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

亚急性硬化性全脑炎（SSPE）病毒，即从SSPE患者脑中分离出的麻疹病毒缺陷变体，其M基因发生偏性高突变，在基因组意义上发现腺嘌呤频繁替换为鸟嘌呤。虽然近年来作用于rna的腺苷脱氨酶（adenosine deaminases, ADARs）可能参与了这种现象，但尚未有直接证据表明ADAR导致麻疹病毒M基因偏态高突变。因此，我们研究了外部引入基因在神经细胞中组成性表达的ADAR是否能诱导麻疹病毒感染细胞的偏置高突变。我们利用来源于人胶质母细胞瘤的A172细胞建立了A172/ADAR+细胞，这是一种ADAR组成型表达细胞系。这些细胞分别感染rMV323病毒（来源于插入GFP基因的Ichinose B株cDNA）或Edm-B病毒（来源于Edmonston-B株cDNA），在35℃下孵卵1周。将一半感染的A172/ADAR+细胞与5 × 10^5个新鲜的A172/ADAR+细胞共培养，进一步孵育。另一半感染细胞储存在-80℃进行病毒RNA分析（RT-PCR和M基因直接测序）。这种传递方式重复10次。在rMV323病毒1 ~ 2代和Edm-B病毒1 ~ 7代样品中检测到M基因cDNA扩增。但目前未发现M基因阳性突变。

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.