Dong Xiaonan, Feng Pinghui
Center for Autophagy Research, Department of Internal Medicine, UT Southwestern Medical Center, USA.
J Vis Exp. 2011 Oct 7(56):3140. doi: 10.3791/3140.
In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production. 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 γHV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus. γHV68 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 γHV68 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 γHV68 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 γHV68 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 γHV68 carrying these mutations can be used to recapitulate the phenotypes of γHV68 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 γHV68 usurps an innate immune signaling pathway to promote viral lytic replication. Specifically, γHV68 de novo infection activates the immune kinase IKKβ and activated IKKβ phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, γHV68 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.
针对病毒感染，宿主会产生多种防御反应，例如激活导致抗病毒细胞因子产生的固有免疫信号通路。为了在宿主体内定植，病毒必须逃避宿主的抗病毒反应并操纵信号通路。阐明宿主与病毒的相互作用将有助于开发针对病毒感染的新型治疗策略。小鼠γ疱疹病毒68（Murine γHV68）与人类致癌性卡波西肉瘤相关疱疹病毒和爱泼斯坦-巴尔病毒密切相关。在实验室小鼠中进行γHV68感染可提供一个易于处理的小动物模型，用于在体内研究宿主反应和病毒感染的整个过程，而这对于人类疱疹病毒来说是无法实现的。在本方案中，我们介绍了一系列用于在体内和体外对γHV68裂解复制过程中的宿主信号成分进行表型特征分析和分子剖析的方法。基因工程改造小鼠品系的可用性使得我们能够研究宿主信号通路在γHV68体内急性感染过程中的作用。此外，从这些缺陷小鼠品系中分离出的小鼠胚胎成纤维细胞（MEFs）可用于在体外进一步剖析这些分子在γHV68裂解复制过程中的作用。通过病毒学和分子生物学检测，我们可以确定宿主与病毒相互作用的分子机制，并鉴定出病毒裂解复制所必需的宿主和病毒基因。最后，细菌人工染色体（BAC）系统有助于将突变引入特定干扰宿主与病毒相互作用的病毒因子中。携带这些突变的重组γHV68可用于在缺乏关键宿主信号成分的MEFs中重现γHV68裂解复制的表型。本方案提供了一个在体内和体外多个干预层面研究宿主-病原体相互作用的优秀策略。最近，我们发现γHV68利用一种固有免疫信号通路来促进病毒裂解复制。具体而言，γHV68的初次感染激活免疫激酶IKKβ，激活的IKKβ使主要病毒转录因子复制与反式激活因子（RTA）磷酸化，从而促进病毒转录激活。通过这种方式，γHV68有效地将其转录激活与宿主固有免疫激活联系起来，从而促进病毒转录和裂解复制。这项研究提供了一个可应用于其他病毒以研究宿主-病毒相互作用的优秀范例。
