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
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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>
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			<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>
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			<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>
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			<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>
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			<td colspan="2" height="21" style="height:21px;">CCL5 quantitative real-time PCR primers</td>
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			<td height="20" style="height:20px;">CCTGCTGCTTTGCCTACCTCTC</td>
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			<td height="20" style="height:20px;">ACACACTTGGCGGTTCCTTCGA</td>
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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. ...

dissecting-innate-immune-signaling-viral-evasion-cytokine

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JoVE Journal

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...