Conditional mutagenesis in vivo reveals cell type- and infection stage-specific requirements for LANA in chronic MHV68 infection.
Conditional mutagenesis in vivo reveals cell type- and infection stage-specific requirements for LANA in chronic MHV68 infection.
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DOI：
10.1371/journal.ppat.1006865
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发表时间：
2018-01
期刊：
影响因子：
6.7
通讯作者：
Forrest JC
中科院分区：
文献类型：
--
作者：
Salinas E;Gupta A;Sifford JM;Oldenburg DG;White DW;Forrest JC
伽玛疱疹病毒（GHV）的发病机制是一个复杂的过程，包括病毒增殖、传播到具有终身潜伏感染的组织，以及从潜伏期重新激活到增殖复制周期。使用小鼠γ疱疹病毒68 (MHV68)（一种允许在体内检查GHV发病机制的模型）对小鼠进行传统的功能丧失诱变方法，对于确定GHV感染中特定病毒基因产物的需求具有宝贵的价值。但是，这些方法不足以充分揭示当编码蛋白促进感染周期中的多个过程以及这些功能随时间和从一个宿主组织到另一个宿主组织变化时，病毒基因产物是如何起作用的。为了解决这一复杂性，我们开发了一个MHV68遗传平台，可以在体内进行细胞类型特异性和可诱导的病毒基因删除。我们使用该系统重新评估MHV68潜伏期相关核抗原（mLANA）的功能，mLANA是一种在病毒复制和潜伏期中都起作用的蛋白质。cre介导的小鼠loxp侧ORF73缺失证明了B细胞中mLANA对MHV68潜伏期建立的必要性。在mLANA缺失后，还观察到从引流淋巴结到血液过渡的潜伏期受损，这支持了B细胞是病毒传播主要渠道的假设。在感染的生发中心（GC） B细胞中消融mLANA严重损害了病毒潜伏期，表明病毒通过GC对潜伏期建立的重要性。最后，在潜伏期诱导消融mLANA导致受影响的病毒基因组完全丢失，这表明mLANA对病毒基因组在稳定潜伏期的维持至关重要。总的来说，这些实验提供了LANA同源功能在GHV定植宿主中的新见解，并强调了新的MHV68遗传平台的潜力，以促进对GHV发病过程中离散阶段病毒基因功能的更全面了解。伽玛疱疹病毒（ghv），包括人类病原体爱泼斯坦-巴尔病毒和卡波西肉瘤相关疱疹病毒，建立可导致癌症的终身感染。确定病毒基因产物在急性复制和慢性感染中的功能对于了解这些病毒如何引起疾病非常重要。用相关GHV小鼠γ疱疹病毒68 （MHV68）感染小鼠，为确定病毒基因产物在慢性感染中的作用和了解宿主因素如何限制疾病提供了一种可处理的小动物模型。在这里，我们描述了一种新的病毒遗传平台的发展，该平台能够在离散宿主细胞或感染期间的不同时间从病毒基因组中靶向删除特定基因。我们利用该系统来更好地定义小鼠MHV68裂解复制和潜伏期中对保守性潜伏期相关核抗原的要求。这项工作强调了MHV68遗传平台在确定GHV感染和疾病机制方面的实用性。
Gammaherpesvirus (GHV) pathogenesis is a complex process that involves productive viral replication, dissemination to tissues that harbor lifelong latent infection, and reactivation from latency back into a productive replication cycle. Traditional loss-of-function mutagenesis approaches in mice using murine gammaherpesvirus 68 (MHV68), a model that allows for examination of GHV pathogenesis in vivo, have been invaluable for defining requirements for specific viral gene products in GHV infection. But these approaches are insufficient to fully reveal how viral gene products contribute when the encoded protein facilitates multiple processes in the infectious cycle and when these functions vary over time and from one host tissue to another. To address this complexity, we developed an MHV68 genetic platform that enables cell-type-specific and inducible viral gene deletion in vivo. We employed this system to re-evaluate functions of the MHV68 latency-associated nuclear antigen (mLANA), a protein with roles in both viral replication and latency. Cre-mediated deletion in mice of loxP-flanked ORF73 demonstrated the necessity of mLANA in B cells for MHV68 latency establishment. Impaired latency during the transition from draining lymph nodes to blood following mLANA deletion also was observed, supporting the hypothesis that B cells are a major conduit for viral dissemination. Ablation of mLANA in infected germinal center (GC) B cells severely impaired viral latency, indicating the importance of viral passage through the GC for latency establishment. Finally, induced ablation of mLANA during latency resulted in complete loss of affected viral genomes, indicating that mLANA is critically important for maintenance of viral genomes during stable latency. Collectively, these experiments provide new insights into LANA homolog functions in GHV colonization of the host and highlight the potential of a new MHV68 genetic platform to foster a more complete understanding of viral gene functions at discrete stages of GHV pathogenesis. Gammaherpesviruses (GHVs), including the human pathogens Epstein-Barr virus and Kaposi sarcoma-associated herpesvirus, establish lifelong infections that can lead to cancer. Defining the functions of viral gene products in acute replication and chronic infection is important for understanding how these viruses cause disease. Infection of mice with the related GHV, murine gammaherpesvirus 68 (MHV68), provides a tractable small animal model for defining how viral gene products function in chronic infection and understanding how host factors limit disease. Here we describe the development of a new viral genetic platform that enables the targeted deletion of specific genes from the viral genome in discrete host cells or at distinct times during infection. We utilize this system to better define requirements for the conserved latency-associated nuclear antigen in MHV68 lytic replication and latency in mice. This work highlights the utility of this MHV68 genetic platform for defining mechanisms of GHV infection and disease.
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