Chen L K, Liao C L, Lin C G, Lai S C, Liu C I, Ma S H, Huang Y Y, Lin Y L
Institute of Preventive Medicine, Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Virology. 1996 Mar 1;217(1):220-9. doi: 10.1006/viro.1996.0109.
Persistent infection with Japanese encephalitis virus (JEV) was established in murine neuroblastoma N18 cells, and the persistency has been maintained in cell culture for over 6 months. From the persistently infected cells, a clone named C2-2 was selected and expanded to form a stable cell line. The vast majority of C2-2 cells showed viral protein staining by immunofluorescence and continuously produced low levels of virus (10(3) to 10(4) PFU/ml) without marked cytopathic effects or cyclic variations. In addition to the wild-type viral proteins, truncated forms of the viral nonstructural protein 1 (NS1) as well as its derivative NS1' were produced in C2-2 cells. Both truncated NS1 and NS1' contain deletions at their N-termini; however, the analyses by RT-PCR and direct sequencing of the viral RNA failed to detect any truncations or mutations within the NS1 region, suggesting that NS1 truncation was a result of a unique posttranslational proteolytic cleavage of NS1 in the persistently infected cells. Similar but not identical truncation of NS1 was also observed in two other persistently infected cell lines established in Vero and DBT (murine astrocytoma) cells. However, viruses released from C2-2 cells did not produce truncated NS1 upon infection of N18 cells, suggesting that NS1 truncations were the result of virus-cell interaction in persistently infected cells. These data indicate a strong association between abnormal NS1 expression and JEV persistency. A probable involvement of dysfunctional NS1 in the establishment and/or maintenance of JEV persistency in tissue culture is discussed.
日本脑炎病毒（JEV）在鼠神经母细胞瘤N18细胞中建立了持续感染，并且这种持续性在细胞培养中已维持了6个多月。从持续感染的细胞中，挑选出一个名为C2-2的克隆并进行扩增，形成了一个稳定的细胞系。绝大多数C2-2细胞通过免疫荧光显示病毒蛋白染色，并持续产生低水平的病毒（10³至10⁴ PFU/ml），没有明显的细胞病变效应或周期性变化。除了野生型病毒蛋白外，C2-2细胞中还产生了病毒非结构蛋白1（NS1）的截短形式及其衍生物NS1'。截短的NS1和NS1'在其N端均有缺失；然而，通过RT-PCR和病毒RNA直接测序分析未能检测到NS1区域内的任何截短或突变，这表明NS1截短是持续感染细胞中NS1独特的翻译后蛋白水解切割的结果。在Vero细胞和DBT（鼠星形细胞瘤）细胞中建立的另外两个持续感染细胞系中也观察到了类似但不完全相同的NS1截短。然而，C2-2细胞释放的病毒在感染N18细胞时不会产生截短的NS1，这表明NS1截短是持续感染细胞中病毒-细胞相互作用的结果。这些数据表明异常的NS1表达与JEV持续性之间存在密切关联。文中讨论了功能失调的NS1在组织培养中JEV持续性的建立和/或维持中可能的作用。
