Mackenzie J M, Khromykh A A, Westaway E G
Clinical Medical Virology Centre, University of Queensland, St. Lucia, Brisbane, Queensland, 4072, Australia.
Virology. 2001 Jan 5;279(1):161-72. doi: 10.1006/viro.2000.0691.
This report focuses mainly on the characterization of a Vero cell line stably expressing the flavivirus Kunjin (KUN) replicon C20SDrep (C20SDrepVero). We showed by immunofluorescence and cryoimmunoelectron microscopy that unique flavivirus-induced membrane structures, termed convoluted membranes/paracrystalline structures, were induced in the C20SDrepVero cells. These induced cytoplasmic foci were immunolabeled with KUN virus anti-NS3 antibodies and with antibodies to the cellular markers ERGIC53 (for the intermediate compartment) and protein disulfide isomerase (for the rough endoplasmic reticulum). However, in contrast to the large perinuclear inclusions observed by immunofluorescence with anti-double-stranded (ds)RNA antibodies in KUN virus-infected cells, the dsRNA in C20SDrepVero cells was localized to small isolated foci scattered throughout the cytoplasm, which were coincident with small foci dual-labeled with the trans-Golgi specific marker GalT. Importantly, persistent expression of the KUN replicons in cells did not produce cytopathic effects, and the morphology of major host organelles (including Golgi, mitochondria, endoplasmic reticulum, and nucleus) was apparently unaffected. The amounts of plus- and minus-sense RNA synthesis in replicon cells were similar to those in KUN virus-infected cells until near the end of the latent period, but subsequently increases of about 10- and fourfold, respectively, occurred in infected cells. Virus-specified protein synthesis in C20SDrepVero cells was also about 10-fold greater than that in infected cells. When several KUN replicon cell lines were compared with respect to membrane induction, the relative efficiencies increased in parallel with increases in viral RNA and protein synthesis, consistent with the increases observed during the virus infectious cycle. Based on these observations, cell lines expressing less-efficient replicons may provide a useful tool to study early events in flavivirus RNA replication, which are difficult to assess in virus infections.
本报告主要聚焦于稳定表达黄病毒库京（KUN）复制子C20SDrep的Vero细胞系（C20SDrepVero）的特性。我们通过免疫荧光和冷冻免疫电子显微镜显示，在C20SDrepVero细胞中诱导出了独特的黄病毒诱导膜结构，即卷曲膜/类晶体结构。这些诱导产生的细胞质病灶用KUN病毒抗NS3抗体以及针对细胞标记物ERGIC53（用于中间区室）和蛋白二硫键异构酶（用于粗面内质网）的抗体进行免疫标记。然而，与KUN病毒感染细胞中用抗双链（ds）RNA抗体通过免疫荧光观察到的大的核周包涵体不同，C20SDrepVero细胞中的dsRNA定位于散布在整个细胞质中的小的孤立病灶，这些病灶与用反式高尔基体特异性标记物GalT进行双重标记的小病灶重合。重要的是，细胞中KUN复制子的持续表达未产生细胞病变效应，主要宿主细胞器（包括高尔基体、线粒体、内质网和细胞核）的形态显然未受影响。直到潜伏期接近结束，复制子细胞中正义和反义RNA合成的量与KUN病毒感染细胞中的量相似，但随后感染细胞中分别出现了约10倍和4倍的增加。C20SDrepVero细胞中病毒特异性蛋白合成也比感染细胞中的大约高10倍。当比较几个KUN复制子细胞系的膜诱导情况时，相对效率随着病毒RNA和蛋白合成的增加而平行增加，这与病毒感染周期中观察到的增加情况一致。基于这些观察结果，表达效率较低的复制子的细胞系可能为研究黄病毒RNA复制的早期事件提供一个有用的工具，而这些事件在病毒感染中难以评估。
