We thank Alejandro Brun from CISA-INIA for providing anti-Rift Valley fever Virus sera. Work in our laboratories is supported by Forschungsf&#246;rderung gem. &#167;2 Abs. 3 Kooperationsvertrag Universit&#228;tsklinikum Giessen und Marburg, the Leibniz Graduate School for Emerging viral diseases (EIDIS), the DFG Sonderforschungsbereich (SFB) 1021, and the DFG Schwerpunktprogramm (SPP) 1596.

1. Seeding of A549 Cells for Infection
<ol>
	<li>Cultivate a T75 flask of A549 cells at 37 &#176;C and 5% CO2 in cell culture medium (DMEM supplemented with 10% FCS, 526.6 mg/l L-glutamine, 50.000 U/l penicillin, and 50 mg/l streptomycin).</li>
	<li>Before starting to harvest the cells, warm up cell culture medium, PBS and 0.05% trypsin-EDTA in a waterbath heated to 37 &#176;C.</li>
	<li>Remove the medium and wash the cells with 10 ml PBS. Remove the PBS again.</li>
	<li>Add 3 ml of trypsin-EDTA and distribu...

No conflicts of interest declared.

Sensing the presence of viruses and activation of the antiviral type I IFN system are crucial for successful innate immune responses22. Virus detection is thereby mediated by pathogen recognition receptors (PRRs) like RIG-I and PKR, enabling a rapid response and activation of antiviral defense mechanisms. Here, we describe two methods to directly evaluate the activation status of RIG-I and PKR.
Limited protease digestion as a tool to monitor conformational changes of RIG-I and PKR w...

A crucial event in antiviral host defense is the rapid detection of the pathogen by the so-called pattern recognition receptors (PRRs)1,2. Intracellular detection of RNA virus infection is dependent on two cytoplasmic RNA helicases, RIG-I (retinoic acid inducible gene I) and MDA5 (melanoma differentiation associated protein 5)3-5. RIG-I is composed of two N-terminal caspase recruitment domains (CARDs), a central DECH-box type RNA helicase domain, and a C-terminal domain (CTD)4,6. Whereas the CTD and the helicase domain are required for recognition of non-self (viral) RNAs, the CARDs mediate downstream signaling leading to establishment of an antiviral host status.
If RIG-I is in the silent state, i.e. in the absence of a specific RNA ligand, the second CARD interacts with the central helicase domain and keeps RIG-I in an auto-inhibitory conformation7-11. RIG-I binds to short double-strand (ds) RNA bearing a 5&#8217;-triphosphate (5&#8217;PPP), long dsRNA, and polyU/UC-rich RNA, classic signature structures which are present on the genomes of many RNA viruses12-16. Two major characteristics of RIG-I activation are a switch to a closed conformation6,17 and the homo-oligomerization6,18,19. The conformational switch enhances RNA binding, exposes the CARDs for downstream signaling, and reconstitutes an active ATPase site8,9,11,20. The formation of oligomeric RIG-I complexes leads to enhanced recruitment of downstream signaling adaptor molecules to form a platform for antiviral signal transduction11. The RIG-I-regulated signaling chain eventually activates the transcription factor IRF-3 for up-regulation of interferon (IFN-alpha/beta) genes and hence the gene expression of interferon stimulated genes (ISGs) for a full antiviral response21,22. One of the best characterized ISGs is the RNA-activated protein kinase (PKR) 23. PKR belongs to the family of eukaryotic translation initiation factor 2 alpha (eIF2&#945;) kinases and is composed of an N-terminal double-stranded RNA binding domain and a C-terminal kinase domain. The kinase domain constitutes the dimerization interface crucial for PKR activation and carries out the catalytic functions of the protein. Binding of PKR to viral dsRNA leads to its conformational change permitting dimerization and auto-phosphorylation at Thr 446 among other residues. PKR then mediates phosphorylation of eIF2&#945;, thereby blocking the translation of viral mRNAs23-27.
Both RIG-I and PKR undergo major structural rearrangements, form oligomeric complexes and are post-translationally modified by phosphorylation/dephosphorylation and ubiquitination10,11,19,23,24,26-29. For a better understanding of which viral RNA structures are activating RIG-I and PKR (and at what stage viral antagonists could be interfering), it is important to precisely determine the activation status. For both PRRs it was previously described that activation leads to the emergence of trypsin-resistant protein fragments6,17,30 and higher-order oligomers6,18,19. However, given the wealth of literature on these key factors of the antiviral host response1,2,24, application of direct methods seems comparatively rare. In the hope of stimulating broader usage, we provide convenient and sensitive protocols to robustly analyze the activation states of RIG-I and PKR. The IFN competent human cell line A549 is infected with an established activator of RIG-I and PKR, the attenuated Rift Valley fever virus mutant clone 13 (Cl 13)31,32. After a simple lysing procedure, the extracts of infected cells are tested by limited trypsin digestion/western blot analysis to evaluate conformational switching, and by blue native polyacrylamide gel electrophoresis (PAGE) /Western blot analysis to measure formation of oligomers.

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			<td class="xl66" height="43" style="height:32.25pt;width:60pt" width="80">Name</td>
			<td class="xl67" style="width:60pt" width="80">Company</td>
			<td class="xl67" style="width:60pt" width="80">Catalog Number</td>
			<td class="xl67" style="width:60pt" width="80">Comments</td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl68" height="22" style="height:16.5pt;width:60pt" width="80">cell culture</td>
			<td class="xl76" colspan="3" style="border-right:1.0pt solid black;
 border-left:none;width:180pt" width="240"></td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl69" height="106" style="height:79.5pt;width:60pt" width="80">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td class="xl70" style="width:60pt" width="80">Gibco</td>
			<td class="xl65" style="width:60pt" width="80">21969-035</td>
			<td class="xl71" style="width:60pt" width="80"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl72" height="22" style="height:16.5pt;width:60pt" width="80">OptiMEM</td>
			<td class="xl70" style="width:60pt" width="80">Gibco</td>
			<td class="xl65" style="width:60pt" width="80">31985-47</td>
			<td class="xl71" style="width:60pt" width="80"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl72" height="22" style="height:16.5pt;width:60pt" width="80">L-glutamine</td>
			<td class="xl70" style="width:60pt" width="80">PAA</td>
			<td class="xl65" style="width:60pt" width="80">25030-024</td>
			<td class="xl71" style="width:60pt" width="80"></td>
		</tr>
		<tr height="61" style="height:45.75pt">
			<td class="xl72" height="61" style="height:45.75pt;width:60pt" width="80">penicillin-streptomycin</td>
			<td class="xl70" style="width:60pt" width="80">PAA</td>
			<td class="xl65" style="width:60pt" width="80">15070-063</td>
			<td class="xl71" style="width:60pt" width="80"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl73" height="64" style="height:48.0pt;width:60pt" width="80">fetal calf serum (FCS)</td>
			<td class="xl70" style="width:60pt" width="80">PAA</td>
			<td class="xl65" style="width:60pt" width="80">10270</td>
			<td class="xl71" style="width:60pt" width="80"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl69" height="64" style="height:48.0pt;width:60pt" width="80">0.05% trypsin-EDTA</td>
			<td class="xl70" style="width:60pt" width="80">Gibco</td>
			<td class="xl65" style="width:60pt" width="80">25300-054</td>
			<td class="xl71" style="width:60pt" width="80"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl68" height="22" style="height:16.5pt;width:60pt" width="80">chemicals</td>
			<td class="xl76" colspan="3" style="border-right:1.0pt solid black;
 border-left:none;width:180pt" width="240"></td>
		</tr>
		<tr height="190" style="height:142.5pt">
			<td class="xl73" height="190" style="height:142.5pt;width:60pt" width="80">L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated (TPCK) trypsin</td>
			<td class="xl65" style="width:60pt" width="80">Sigma Aldrich</td>
			<td class="xl65" style="width:60pt" width="80">T1426</td>
			<td class="xl65" style="width:60pt" width="80"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl68" height="22" style="height:16.5pt;width:60pt" width="80">antibodies</td>
			<td class="xl78" colspan="3" style="border-right:1.0pt solid black;
 border-left:none;width:180pt" width="240"></td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl69" height="106" style="height:79.5pt;width:60pt" width="80">mouse monoclonal anti-RIG-I antibody (ALME-1)</td>
			<td class="xl74" style="width:60pt" width="80">Enzo Life Sciences</td>
			<td class="xl65" style="width:60pt" width="80">ALX-804-849-C100</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:500 in 1% skim milk in TBS</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl69" height="85" style="height:63.75pt;width:60pt" width="80">mouse monoclonal anti-PKR (B10)</td>
			<td class="xl65" style="width:60pt" width="80">Santa Cruz</td>
			<td class="xl65" style="width:60pt" width="80">sc-6282</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:500 in 1% skim milk in TBS</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl69" height="85" style="height:63.75pt;width:60pt" width="80">rabbit monoclonal anti-P-PKR (Thr446)</td>
			<td class="xl65" style="width:60pt" width="80">Epitomics</td>
			<td class="xl65" style="width:60pt" width="80">1120-1</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:1.000 in 5% BSA in TBS</td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl69" height="64" style="height:48.0pt;width:60pt" width="80">rabbit polyclonal anti-IRF-3</td>
			<td class="xl65" style="width:60pt" width="80">Santa Cruz</td>
			<td class="xl65" style="width:60pt" width="80">sc-9082</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:500 in 1% skim milk in TBS</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl69" height="85" style="height:63.75pt;width:60pt" width="80">rabbit monoclonal anti-P-IRF-3 (Ser386)</td>
			<td class="xl65" style="width:60pt" width="80">IBL</td>
			<td class="xl65" style="width:60pt" width="80">og-413</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:100 in 1% skim milk in TBS</td>
		</tr>
		<tr height="126" style="height:94.5pt">
			<td class="xl81" height="211" rowspan="2" style="border-bottom:1.0pt solid black;
 height:158.25pt;border-top:none;width:60pt" width="80">rabbit anti-RVFV hyperimmune serum &#34;C2&#34; (MP-12)</td>
			<td class="xl82" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:60pt" width="80"></td>
			<td class="xl82" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:60pt" width="80"></td>
			<td class="xl75" style="width:60pt" width="80">Kindly provided by Alejandro Brun (CISA-INIA)</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl65" height="85" style="height:63.75pt;width:60pt" width="80">WB: 1:2.000 in 1% skim milk in TBS</td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl69" height="106" style="height:79.5pt;width:60pt" width="80">mouse monoclonal anti-beta-actin (8H10D10)</td>
			<td class="xl65" style="width:60pt" width="80">Cell Signalling</td>
			<td class="xl65" style="width:60pt" width="80">3700</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:1.000 in 1% skim milk in TBS</td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl69" height="106" style="height:79.5pt;width:60pt" width="80">polyclonal peroxidase-conjugated goat anti-rabbit</td>
			<td class="xl65" style="width:60pt" width="80">Thermo Fisher</td>
			<td class="xl65" style="width:60pt" width="80">0031460 1892914</td>
			<td class="xl65" style="width:60pt" width="80">WB: 1:20.000 in 1% skim milk in TBS</td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl64" height="106" style="height:79.5pt;width:60pt" width="80">polyclonal peroxidase-conjugated goat anti-mouse</td>
			<td class="xl63" style="width:60pt" width="80">Thermo Fisher</td>
			<td class="xl63" style="width:60pt" width="80">0031430 1892913</td>
			<td class="xl63" style="width:60pt" width="80">WB: 1:20.000 in 1% skim milk in TBS</td>
		</tr>
	</tbody>
</table>

monitoring activation of the antiviral pattern recognition receptors rig-i and pkr by limited protease digestion and native page

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Recognition of a viral agonist by RIG-I or PKR triggers conformational switching6,17,30 and oligomerization6,18,27. We assayed these two activation markers by limited protease digestion and native polyacrylamide gel electrophoresis (PAGE), respectively.
Human A549 cells were infected with Rift Valley fever virus clone 13 (Cl 13), which is characterized by a mutation of the IFN antagonist NSs35,36. Due to the absence of functional NSs, Clone 13 strongly induces ...

Watch this Scientific Journal Video about Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE at JoVE.com

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

monitoring-activation-antiviral-pattern-recognition-receptors-rig-i

Research

JoVE Journal

Immunology and Infection

12.0K Views.  Philipps-University Marburg. Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.