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Involvement of host ADAR in biased hypermutation of intracelebral measles virus causing SSPE

宿主 ADAR 参与脑内麻疹病毒导致 SSPE 的偏向超突变

基本信息

批准号：
14570271
负责人：
OGURA Hisashi
金额：
$ 2.24万
依托单位：
Osaka City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570271/
关键词：
Subacute sclerosing panencephalitis SSPE virus Measles virus Biased hypermutation ADAR

项目摘要

It has been clarified that SSPE (subacute sclerosing panencephalitis) viruses, measles virus defective variants isolated from brains of SSPE patients, acquire biased hypermutation in their M genes, in which frequent replacements from adenine to guanine in genome sense are found. Although in recent years adenosine deaminases that act on RNAs (ADARs) might be involved in its phenomenon, there has not yet been any report showing direct evidences that ADAR causes biased hypermutation of measles virus M gene. Therefore, we examined if ADAR constitutively expressed in neural cells from the externally introduced gene can induce biased hypermutation in the measles virus infected cells.We established A172/ADAR+ cells, an ADAR constitutively expressed cell line using A172 cells, derived from human glioblastoma. These cells were infected rMV323 virus (derived from the Ichinose B strain cDNA inserted with GFP gene) or Edm-B virus (derived from the Edmonston-B strain cDNA) and incubated at 35℃ for 1 week. One half of the infected A172/ADAR+ cells were cocultured with 5 x 10^5 fresh A172/ADAR+ cells and further incubated. The other half of the infected cells were stocked at -80℃ for viral RNA analysis (RT-PCR and direct sequencing of the M gene). This way of passage were repeated 10 times. Amplification of the M gene cDNA was detected in samples of the passage 1 to 2 of rMV323 virus and of the passage 1 to 7 of Edm-B virus. At present, however, positive mutation is not suggested in the M gene.

已经澄清，SSPE亚急性硬化性全脑炎（SSPE）病毒是从SSPE患者脑组织中分离的麻疹病毒缺陷型，其M基因发生了偏性超突变，在基因组意义上经常发生腺嘌呤到鸟嘌呤的替换，虽然近年来研究表明作用于RNA的腺苷脱氨酶（ADARs）可能参与了这一现象，目前还没有任何报道显示阿达尔引起麻疹病毒M基因的偏性超突变的直接证据。因此，我们研究了阿达尔在神经细胞中组成型表达的外部导入基因是否可以在麻疹病毒感染的细胞中诱导偏向性超突变。我们建立了A172/阿达尔+细胞，这是一种由人胶质母细胞瘤来源的A172细胞组成型表达的阿达尔细胞系。这些细胞被rMV 323病毒（来自插入GFP基因的Ichinose B株cDNA）或Edm-B病毒（来自Edmonston-B株cDNA）感染，并在35℃下孵育1周。将一半感染的A172/阿达尔+细胞与5 x 10^5个新鲜A172/阿达尔+细胞共培养，并进一步孵育。另一半感染细胞在-80℃下储存，用于病毒RNA分析（RT-PCR和M基因直接测序）。这种通过方式重复了10次。在rMV 323病毒第1 ~ 2代和Edm-B病毒第1 ~ 7代的样品中检测到M基因cDNA的扩增。然而，目前，在M基因中未提示正突变。