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

Авторы хотели бы поблагодарить д-ра Джеймса (Zhijian) Чэнь (UT Southwestern, молекулярная биология) для обеспечения основных реагентов, в том числе Mavs - / - мышей, и д-р Рене Sun (Калифорнийский университет в Лос-Анджелесе, фармакологии и молекулярной медицины ) для обеспечения бактериальные искусственные хромосомы γHV68 для данного исследования.

1. Мышь инфекции γHV68 <ol><li> Шесть-восемь недель старые, соответствующего пола однопометница мышей (8 до 12 мышей / группа) используются для вирусной инфекции. Разрешить мышей, чтобы акклиматизироваться в течение четырех суток (96 часов) после отгрузки. </li><li> Протокол шаги с помощью вируса до?...

<ol>
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В ответ на вирусную инфекцию, MAVS зависит от врожденной иммунной сигнальных путей, которые активируются, чтобы содействовать производству антивирусных цитокинов 10-14. Использование мышиный γHV68 в качестве модели вируса саркомы связанных вирус герпеса человека онкогенных Капоши и ...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td></tr><tr><td> Липофектамина 2000 </td><td> Invitrogen </td><td> 11668-019 </td></tr><tr><td> Электро-MAX DH10B компетентных клеток </td><td> Invitrogen </td><td> 18290-015 </td></tr><tr><td> Метилцеллюлоза </td><td> Сигма </td><td> M0512 </td></tr><tr><td> POWERPREP HP плазмиды системы Miniprep </td><td> OriGene </td><td> NP100004 </td></tr><tr><td> POWERPREP HP плазмиды системы Midiprep </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

Мы описываем протокол определить ключевые роли принимающей сигнальных молекул в литическая репликация вируса герпеса модели, гамма-герпесвирусов 68 (γHV68). Использование генетически модифицированных мышей штамма эмбриональных фибробластов и для γHV68 литической репликации, протокол позволяет как фенотипическая характеристика и молекулярно допроса вирус-хозяин взаимодействий в вирусную литической репликации.

Пройдя Host-вирус Взаимодействие в Литические репликации Модель Herpesvirus

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

Research

JoVE Journal

Immunology and Infection

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV

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Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

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