MUTATIONS AND DELETIONS OF TMEV SURFACE RESIDUES

这项拟议研究的总体目标是阐明分子 泰勒氏小鼠脑脊髓炎病毒-细胞相互作用的基础 病毒(TMEV)诱导的脱髓鞘疾病。髓鞘分解一直是 被证明是由免疫介导的，而不是由于 病毒和病毒的持久性是持续脱髓鞘所必需的 进程。一种主要的病毒抗原负荷驻留在巨噬细胞中 我们相信是TMEV坚持的优先地点。病毒 中枢神经系统巨噬细胞的复制受到高度限制。的特点 这种疾病是病毒特异性T细胞水平的慢性升高 针对病毒表位而不是宿主神经抗原的反应，在 尤其是在感染的早期。病毒特异性迟发型超敏反应的核心作用 主要组织相容类(MHC)Il限制性CD4+Th1 T细胞介导 细胞脱髓鞘已被提出。人们对其作用知之甚少 TMEV感染中的抗体反应。 我们计划：(1)利用最近的技术鉴定TMEV的细胞受体 开发的阻断病毒感染的单抗将被用来分离 从lambdagt11基因表达文库中获得编码受体的基因 取自BHK-21细胞或小鼠肠道刷状缘(IBBM)基因。另一个 克隆技术CELICS将作为一种替代方法用于 受体识别。克隆的受体基因将在 哺乳动物表达系统，该受体的特征是 它与其他已知蛋白质的同源性、生物学功能和分布 在小鼠组织和中枢神经系统细胞中。(2)表征TMEV诱导的程序化 不同类型巨噬细胞系的细胞死亡(凋亡) 激活/分化状态；确定角色(如果有的话) Bcl2癌基因或bcl2同源基因抑制细胞凋亡的研究 巨噬细胞；并使用一组 已构建的TMEV重组病毒。(3)绘制地图 中和泰勒氏病毒粒子免疫原点的研究 中和单抗(NmAb)筛选中和逃逸突变体 将其中的氨基酸序列进行测序，以鉴定突变的氨基酸序列(S)； 并分析了体外中和动力学和体外中和能力。 对代表不同NLMS的nmAb进行体内保护。和(4)完成 特定病毒体表面定点突变的研究进展 氨基酸在病毒附着细胞中的作用 受体。

The overall goal of the proposed research is to elucidate the molecular basis of virus-cell interactions in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. Myelin breakdown has been shown to be immune-mediated rather than due to a cytolytic effect of the virus and virus persistence is required to perpetuate the demyelinating process. A predominant viral antigen burden resides in macrophages which we believe to be the preferential site of TMEV persistence. Virus replication in CNS macrophages is highly restricted. Characteristic of this disease are chronic elevated levels of virus-specific T cell responses directed at viral epitopes rather than host neuroantigens, at least early in the infection. A central role for virus-specific DTH mediated by major histocompatibility class (MHC) Il-restricted CD4+ Th1 T cells in demyelination has been proposed. Less is known about the role of antibody responses in TMEV infection. We plan to: (1) Identify the cellular receptor for TMEV using recently developed MAbs blocking virus infection which will be used to isolate the gene encoding the receptor from a lambdagt11 cDNA expression library made from BHK-21 cell or mouse intestinal brush border (IBBM) mRNA. Another cloning technique, CELICS, will be used as an alternative method of receptor identification. The cloned receptor gene will be expressed in a mammalian expression system, and the receptor characterized in terms of its homology with other known proteins, biologic function and distribution in mouse tissues and CNS cells. (2) Characterize TMEV-induced programmed cell death (apoptosis) in macrophage cell lines representing different states of activation/differentiation; determine a role, if any, of the bcl-2 oncogene or a bcl-2 homologue in inhibition of apoptosis in macrophages; and map this determinant within the capsid using a panel of TMEV recombinant viruses which have already been constructed. (3)Map the neutralizing immunogenic sites (nlMs) on the Theiler's virion using neutralizing mAbs (nmAb) to select neutralizing escape mutants, the RNAs of which will be sequenced to identify the mutated amino acid sequence(s); and analyze the kinetics of neutralization in vitro and the ability to protect in vivo for nmAb representing different nlMs. and (4) Finish ongoing studies of site-specific mutagenesis of selected virion surface amino acids to demonstrate a role in virus attachment to the cell receptor.