Mechanisms of Neuronal Spread of Neurotropic Mouse Hepatitis Virus

描述(申请人提供)：小鼠肝炎病毒(MHV)感染小鼠中枢神经系统(CNS)被广泛用于模拟人类脑炎和多发性硬化症。与神经毒力弱的MHV A59毒株从大脑传播到身体其他部位不同，中枢神经系统适应的JHM毒株具有极强的神经毒力，但在中枢神经系统外复制能力很差，有两个毒株特有且明显独立的神经毒力特征：能够在缺乏规范MHV受体Ceacam1a的神经元之间传播，以及能够在大脑中复制，而不会引发抗原特异性CD8 T细胞反应。根据初步数据，我推测这两个特征都源于JHM对神经元间传播的专门化，JHM通过将病毒传播限制在中枢神经系统的抗原提呈细胞和/或引流淋巴结来降低CD8T细胞的反应。我建议更好地定义这种特化的机制，以便更好地识别潜在的神经毒力病毒，研究JHM分离株用于神经元回路追踪的潜力，并提高我们对有效呈递中枢神经系统衍生抗原的要求的理解。我将首先检测C57BL/6和ceacam1a-/-小鼠的神经毒力，在C57BL/6和ceacam1a-/-神经元培养中的传播能力，以及鼻腔内传播的方式。在C57BL/6小鼠中接种在非小鼠细胞中存在受体非依赖性传播(RIS)缺陷的突变JHM病毒，以确定Ceacam1a在神经元中的非依赖性传播是否仅需要RIS或神经元特有的传播机制。然后，我将检测跨突触的传播，隔室培养中神经元细胞体和轴突中病毒的释放，以及JHM、A59、rA59/SJHM(嵌合A59病毒和JHM刺突基因的嵌合病毒)感染神经元的膜融合，以及培养的原代C57BL/6神经元中的RIS突变体，以确定JHM是否通过神经元间传播。接下来，我将调查JHM在颅内(I.C.)后是否未能在大脑中诱导CD8 T细胞反应。接种，因为它不会扩散到颅内外的黑素呈现细胞。首先，我将使用各种RNA、蛋白质和功能分析来证实JHM感染激活了骨髓来源的树突状细胞(DC)，用于抗原呈递。接下来，我将感染过继转移淋巴脉络膜脑膜炎病毒(LCMV)gp33抗原特异性T细胞的C57BL/6小鼠。和腹膜内注射(Ip)。用表达LCMV gp33表位的JHM(JHM-gp33)检测脑内抗gp33 CD8 T细胞应答，以确定颅外病毒复制是否挽救了颅内CD8 T细胞应答。最后，我将过继转移的小鼠免疫接种。使用野生型JHM，注射DC 感染JHM-gp33I.C.，并评估脑内gp33特异性T细胞反应，以确定受感染的DC呈递颅内抗原是否拯救了颅内CD8反应。这些实验将极大地提高我们对神经毒力的理解，并为探索神经元间病毒传播和中枢神经系统抗原递呈开辟新的途径。 公共卫生相关性：小鼠肝炎病毒(MHV)感染小鼠中枢神经系统(CNS)被广泛用于模拟人类的脑炎和多发性硬化症。这两种疾病过程都受到大脑中神经元间传播的效率和抗病毒CD8 T细胞反应的影响。这项拟议的项目将通过定义MHV极具神经毒力的分离株在中枢神经系统传播的适应影响CD8 T细胞反应的神经元间传播和启动的方式来增强我们对这些模型系统的理解。

DESCRIPTION (provided by applicant): Mouse hepatitis virus (MHV) infection of the mouse central nervous system (CNS) is widely used to model both encephalitis and multiple sclerosis in humans. Unlike the weakly neurovirulent A59 strain of MHV, which spreads from the brain to other parts of the body, the CNS-adapted JHM isolates, which are extremely neurovirulent but replicate poorly outside the CNS, share two strain-specific and apparently independent neurovirulence traits: the ability to spread among neurons lacking the canonical MHV receptor Ceacam1a and the ability to replicate in the brain without eliciting an antigen-specific CD8+ T-cell response. Based on preliminary data, I hypothesize that both of these traits stem from the specialization of JHM for interneuronal spread, which reduces the CD8+ T-cell response by limiting viral spread to antigen-presenting cells in the CNS and/or draining lymph nodes. I propose to better define the mechanisms of this specialization in order to improve identification of potentially neurovirulent viruses, investigate the potential of JHM isolates for neuronal circui tracing, and improve our understanding of the requirements for effective presentation of CNS-derived antigen. I will first examine the neurovirulence in C57BL/6 and ceacam1a-/- mice, the abilities to spread in C57BL/6 and ceacam1a-/- neuron cultures, and the patterns of spread following intranasal (i.n.) inoculation in C57BL/6 mice of mutant JHM viruses defective for receptor-independent spread (RIS) in non- murine cells in order to determine whether Ceacam1a-independent spread in neurons requires merely RIS or a neuron-specific mechanism of spread. I will then examine the spread across synapses, release of virus from neuronal cell bodies and axons in compartmented cultures, and membrane fusion of infected neurons of JHM, A59, rA59/SJHM (a chimeric A59 virus with the JHM spike gene), and, if relevant, the RIS mutants in cultured primary C57BL/6 neurons to determine whether JHM spreads interneuronally. I will next investigate whether JHM fails to induce a CD8+ T-cell response in the brain after intracranial (i.c.) inoculation because it fails to spread to intra- or extracranial anigen-presenting cells. First, I will use a variety of RNA, protein, and functional assays to confirm tha JHM infection activates bone marrow-derived dendritic cells (DCs) for antigen presentation. Next, I will infect C57BL/6 mice adoptively transferred with lymphochoriomeningitis virus (LCMV) gp33 antigen-specific T cells simultaneously i.c. and intraperitoneally (i.p.) with LCMV gp33 epitope-expressing JHM (JHM-gp33) and evaluate the anti-gp33 CD8+ T cell response in the brain to determine whether extracranial viral replication rescues the intracranial CD8+ T cell response. Finally, I will inoculate adoptively transferred mice i.n. with wild-type JHM, inject DCs infected with JHM-gp33 i.c., and evaluate the gp33-specific T cell response in the brain to determine whether intracranial antigen presentation by infected DCs rescues the intracranial CD8+ response. These experiments will greatly enhance our understanding of neurovirulence and open new avenues of exploration of interneuronal viral spread and CNS antigen presentation. PUBLIC HEALTH RELEVANCE: Mouse hepatitis virus (MHV) infection of the mouse central nervous system (CNS) is widely used to model encephalitis and multiple sclerosis in humans. Both of these disease processes are affected by the efficiency of interneuronal spread and the antiviral CD8+ T cell response in the brain. The proposed project will enhance our understanding of these model systems by defining the ways in which the adaptations of an extremely neurovirulent isolate of MHV to spread in the CNS influence both interneuronal spread and priming of the CD8+ T cell response.