Wild-type (Mavs+/+) and knockout (Mavs&#8722;/&#8722;) mice, Wild-type (Mavs+/+) and knockout (Mavs&#8722;/&#8722;) MEFs were kindly provided by Dr. Zhijian J. Chen (University of Texas Southwestern Medical Center)13. This publication is based on work funded by grants from NIH (R01 CA134241 and R01 DE021445) and American Cancer Society (RSG-11-162-01-MPC).

Ethics Statement: All animal work was performed under strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Southern California.
1.&#160;Cytokine Gene Expression by Quantitative Real-time PCR and Secretion by ELISA&#160;in &#947;HV68-infected Mice
<ol>
	<li>Anesthetize gender-matched, 6-8&#160;week-old C57BL/6 (B6) littermates (8-12 mice/group) via intraperitoneally injecting 1.5 mg of ketamine/0.12 mg of xylazine, and inoculate intranasally with 40 PFU of &#947;HV68 in 40 &#181;l (infection detailed in Dong and Feng11). At different time points post-infection, euthanize the mice by using&#160;CO2 inhalation&#160;followed by cervical dislocation.</li>
	<li>Open the chest with a left lateral cut along the sternum and cut through the ribs with sharp scissors. Collect the lung tissues.</li>
	<li>Collect half of the lung tissue (left lobe) into sterile 1.5 ml screw-capped tubes containing 500 &#181;l 1.0 mm Zirconia/Silica beads. Add 1 ml of cold serum-free DMEM into the tube and homogenize the lung tissue by bead-beating for 30 sec.&#160;Chill tubes on ice for 2 min and repeat this process once.</li>
	<li>Centrifuge the tubes (15,000 x g for 10 min) at 4 &#176;C, collect the supernatant and measure the cytokine production as described below.</li>
	<li>Measure cytokines using commercially available cytokine ELISA kits according to the manufacturer&#39;s instruction.</li>
	<li>Collect the other half of the lung tissue (right lobe) into another bead-containing tube and add 1 ml TRIzol. Homogenize and collect supernatant as described in step 1.3 and extract RNA according to the manufacture&#39;s instruction. Determine RNA concentration by taking absorbance readings at 260 and 280 nm.</li>
	<li>Prepare cDNA with 1 &#181;g of total RNA and reverse transcriptase according to the manufacture&#39;s instruction (the total final volume was 20 &#181;l). Dilute cDNA 50 times with DNAse- and RNAse-free water and perform quantitative real-time PCR.</li>
	<li>Perform the program on a quantitative real-time PCR machine. Each reaction contains 0.5 &#181;l&#160;of a pair of gene-specific primers or control primers of GAPDH (the working concentration of the primers were 10 &#181;M, CE 500 nM for each primer), 5 &#181;l&#160;of the SYBR master mix and 4 &#181;l&#160;of the diluted cDNA. Calculate &#916;&#916;Ct to determine the relative quantity of mRNA transcripts of antiviral cytokines.</li>
</ol>
2.&#160;MEF Cell Infection and Cytokine Quantification by ELISA and qRT-PCR
<ol>
	<li>Grow Mavs+/+ and Mavs-/- MEF cells to sub-confluent (approximately 80%) density before plating cells.</li>
	<li>Split MEF cells into 12-well plates at 100,000 cells/well (the cell density will be around 70% on the second day). Carry out &#947;HV68 infection in triplicates, with a multiplicity of infection (MOI) of 5-10 to synchronize and maximize cytokine induction.</li>
	<li>Prepare &#947;HV68 suspension in complete DMEM medium containing appropriate amount of virus (1 ml for each well).</li>
	<li>Remove medium and add &#947;HV68-containing suspension to MEF cells.</li>
	<li>Place the plate in tissue culture incubator, rock every 30 min, and allow a total of 2 hr of incubation.</li>
	<li>Remove &#947;HV68-containing medium, wash once with PBS, and replace with fresh complete DMEM.</li>
	<li>At various time points post-infection, harvest medium into 1.5 ml Eppendorf tubes. Wash cells with cold PBS and collect infected cells by trypsin digestion.</li>
	<li>Measure antiviral cytokines in medium as described in step 1.5.</li>
	<li>Extract RNA, prepare cDNA and perform qRT-PCR to determine antiviral cytokine gene expression as described in steps 1.5-1.7.</li>
</ol>
3.&#160;Lentivirus Production, Infection, and Generation of Stable Cell Lines
<ol>
	<li>Split 293T cells one day before transfection and allow the cells to reach ~40% confluency at the time of transfection.</li>
	<li>Transfect 10 cm dish of 293T cells with the packaging plasmids (1.2 &#181;g of pVSV-G and 6 &#181;g of pDR8.9) and 7.2 &#181;g of pCDH-FLAG-MAVS or pCDH control by calcium phosphate precipitation.</li>
	<li>Replace medium at 6 to 8 hr post-transfection with fresh complete DMEM.</li>
	<li>At 72 hr post-transfection, collect medium containing lentivirus, centrifuge at 4,000 x g for 15 min, and filter with 0.22 &#181;m membrane. To increase the lentivirus titer, we centrifuge the virus-containing medium at 110,000 x g for 2.5 hr at 4 &#176;C. Carefully resuspend the viral pellet in small volume of medium of interest.</li>
	<li>Seed Mavs-/- MEF cells or other knockout MEF cells one day before infection in 6-well&#160;plates at ~30-40% confluency.</li>
	<li>Infect MEF cells with 1 ml lentivirus and 2 ml fresh complete DMEM, supplemented with polybrene to a final concentration of 10 &#181;g/ml.</li>
	<li>Centrifuge the plate at 500 x g at 30 &#176;C for 30 min and transfer the plate to a tissue culture incubator.</li>
	<li>Replace medium at 6 hr post-infection with fresh complete DMEM.</li>
	<li>Split MEF cells at 24 hr post-infection and add puromycin to a final concentration of 1 &#181;g/ml at 48 hr post-infection to select cells stably expressing MAVS.</li>
	<li>Maintain MEF cells in puromycin-containing medium and verify protein expression by immunoblotting with corresponding antibodies.</li>
	<li>Cells can be used for viral infection, cytokine gene expression and secretion experiments as described in Protocol 2.</li>
</ol>

<ol>
	<li>Akira, S., Uematsu, S., Takeuchi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+recognition+and+innate+immunity.">Pathogen recognition and innate immunity.</a> Cell. 124, 783-801 (2006).</li><li>Kato, H., Takahasi, K., Fujita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIG-I-like+receptors:+cytoplasmic+sensors+for+non-self+RNA.">RIG-I-like receptors: cytoplasmic sensors for non-self RNA.</a> Immunol. Rev. 243, 91-98 (2011).</li><li>Kawai, T., 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=IPS-1,+an+adaptor+triggering+RIG-I-+and+Mda5-mediated+type+I+interferon+induction.">IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.</a> Nat. Immunol. 6, 981-988 (2005).</li><li>Seth, R. B., Sun, L., Ea, C. K., Chen, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+MAVS,+a+mitochondrial+antiviral+signaling+protein+that+activates+NF-kappaB+and+IRF+3.">Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.</a> Cell. 122, 669-682 (2005).</li><li>Xu, L. G., 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=VISA+is+an+adapter+protein+required+for+virus-triggered+IFN-beta+signaling.">VISA is an adapter protein required for virus-triggered IFN-beta signaling.</a> Mol. Cell. 19, 727-740 (2005).</li><li>Meylan, E., 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=Cardif+is+an+adaptor+protein+in+the+RIG-I+antiviral+pathway+and+is+targeted+by+hepatitis+C+virus.">Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.</a> Nature. 437, 1167-1172 (2005).</li><li>Speck, S. H., Virgin, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+and+viral+genetics+of+chronic+infection:+a+mouse+model+of+gamma-herpesvirus+pathogenesis.">Host and viral genetics of chronic infection: a mouse model of gamma-herpesvirus pathogenesis.</a> Curr. Opin. Microbiol. 2, 403-409 (1999).</li><li>Dong, X., 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=Murine+gamma-herpesvirus+68+hijacks+MAVS+and+IKKbeta+to+initiate+lytic+replication.">Murine gamma-herpesvirus 68 hijacks MAVS and IKKbeta to initiate lytic replication.</a> PLoS Pathog. , (2010).</li><li>Dong, X., Feng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gamma+herpesvirus+68+hijacks+MAVS+and+IKKbeta+to+abrogate+NFkappaB+activation+and+antiviral+cytokine+production.">Murine gamma herpesvirus 68 hijacks MAVS and IKKbeta to abrogate NFkappaB activation and antiviral cytokine production.</a> PLoS Pathog. 7, (2011).</li><li>Dong, X., 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=Murine+gammaherpesvirus+68+evades+host+cytokine+production+via+replication+transactivator-induced+RelA+degradation.">Murine gammaherpesvirus 68 evades host cytokine production via replication transactivator-induced RelA degradation.</a> J. Virol. 86, 1930-1941 (2012).</li><li>Dong, X., Feng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+Host-virus+Interaction+in+Lytic+Replication+of+a+Model+Herpesvirus.">Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus.</a> J. Vis. Exp. , (2011).</li><li>He, 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=Receptor+interacting+protein+kinase-3+determines+cellular+necrotic+response+to+TNF-alpha.">Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.</a> Cell. 137, 1100-1111 (2009).</li><li>Sun, Q., 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+specific+and+essential+role+of+MAVS+in+antiviral+innate+immune+responses.">The specific and essential role of MAVS in antiviral innate immune responses.</a> Immunity. 24, 633-642 (2006).</li></ol>

The authors declare no conflicts of interest.

Viral immune evasion is one of the most dynamic and fascinating interactions interfacing viral offense and host defense9. The host innate immune components are structured such that signal transduction is effectively initiated and faithfully transmitted. Delineating the hierarchy and regulation of signaling cascades is a preeminent topic of innate immunity. Here, we introduce a protocol to identify the regulatory roles of an innate immune component, MAVS, in viral evasion of cytokine production. The protocol co...

Recent studies have outlined the overall signaling cascades in mounting host innate immune responses. Residing within distinct compartments, pattern recognition receptors (PRRs) detect pathogen-associated molecular patterns (PAMPs) of diverse origin to trigger innate immune signaling1.&#160;The retinoic acid-induced gene I (RIG-I) and melanoma differentiation antigen 5 (MDA5) proteins are cytosolic sensors that recognize RNA species with specific structural features2. Upon activation, RIG-I interacts with its downstream MAVS (also known as IPS-1, VISA, and CARDIF) adaptor that, in turn, activates the IKK (IKK&#945;&#946;&#947;) and IKK-related kinase (TBK1 and IKK&#949;, also known as IKKi) complexes3-6. Activated innate immune kinases phosphorylate key regulators of gene expression, including transcription factors and inhibitors thereof, and enable transcriptional activation of host antiviral genes (e.g.&#160;IL6, TNF&#945;, CCL5, and IFN&#946;). These signaling cascades constitute potent intrinsic innate immune responses that establish an anti-microbial state to restrict pathogen propagation during early stages of infection.
Murine &#947;HV68 is closely related to human oncogenic KSHV and EBV. Thus, &#947;HV68 infection of mice provides a tractable small animal model to examine the host immune response to gamma herpes virus infection&#160;in vivo7. Using &#947;HV68, our lab has uncovered an intricate strategy whereby &#947;HV68 hijacks host innate immune signaling to enable viral infection. On one hand, &#947;HV68 activates the MAVS-IKK&#946; pathway to promote viral transcriptional activation via directing activated IKK&#946; to phosphorylate replication transactivator (RTA), the viral transcription factor key for &#947;HV68 replication8. On the other hand, the IKK&#946;-mediated phosphorylation primes RelA for degradation and terminates NF&#922;B activation9. As such, &#947;HV68 infection effectively avoids antiviral cytokine production. Interestingly, a screening utilizing an expression library of &#947;HV68 identified RTA as an E3 ligase to induce RelA degradation and abrogate NF&#922;B activation10. These findings uncover an intricate immune evasion strategy whereby the upstream immune signaling events are harnessed by &#947;HV68 to negate the ultimate antiviral cytokine production.
Here we describe a protocol to measure the antiviral cytokine production in mice infected with &#947;HV68 both in vivo and ex vivo. In the protocol we further explore the &#34;reconstituted&#34; expression of innate immune components in the knockout embryonic fibroblasts by lentiviral transduction, which pinpoints the function of the specific innate immune molecules in regulating the antiviral cytokine production. This protocol can be easily adapted to other viruses and signaling pathways.

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			<td height="20" style="height:20px;width:251px;">Mouse CCL5 ELISA kit</td>
			<td style="width:211px;">R&#38;D systems</td>
			<td style="width:223px;"><a href="http://www.rndsystems.com/Products/DY478">DY478</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">1.0 mm Zirconia/Silica beads</td>
			<td style="width:211px;">BioSpec Products</td>
			<td style="width:223px;">11079110z</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">TRIzol</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:223px;"><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=15596018">15596-018</a></td>
		</tr>
		<tr height="79">
			<td height="98" rowspan="2" style="height:98px;width:251px;">SuperScript II Reverse Transcriptase</td>
			<td rowspan="2" style="width:211px;">Invitrogen</td>
			<td rowspan="2" style="width:223px;">18064-014</td>
		</tr>
		<tr height="19">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"> </td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">CCL5 quantitative real-time PCR primers</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCTGCTGCTTTGCCTACCTCTC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ACACACTTGGCGGTTCCTTCGA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dissecting innate immune signaling in viral evasion of cytokine production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

Three representative figures are shown here, including cytokine production in the lung of &#947;HV68-infected Mavs+/+ and Mavs-/- mouse, cytokine secretion and gene expression level of &#947;HV68-infected Mavs+/+ and Mavs-/- MEFs, and cytokine mRNA levels of &#947;HV68-infected Mavs-/- MEFs &#34;reconstituted&#34; with MAVS. These representative experiments utilize gene knockout mice to investigate antiviral cytokine produ...

Watch this Scientific Journal Video about Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production at JoVE.com

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

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Immunology and Infection

10.4K Views.  Keck School of Medicine, University of Southern California. We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (γHV68) that is closely-related to human Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine p...