Name: dissecting host-virus interaction in lytic replication of a model herpesvirus
Uploaded: 2011-10-07T12:00:00
Description: Watch this Scientific Journal Video about Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus at JoVE.com

The authors would like to thank Dr. James (Zhijian) Chen (UT Southwestern, Molecular Biology) for providing essential reagents, including the Mavs-/- mice, and Dr. Ren Sun (University of California-Los Angeles, Pharmacology and Molecular Medicine) for providing the bacterial artificial chromosome of &gamma;HV68 for this study.

1. Mouse infection with &gamma;HV68
<ol>
	<li>Six-to-eight-week old, gender-matched littermate mice (8 to 12 mice/group) are used for viral infection. Allow mice to acclimate over four full days (96 hours) after shipment.</li>
	<li>Protocol steps using virus should be carried out in a cabinet of biosafety level 2 (BSL2) using standard BSL2 precautions.</li>
	<li>Prepare viral suspension (40 to 1 x 105 plaque-forming units [PFU]) of &gamma;HV68 in 30 &mu;l of sterile PBS per mouse just before the experiment. ...

<ol>
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In response to viral infection, the MAVS-dependent innate immune signaling pathways are activated to promote the production of antiviral inflammatory cytokines10-14. Using murine &gamma;HV68 as a model virus for human oncogenic Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus3,4, we discovered that &gamma;HV68 usurps the MAVS-IKK&beta; pathway to promote viral lytic replication via transcriptional activation5. Employing genetically modified MEFs and techniques in molecular v...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
		</tr>
		<tr>
			<td>Electro-MAX DH10B competent cells</td>
			<td>Invitrogen</td>
			<td>18290-015</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Sigma</td>
			<td>M0512</td>
		</tr>
		<tr>
			<td>POWERPREP HP Plasmid Miniprep System</td>
			<td>OriGene</td>
			<td>NP100004</td>
		</tr>
		<tr>
			<td>POWERPREP HP Plasmid Midiprep System</td>
			<td>OriGene</td>
			<td>NP100006</td>
		</tr>
	</tbody>
</table>

dissecting host-virus interaction in lytic replication of a model herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

Watch this Scientific Journal Video about Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus at JoVE.com

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

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