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 S...

<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. G., 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=Functional+association+between+viral+and+cellular+transcription+during+influenza+virus+infection.">Functional association between viral and cellular transcription during influenza virus infection.</a> Rev. Med. Virol. 16, 329-345 (2006).</li><li>Schwartz, L. B., Sklar, V. E., Jaehning, J. A., Weinmann, R., Roeder, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+partial+characterization+of+the+multiple+forms+of+deoxyribonucleic+acid-dependent+ribonucleic+acid+polymerase+in+the+mouse+myeloma,+MOPC+315.">Isolation and partial characterization of the multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in the mouse myeloma, MOPC 315.</a> J. Biol. Chem. 249, 5889 (1974).</li><li>Lambert, J. 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 vues.  Universitätsklinikum Freiburg. Les virus grippaux répliquer leur génome à ARN en association avec la cellule hôte chromatine. Ici, nous présentons une méthode pour purifier intactes complexes ribonucléoprotéiques virales de la chromatine des cellules infectées. Purifiée des complexes viraux peuvent être analysés à la fois par Western blot et par extension d'amorce de la teneur en protéines et d'ARN, respectivement.