 <td>Gibco</td>
 <td>11965-092</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A9418</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Protease inhibitor Mix G</td>
 <td>Serva</td>
 <td>39101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Benzonase Nuclease</td>
 <td>Novagen</td>
 <td>71206</td>
 <td>25 U/&mu;l</td>
 </tr>
 <tr>
 <td>DNase I, RNase-free</td>
 <td>ThermoScientific</td>
 <td>EN0523</td>
 <td>50 U/&mu;l</td>
 </tr>
 <tr>
 <td>Dounce homogenizer</td>
 <td>Wheaton</td>
 <td>432-1271</td>
 <td>Use type "B" 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>&nbsp;</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

Research

JoVE Journal

Immunology and Infection

13.0K visualizações.  Universitätsklinikum Freiburg. Os vírus da gripe replicar seu genoma de RNA em associação com a célula hospedeira da cromatina. Aqui, apresentamos um método para purificar intactos os complexos de ribonucleoproteínas virais da cromatina das células infectadas. Purificou complexos virais podem ser analisados ​​por Western blot e tanto de extensão do primer de proteína e do conteúdo de ARN, respectivamente.

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

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 ...

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, ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

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 ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...