MUTATIONS AND DELETIONS OF TMEV SURFACE RESIDUES

拟议研究的总体目标是阐明 Theiler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。髓磷脂的分解 显示是免疫介导的，而不是由于 需要病毒和病毒持久性来使脱髓鞘 过程主要的病毒抗原负荷存在于巨噬细胞中， 我们认为这是TMEV持续存在的优先位点。 病毒 CNS巨噬细胞中的复制受到高度限制。特性 这种疾病是慢性病毒特异性T细胞水平升高 针对病毒表位而不是宿主神经抗原的反应， 至少在感染初期。病毒特异性DTH的核心作用 由主要组织相容性（MHC）II类限制性CD 4 + Th 1 T介导 脱髓鞘的细胞。对这一角色的了解较少， TMEV感染中的抗体应答。 本研究拟：（1）利用近年来的研究成果，鉴定TMEV的细胞受体 开发了阻断病毒感染的单克隆抗体， 从制备的cDNA表达文库中编码受体的基因 从BHK-21细胞或小鼠肠刷状缘（IBBM）mRNA。另一 克隆技术，CELICS，将被用作一种替代方法， 受体鉴定克隆的受体基因将在 哺乳动物表达系统，并且所述受体的特征在于 它与其它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诱导程控 巨噬细胞系中的细胞死亡（凋亡）代表不同的 激活/分化的状态;确定激活/分化的作用，如果有的话， bcl-2癌基因或bcl-2同源物在抑制细胞凋亡中的作用 巨噬细胞;并使用一组 已经构建的TMEV重组病毒。（3）映射 中和泰勒病毒体上的免疫原性位点（nIM）， 中和mAb（nmAb）以选择中和逃逸突变体，所述RNA 其中的氨基酸序列将被测序以鉴定突变的氨基酸序列; 并分析体外中和动力学和 在体内保护代表不同nIM的nmAb。（4）完成 正在进行的对选定的病毒体表面进行定点诱变的研究 氨基酸，以证明病毒附着于细胞的作用 受体的

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.