Westaway E G, Khromykh A A, Kenney M T, Mackenzie J M, Jones M K
Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Australia.
Virology. 1997 Jul 21;234(1):31-41. doi: 10.1006/viro.1997.8629.
The subcellular locations in infected Vero cells of Kunjin (KUN) virus core protein C and NS4B were analyzed by immunofluorescence (IF) and by immunoelectron microscopy using monospecific antibodies. Selection of appropriate fixation methods for IF showed that both proteins were associated at all times with perinuclear membranes spreading outward in a reticular pattern and they entered the nucleus late during the latent period. Subsequently NS4B was also dispersed through the nucleoplasm, while C appeared in the nucleolus and the nucleoplasm. These nuclear locations were confirmed by immunogold labeling of cryosections of infected cells at 24 hr postinfection. Labeling of NS4B in cryosections was especially enriched in the perinuclear membranes of the endoplasmic reticulum. When C and NS4B were each expressed separately in stably transformed cell lines, both cytoplasmic and nuclear localization was observed by IF and confirmed by immunoelectron microscopy. Thus the two proteins translocated to the nucleus independently of each other and of other viral proteins. Dual IF with antibodies to double-stranded RNA showed that cytoplasmic locations of C and NS4B were apparently associated in part with the sites of viral RNA synthesis which were resistant to solubilization by Triton X-100.
使用单特异性抗体，通过免疫荧光（IF）和免疫电子显微镜分析了库京（KUN）病毒核心蛋白C和NS4B在受感染的非洲绿猴肾（Vero）细胞中的亚细胞定位。IF合适固定方法的选择表明，两种蛋白始终与以网状模式向外扩散的核周膜相关联，并且它们在潜伏期后期进入细胞核。随后，NS4B也分散在核质中，而C出现在核仁和核质中。这些核定位通过感染后24小时对感染细胞冷冻切片的免疫金标记得到证实。冷冻切片中NS4B的标记在内质网的核周膜中特别丰富。当C和NS4B分别在稳定转化的细胞系中单独表达时，通过IF观察到细胞质和细胞核定位，并通过免疫电子显微镜得到证实。因此，这两种蛋白彼此独立且独立于其他病毒蛋白转运到细胞核。用双链RNA抗体进行的双重IF显示，C和NS4B的细胞质定位显然部分与对Triton X-100溶解有抗性的病毒RNA合成位点相关。
