Chung Siu-Wah, Arnott John A, Yang Yizeng, Wong Peter M C
Department of Pathology & Laboratory Medicine, Fels Institute for Cancer Research & Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
J Biol Chem. 2003 Dec 12;278(50):50635-40. doi: 10.1074/jbc.M309945200. Epub 2003 Sep 30.
Ran GTPase has been shown to be involved in host innate immune response, and two alleles, RanT/n and RanC/d, which differ from each other by a single nucleotide, have opposite effects on host innate immune response. In this study, we showed that although intravenous administration in mice with either Ran cDNA using an identical adenovirus (Ad) vector resulted in no significant difference in vector tissue distribution, intraperitoneal administration resulted in effective vector transduction into peritoneal macrophages, coupled with a striking difference in vector tissue distribution in 2 h or less. We further demonstrated the presence of prepackaged RNA in virions of Ad vectors, in cells actively producing Ad virus particles, and in cells very shortly after Ad infection. Real-time PCR analysis confirmed the presence of prepackaged RNA and estimated the copy number to be one per viral genome. The prepackaged viral mRNA could be used for translation into proteins, as shown by experiments in which the transcriptional inhibitor actinomycin-D was used. Hence, translation of Ran proteins from prepackaged viral mRNA immediately after virus uncoating in the cytoplasm is one mechanism that would account for an early difference in Ad-vector tissue distribution after efficient gene transfer into macrophages.
Ran GTP酶已被证明参与宿主先天免疫反应，并且两个仅相差一个核苷酸的等位基因RanT/n和RanC/d，对宿主先天免疫反应具有相反的作用。在本研究中，我们发现，虽然使用相同腺病毒（Ad）载体向小鼠静脉注射任一种Ran cDNA时，载体组织分布没有显著差异，但腹腔注射会导致载体有效转导至腹腔巨噬细胞，并且在2小时或更短时间内载体组织分布存在显著差异。我们进一步证明，在Ad载体的病毒粒子中、在活跃产生Ad病毒颗粒的细胞中以及在Ad感染后不久的细胞中存在预包装的RNA。实时PCR分析证实了预包装RNA的存在，并估计每个病毒基因组的拷贝数为一个。如使用转录抑制剂放线菌素-D的实验所示，预包装的病毒mRNA可用于翻译蛋白质。因此，病毒在细胞质中脱壳后立即从预包装的病毒mRNA翻译Ran蛋白，是一种可以解释有效基因转移到巨噬细胞后Ad载体组织分布早期差异的机制。
