Mechanisms of Neuronal Spread of Neurotropic Mouse Hepatitis Virus

描述（由申请人提供）：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。与从大脑扩散到身体其他部位的弱神经毒性的MHV A59株不同，CNS适应性的JHM分离株具有极强的神经毒性，但在CNS外复制能力差，具有两个菌株特异性和明显独立的神经毒性特征：在缺乏典型MHV受体Ceacam1a的神经元中传播的能力，以及在大脑中复制而不引起抗原特异性CD8+ t细胞反应的能力。根据初步数据，我假设这两种特征都源于JHM对神经元间扩散的特化，通过限制病毒传播到中枢神经系统和/或引流淋巴结的抗原呈递细胞，减少了CD8+ t细胞反应。我建议更好地定义这种专门化的机制，以提高对潜在神经毒性病毒的识别，研究JHM分离株在神经回路追踪方面的潜力，并提高我们对中枢神经系统衍生抗原有效呈递要求的理解。我将首先检查C57BL/6和ceacam1a-/-小鼠的神经毒力，在C57BL/6和ceacam1a-/-神经元培养物中的传播能力，以及在C57BL/6小鼠鼻内接种在非小鼠细胞中具有受体非依赖性传播（RIS）缺陷的突变型JHM病毒后的传播模式，以确定ceacam1a非依赖性神经元传播是否仅需要RIS或神经元特异性传播机制。然后，我将检查突触间的传播，隔离培养中神经元细胞体和轴突的病毒释放，以及JHM、A59、rA59/SJHM（与JHM刺突基因嵌合的A59病毒）感染神经元的膜融合，如果相关，还将检查培养的原代C57BL/6神经元中的RIS突变体，以确定JHM是否在神经元间传播。接下来，我将研究是否JHM在颅内（i.c）接种后不能在大脑中诱导CD8+ t细胞反应，因为它不能扩散到颅内或颅外的抗原呈递细胞。首先，我将使用各种RNA，蛋白质和功能分析来确认JHM感染激活骨髓来源的树突状细胞（dc）进行抗原呈递。接下来，我将采用淋巴脉络丛脑膜炎病毒（LCMV） gp33抗原特异性T细胞转染C57BL/6小鼠，同时腹腔注射表达LCMV gp33表位的JHM (JHM-gp33)，并评估脑内抗gp33 CD8+ T细胞反应，以确定颅外病毒复制是否能挽救颅内CD8+ T细胞反应。最后，我将过继转移小鼠接种野生型JHM，注射dc

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.