Department of Microbiology and Immunology and Center for Microbial Pathogenesis and Host Inflammatory Responses, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
Gundersen Health System, La Crosse, Wisconsin, United States of America.
PLoS Pathog. 2018 Jan 24;14(1):e1006865. doi: 10.1371/journal.ppat.1006865. eCollection 2018 Jan.
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.
γ疱疹病毒 (GHV) 的发病机制是一个复杂的过程，涉及病毒的有效复制、传播到潜伏感染的组织以及从潜伏状态重新激活到有效复制周期。利用小鼠γ疱疹病毒 68 (MHV68) 在小鼠中进行的传统功能丧失性诱变方法是一种模型，可以在体内研究 GHV 的发病机制，对于确定 GHV 感染中特定病毒基因产物的要求非常有价值。但是，这些方法不足以充分揭示病毒基因产物在编码蛋白促进感染周期中的多个过程时，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而变化时如何发挥作用。为了解决这个复杂性，我们开发了一种 MHV68 遗传平台，使体内能够进行细胞类型特异性和诱导性病毒基因缺失。我们利用该系统重新评估了 MHV68 潜伏相关核抗原 (mLANA) 的功能，该蛋白在病毒复制和潜伏中都有作用。在小鼠中，通过 Cre 介导的loxP 侧翼 ORF73 的缺失证明了 mLANA 在 B 细胞中对于 MHV68 潜伏期建立的必要性。在 mLANA 缺失后，从引流淋巴结到血液的潜伏期转变也受到了损害，这支持了 B 细胞是病毒传播的主要途径的假说。在感染的生发中心 (GC) B 细胞中缺失 mLANA 严重损害了病毒的潜伏，这表明病毒通过 GC 传播对于潜伏期的建立非常重要。最后，在潜伏期期间诱导性缺失 mLANA 导致受影响的病毒基因组完全丢失，表明 mLANA 对于稳定潜伏期间病毒基因组的维持至关重要。总之，这些实验为 LANA 同源物在 GHV 宿主定殖中的功能提供了新的见解，并突出了新的 MHV68 遗传平台在促进对 GHV 发病机制的不同阶段的病毒基因功能的更全面理解方面的潜力。
