Name: co-immunoprecipitation of the mouse mx1 protein with the influenza a virus nucleoprotein
Uploaded: 2015-04-21T15:00:00.000+00:00
Duration: 9 min 39 s
Description: Watch this Scientific Journal Video about Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein at JoVE.com

This work was supported by FWO-Vlaanderen, the IOF project IOF10/StarTT/027 and Ghent University Special Research Grant BOF12/GOA/014.

Note: The following transfection and co-immunoprecipitation protocol is established for a 9 cm Petri dish format. Other formats are also possible after scaling the protocol.
1. Seeding the Human Embryonic Kidney (HEK) 293T Cells
<ol>
	<li>Seed the HEK293T cells one day before transfection at 1.2 x 106 cells per 9 cm Petri dish in 12 ml of Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.4 mM Na-pyruvate, 0.1 mM non-essential amino acids, 100 U/ml penicillin and 0.1 mg/ml streptomycin.</li>
	<li>Grow the cells 16 hr at 37 &#176;C and 5% CO2.</li>
	<li>Visually inspect the morphology and viability of the cells with an inverted light microscope before transfection. The cells need to be sub-confluent for optimal transfection efficiency.</li>
</ol>
2. Calcium-phosphate Transfection of HEK293T Cells
Note: Use 0.5-1 &#181;g of pCAXL-NP or empty pCAXL plasmid in combination with 1-3 &#181;g of pCAXL-Mx1 per 9 cm dish. Use an equal amount of total plasmid DNA in all samples; adjust with empty plasmid if necessary.
<ol>
	<li>Prepare the following transfection buffers:
	<ol>
		<li>Prepare Tris-EDTA (TE) with concentrations of 1.0 mM Tris-HCl pH 8.0 and 0.1 mM EDTA pH 8.0.</li>
		<li>Prepare BS/HEPES with concentrations of 25 mM HEPES (5.96 g/L, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 274 mM NaCl (16 g/L), 10 mM KCl (0.74 g/L), 1.5 mM NaHPO4&#183;12H2O (0.5 g/L) and 11.1 mM dextrose (2 g/L). Adjust pH to 7.05.</li>
		<li>Prepare CaCl2/HEPES with concentrations of 1.25 M CaCl2&#183;2H2O (183.8 g/L) and 125 mM HEPES (29.79 g/L). Adjust pH to 7.05 with NaOH.</li>
	</ol>
	</li>
	<li>Warm the transfection buffers at 37 &#176;C before use.</li>
	<li>Prepare the plasmid samples by diluting the plasmid DNA in 600 &#181;l of TE. Prepare these mixtures in wells of a 6-well plate.</li>
	<li>Add 150 &#181;l of CaCl2/HEPES in a drop wise manner to the plasmid samples and mix by pipetting 3 times up and down.</li>
	<li>Prepare the transfection solution by drop wise adding the plasmid solution (TE + DNA + CaCl2/HEPES; 750 &#181;l) to 750 &#181;l of BS/HEPES buffer provided in a fresh 6-well plate. Distribute the plasmid solution evenly over the complete well containing BS/HEPES buffer.</li>
	<li>Shake the transfection solution on a plate shaker for 90 sec at 1,000 RPM.</li>
	<li>Incubate the mixture for 5 min at room temperature.</li>
	<li>Add the transfection solution (1.5 ml) drop wise to the cells. Use a P1000 micropipet to drip the transfection solution on the cells. Disperse the mixture over the complete 9 cm Petri dish and shake the plate very gently.</li>
	<li>Incubate the cells at 37 &#176;C and 5% CO2 for 6 hr. Then remove the medium by aspiration and immediately replace with 12 ml fresh, pre-warmed medium. Gently add the fresh medium to the cells to prevent cell detachment. For this, hold the tip of the pipet against the side of the well and gently push out the medium.</li>
	<li>Incubate the cells for an additional 16 hr at 37 &#176;C and 5% CO2.</li>
</ol>
3. Co-immunoprecipitation
Note: Perform the co-immunoprecipitation 24 hr after transfection.
<ol>
	<li>Preparation of the low salt lysis buffer and high salt wash buffer.
	<ol>
		<li>Prepare a stock solution of 2 M N-ethylmaleimide (NEM) by weighing the amount of NEM and dissolving it in absolute ethanol. Prepare the NEM stock solution fresh before use. 
		CAUTION: NEM is very toxic, prepare and use this stock solution in a fume hood.</li>
		<li>Prepare low salt lysis buffer at concentrations of 50 mM Tris-HCl pH 8, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% NP40 and a protease inhibitor cocktail (dissolve 1 tablet in 50 ml lysis buffer). Add NEM to a final concentration of 25 mM (i.e., dilute 1:80). Keep on ice after adding the protease inhibitors and NEM. 
		Note: Always add the protease inhibitors and NEM freshly before use.</li>
		<li>Prepare a high salt wash buffer at concentrations of 50 mM Tris-HCl pH 8, 500 mM NaCl, 5 mM EDTA and 1% NP40. Note that the high salt wash buffer does not contain NEM.</li>
	</ol>
	</li>
	<li>Preparation of cell lysates.
	<ol>
		<li>Remove the medium and wash the cells with 2 ml of ice cold phosphate buffered saline (PBS). Very gently add the wash buffer, as HEK293T cells detach easily.</li>
		<li>Remove the PBS and add 600 &#181;l of ice cold low salt lysis buffer per 9 cm Petri dish.</li>
		<li>Incubate the plates for 20 min on ice. Make sure that the plates are kept horizontal, to assure complete coverage of the plate surface with lysis buffer. Gently shake the plates every 5 min.</li>
		<li>Collect the cell lysate in a 1.5 ml microcentrifuge tube and centrifuge for 3 min at 4 &#176;C and 16,000 x g to pellet the insoluble fraction.</li>
		<li>Transfer the soluble fraction, i.e. the cell lysate to a fresh 1.5 ml microcentrifuge tube and keep on ice. Immediately continue with the co-immunoprecipitation protocol, to prevent dissociation of the interacting proteins. Perform all the following steps as much as possible on ice or at 4 &#176;C to limit proteolytic activity in the lysates.</li>
	</ol>
	</li>
	<li>Generation of immune-complexes. 
	Note: In this step, the protein of interest is bound by the appropriate antibody. To study the NP&#8211;Mx1 interaction, use a mouse anti-NP monoclonal antibody.
	<ol>
		<li>For each sample, mix 135 &#181;l of lysate with 2 &#181;l of anti-NP monoclonal antibody and 113 &#181;l of low salt lysis buffer (total volume of 250 &#181;l). Store the remaining lysate at -20 &#176;C for further analysis such as western blotting, to document the expression levels of the surmised interaction partners in the transfected cells. 
		Note: Alternatively, measure the protein concentration of the lysate (e.g. with Bradford reagent) and use a fixed amount of total protein, e.g., 400 &#181;g, for each lysate.</li>
		<li>Incubate the antibody-lysate mix for 3 hr on a turning wheel at 4 &#176;C. This step can be extended to an overnight incubation.</li>
	</ol>
	</li>
	<li>Preparation of the protein G beads. 
	Note: The protein G beads are shipped and stored in 20% ethanol for preservation. The bead-slurry normally consists of 50% beads and these beads need to be washed before they are used to immunoprecipitate the immune-complexes.
	<ol>
		<li>Use 50 &#181;l of beads, i.e., 100 &#181;l of bead-slurry, for each sample. Wash the amount of beads needed for all samples in the co-immunoprecipitation assay in one tube. Cut the tip of a 1 ml pipette tip to ease pipetting of the bead-slurry.</li>
		<li>Centrifuge the protein G bead-slurry at 8,000 x g and 4 &#176;C for 30 sec. Remove the ethanol solution and add an equal volume of low salt lysis buffer. Centrifuge the protein G bead-slurry at 8,000 x g and 4 &#176;C for 30 sec and remove the supernatant gently. Repeat this wash step 3 times. 
		Note: The low salt lysis buffer used to wash the beads does not need to contain protease inhibitors or NEM.</li>
		<li>Estimate the volume of protein G beads and add an equal volume of low salt lysis buffer to make a new 50% bead-slurry in low salt lysis buffer.</li>
		<li>For each sample, transfer 100 &#181;l of bead-slurry in a fresh 1.5 ml microcentrifuge tube and store on ice until use. Be careful to resuspend the bead-slurry before dividing, as these beads quickly sediment to the bottom of the tube.</li>
	</ol>
	</li>
	<li>Immunoprecipitation of the immune-complexes by protein G beads and their elution.
	<ol>
		<li>Before using the protein G beads for immunoprecipitation, centrifuge all tubes 30 sec at 8,000 x g and 4 &#176;C and verify by visual inspection that there is an equal amount of beads present in all samples. If necessary adjust the amount of beads in some of the samples and centrifuge again. Discard the supernatants. Be careful not to disturb the pelleted protein G beads.</li>
		<li>Briefly centrifuge the immune-complexes (i.e., lysates with antibody, 250 &#181;l) for 30 sec at 8,000 x g and 4 &#176;C to collect the complete sample at the bottom of the tube. Transfer the immune-complexes to the protein G beads (50 &#181;l).</li>
		<li>Incubate 60 min on a turning wheel at 4 &#176;C. Do not incubate these immune complexes longer than 75 min with the beads to reduce nonspecific binding of proteins to the protein G beads.</li>
		<li>Centrifuge the protein G beads (with bound immune-complexes) for 30 sec at 8,000 x g and 4 &#176;C and remove the supernatants. Be careful not to disturb the pelleted protein G beads. Optional: store this supernatants at 4 &#176;C or -20 &#176;C for later analysis, e.g., to estimate the amount of unbound protein.</li>
		<li>Wash the protein G beads for approximately 5 min with 900 &#181;l of high salt lysis buffer. Make sure that the beads are completely resuspended in the wash buffer for optimal washing. Centrifuge the protein G beads for 30 sec at 8,000 x g and 4 &#176;C and discard the supernatants. Repeat this wash step 4 times. Be careful not to disturb the pelleted protein G beads to avoid loss of immunoprecipitated material.</li>
		<li>After the last wash step, add 50 &#181;l of 2x&#160;Laemmli sample buffer to the beads and heat the suspension for 10 min at 95 &#176;C to elute the (co)immunoprecipitated proteins.
		<ol>
			<li>Prepare 10 ml of 6x Laemmli buffer with 1 g of sodium dodecyl sulfate, 3.5 ml of glycerol, 3.5 ml of 1 M Tris-HCl pH 6.8 and 420 &#181;l of &#946;-mercaptoethanol. Adjust to a total volume of 10 ml by adding distilled water. Dilute 3 times in distilled water to obtain 2x Laemmli buffer. 
			CAUTION: &#946;-mercaptoethanol is toxic, prepare and use Laemmli buffer in a fume hood.</li>
		</ol>
		</li>
		<li>After heating, centrifuge the protein G beads for 30 sec at 8,000 x g and store the samples at 4 &#176;C (short term) or -20 &#176;C (long term).</li>
	</ol>
	</li>
</ol>
4. Analyze the (co-)Immunoprecipitated Proteins
<ol>
	<li>Visualize the proteins present in the cell lysate and eluate of the co-immunoprecipitation by SDS-PAGE20 and western blotting21,22. Typically load half of the Laemmli eluate on gel. Be careful not to disturb the pelleted protein G beads when taking samples for gel loading. Mx1 and NP expression were revealed with an anti-Mx1 and anti-NP antibody, respectively14. The bands were detected with HRP-based chemiluminescence and an X-ray film developer.</li>
</ol>

<ol>
	<li>Verhelst, J., Hulpiau, P., Saelens, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mx+proteins:+antiviral+gatekeepers+that+restrain+the+uninvited.">Mx proteins: antiviral gatekeepers that restrain the uninvited.</a> Microbiol Mol Biol Rev. 77 (4), 551-566 (2013).</li><li>Goujon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+MX2+is+an+interferon-induced+post-entry+inhibitor+of+HIV-1+infection.">Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection.</a> Nature. 502 (7472), 559-562 (2013).</li><li>Kane, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MX2+is+an+interferon-induced+inhibitor+of+HIV-1+infection.">MX2 is an interferon-induced inhibitor of HIV-1 infection.</a> Nature. 502 (7472), 563-566 (2013).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interferon-inducible+MxB+protein+inhibits+HIV-1+infection.">The interferon-inducible MxB protein inhibits HIV-1 infection.</a> Cell Host Microbe. 14 (4), 398-410 (2013).</li><li>Kochs, G., Haller, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-induced+human+MxA+GTPase+blocks+nuclear+import+of+Thogoto+virus+nucleocapsids.">Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids.</a> Proc Natl Acad Sci U S A. 96 (5), 2082-2086 (1999).</li><li>Flohr, F., Schneider-Schaulies, S., Haller, O., Kochs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+central+interactive+region+of+human+MxA+GTPase+is+involved+in+GTPase+activation+and+interaction+with+viral+target+structures.">The central interactive region of human MxA GTPase is involved in GTPase activation and interaction with viral target structures.</a> FEBS Lett. 463 (1-2), 24-28 (1999).</li><li>Kochs, G., Haller, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTP-bound+human+MxA+protein+interacts+with+the+nucleocapsids+of+Thogoto+virus+(Orthomyxoviridae).">GTP-bound human MxA protein interacts with the nucleocapsids of Thogoto virus (Orthomyxoviridae).</a> J Biol Chem. 274 (7), 4370-4376 (1999).</li><li>Mitchell, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution-guided+identification+of+antiviral+specificity+determinants+in+the+broadly+acting+interferon-induced+innate+immunity+factor+MxA.">Evolution-guided identification of antiviral specificity determinants in the broadly acting interferon-induced innate immunity factor MxA.</a> Cell Host Microbe. 12 (4), 598-604 (2012).</li><li>Patzina, C., Haller, O., Kochs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+requirements+for+the+antiviral+activity+of+the+human+MxA+protein+against+Thogoto+and+influenza+A+virus.">Structural requirements for the antiviral activity of the human MxA protein against Thogoto and influenza A virus.</a> J Biol Chem. 289 (9), 6020-6027 (2014).</li><li>Turan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+MxA+proteins+form+a+complex+with+influenza+virus+NP+and+inhibit+the+transcription+of+the+engineered+influenza+virus+genome.">Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome.</a> Nucleic Acids Res. 32 (2), 643-652 (2004).</li><li>Dittmann, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+A+virus+strains+differ+in+sensitivity+to+the+antiviral+action+of+Mx-GTPase.">Influenza A virus strains differ in sensitivity to the antiviral action of Mx-GTPase.</a> J Virol. 82 (7), 3624-3631 (2008).</li><li>Zimmermann, P., Manz, B., Haller, O., Schwemmle, M., Kochs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+viral+nucleoprotein+determines+Mx+sensitivity+of+influenza+A+viruses.">The viral nucleoprotein determines Mx sensitivity of influenza A viruses.</a> J Virol. 85 (16), 8133-8140 (2011).</li><li>Manz, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pandemic+influenza+A+viruses+escape+from+restriction+by+human+MxA+through+adaptive+mutations+in+the+nucleoprotein.">Pandemic influenza A viruses escape from restriction by human MxA through adaptive mutations in the nucleoprotein.</a> PLoS Pathog. 9 (3), e1003279 (2013).</li><li>Verhelst, J., Parthoens, E., Schepens, B., Fiers, W., Saelens, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-inducible+protein+Mx1+inhibits+influenza+virus+by+interfering+with+functional+viral+ribonucleoprotein+complex+assembly.">Interferon-inducible protein Mx1 inhibits influenza virus by interfering with functional viral ribonucleoprotein complex assembly.</a> J Virol. 86 (24), 13445-13455 (2012).</li><li>Brewer, C. F., Riehm, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+possible+nonspecific+reactions+between+N-ethylmaleimide+and+proteins.">Evidence for possible nonspecific reactions between N-ethylmaleimide and proteins.</a> Anal Biochem. 18 (2), 248-255 (1967).</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 (13), 6618-6628 (2011).</li><li>Chen, Z., Zhou, J., Zhang, Y., Bepler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+ribonucleotide+reductase+M1-gemcitabine+interaction+in+vivo+by+N-ethylmaleimide.">Modulation of the ribonucleotide reductase M1-gemcitabine interaction in vivo by N-ethylmaleimide.</a> Biochem Biophys Res Commun. 413 (2), 383-388 (2011).</li><li>Rennie, M. L., McKelvie, S. A., Bulloch, E. M., Kingston, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+dimerization+of+human+MxA+promotes+GTP+hydrolysis,+resulting+in+a+mechanical+power+stroke.">Transient dimerization of human MxA promotes GTP hydrolysis, resulting in a mechanical power stroke.</a> Structure. 22 (10), 1433-1445 (2014).</li><li>Gifford, J. L., Walsh, M. P., Vogel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+and+metal-ion-binding+properties+of+the+Ca2+-binding+helix-loop-helix+EF-hand+motifs.">Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs.</a> Biochem J. 405 (2), 199-221 (2007).</li><li>Gallagher, S., Chakavarti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblot+analysis.">Immunoblot analysis.</a> J Vis Exp. (16), (2008).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J Vis Exp. (44), (2010).</li><li>Pavlovic, J., Haller, O., Staeheli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+mouse+Mx+proteins+inhibit+different+steps+of+the+influenza+virus+multiplication+cycle.">Human and mouse Mx proteins inhibit different steps of the influenza virus multiplication cycle.</a> J Virol. 66 (4), 2564-2569 (1992).</li></ol>

The authors declare that they have no competing financial interests.

Studying the interaction between antiviral proteins and their viral targets is very important to understand the details of the antiviral mechanism of these proteins. This can give new insights into how viruses and their hosts co-evolved and be the basis for the development of new antiviral strategies. The optimized co-immunoprecipitation protocol described here allows to study the interaction between the mouse Mx1 protein and its viral target, the influenza NP protein. The most important aspect of this protocol is the ad...

Myxovirus resistance (Mx) proteins are an important part of the innate immune defense against viral pathogens. These proteins are large dynamin-like GTPases that are induced by type I and type III interferons. The corresponding Mx genes are present in nearly all vertebrates in one or multiple copies and their gene products inhibit a wide range of viruses, including Orthomyxoviridae (e.g., influenza virus), Rhabdoviridae (e.g., vesicular stomatitis virus), Bunyaviridae (e.g., la crosse virus) and Retroviridae (e.g., human immunodeficiency virus-1)1-4. It is unclear how these proteins recognize such a broad array of viruses, without any apparent shared primary sequence motifs in these viruses. Analyzing the interaction of Mx proteins with their viral targets, potentially involving higher order complexes with other host cell factors, will help to understand the molecular mechanisms that have evolved in the arms race between viruses and their hosts.
The interaction between mammalian Mx proteins and viral targets has been studied most extensively for human MxA. Human MxA can inhibit the replication of many viruses, including the orthomyxoviruses influenza A and Thogoto virus. MxA binds the Thogoto virus ribonucleoprotein complexes (vRNPs), thereby preventing their nuclear entry, which results in block of infection5. This interaction between MxA and Thogoto virus vRNPs has been demonstrated with co-sedimentation and co-immunoprecipitation experiments6-9. How Mx proteins hinder influenza A viruses is less clear. One major problem is that it is not straightforward to demonstrate an interaction between an Mx protein and an influenza gene product. One report demonstrated an interaction between human MxA and the NP protein in influenza A virus infected cells10. This interaction could only be shown by co-immunoprecipitation if the cells had been treated with the cross-linking reagent dithiobis (succinimidyl propionate) before lysis, suggesting that the interaction is transient and/or weak. More recent studies have shown that the differential Mx sensitivity of different influenza A strains is determined by the origin of the NP protein11,12. In line with this, influenza A viruses can partly escape from Mx control by mutating specific residues in the NP protein13. This suggests that the main target of influenza A viruses for host Mx is the NP protein, most probably NP assembled in vRNP complexes. However, none of these more recent studies demonstrated an interaction between influenza NP or vRNPs and either human MxA or mouse Mx1.
Recently we showed, for the first time, an interaction between the influenza NP and the mouse Mx1 protein with an optimized co-immunoprecipitation protocol14, which is described here in detail. In general, co-immunoprecipitation is one of the most frequently used biochemical approaches to investigate protein-protein interactions. This technique is often preferred over alternative techniques, e.g., yeast two hybrid, since it allows to investigate protein-protein interactions in their natural environment. Co-immunoprecipitation can be carried out on endogenously expressed proteins if antibodies against the proteins of interest are available. Alternatively, the proteins of interest can be expressed in the cell through transfection or infection and an affinity tag may be used. In addition to the above-mentioned advantages, the described co-immunoprecipitation protocol allows the detection of weak and/or transient protein interactions. The main component in this optimized protocol is the addition of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM is an alkylating reagent that reacts with free thiol groups such as present in cysteines, at a pH of 6.5-7.5, to form a stable thio-ester (Figure 1). At higher pH, NEM can also react with amino groups or undergo hydrolysis15. NEM is typically used to block free thiol groups, in order to prevent disulfide bond formation or inhibit enzymatic activity. For example, NEM is often used to block desumoylating enzymes, which are cysteine proteases. In the described co-immunoprecipitation protocol, NEM was initially included in the lysis buffer because it had been reported that the sumoylation of influenza proteins can influence the interaction between viral proteins16. Unexpectedly, the addition of NEM proved to be key to document the interaction between influenza NP and mouse Mx1 by co-immunoprecipitation. It is unclear why the addition of NEM is crucial to detect the NP&#8211;Mx1 interaction. Possibly the interaction is too transient and/or weak. NEM could stabilize the interaction, e.g., by preserving a specific conformation of Mx1, a viral protein or even an unknown third component. Such a stabilizing effect of NEM has been observed before, e.g., for the interaction between the ribonucleotide reductase M1 and its inhibitor gemcitabine (F2dC)17. Mx1 and NP both contain multiple cysteine residues which could be modified by NEM. For example, a recent study by Rennie et al. demonstrated that a stalkless MxA-variant contains three solvent exposed cysteine residues which can be modified by iodoacetamide. Mutating these residues to serines did not influence the enzymatic activity of MxA, but prevented disulfide-mediated aggregation18. As these cysteines are conserved in Mx1, this suggests that the analogous cysteines in Mx1 can be modified by NEM and as such influence its conformation or solubility. In addition, NEM might also affect the GTPase activity of Mx1, which is essential for the anti-influenza activity of Mx1, and thereby stabilize the interaction between Mx1 and NP. However, a direct effect of NEM on the GTPase activity of Mx1 is unlikely, as NEM is also required to detect the interaction between influenza NP and GTPase inactive mutants of the Mx1 protein14. Clearly, more research is needed to unravel the effect of NEM on the NP&#8211;Mx1 interaction.
In summary, the described co-immunoprecipitation protocol allows to study the interaction between the antiviral Mx1 protein and its viral target, the influenza NP protein. This protocol could also be used to study other weak or transient interactions that depend on the stabilization of specific protein conformations. Protein-protein interaction that depend on specific conformations have been described before, e.g., for calcium-binding proteins such as calmodulin19. Finally, the beneficial role of NEM could also be used in other methods that detect protein-protein interactions, such as co-sedimentation assays.

<table><tbody><tr><td>DMEM high glucose</td><td>Gibco</td><td>52100-047</td><td></td></tr><tr><td>N-Ethylmaleimide</td><td>Sigma</td><td>E-3876</td><td>Toxic</td></tr><tr><td>Igepal CA-630</td><td>Sigma</td><td>I-30212</td><td>also known as NP40</td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche</td><td>11 873 580 001</td><td></td></tr><tr><td>anti-NP monoclonal antibody</td><td>NIH Biodefense and Emerging Infections Research Resources Repository</td><td>NR-4282</td><td>ascites blend of clones A1 and A3</td></tr><tr><td>anti-RNP polyclonal serum</td><td>NIH Biodefense and Emerging Infections Research Resources Repository</td><td>NR-3133</td><td>directed against A/Scotland/840/74 (H3N2)</td></tr><tr><td>Protein G Sepharose 4FF</td><td>GE Healthcare</td><td>17-0618-01</td><td></td></tr><tr><td>Hyperfilm ECL 18 x 24 cm</td><td>GE Healthcare</td><td>28-9068-36</td><td></td></tr><tr><td>ECL western blotting substrate</td><td>Pierce</td><td>32106</td><td></td></tr></tbody></table>

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.

N-ethylmaleimide is an organic compound that can be used to irreversibly modify free thiol groups, e.g. to inhibit cysteine proteases (Figure 1).
The antiviral Mx1 protein inhibits influenza A virus replication by interacting with the viral nucleoprotein. The optimized co-immunoprecipitation protocol described here allows to study this NP&#8211;Mx1 interaction. HEK293T cells were transfected with expression vectors for the antiviral Mx1 protein in the absence or prese...

Watch this Scientific Journal Video about Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein at JoVE.com

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

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 ...

co-immunoprecipitation-mouse-mx1-protein-with-influenza-virus

Research

JoVE Journal

Immunology and Infection

17.7K Views.  VIB. 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.

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

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Biochemistry

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

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, ...

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

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

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

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

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

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.