Ugai Hideyo, Wang Minghui, Le Long P, Matthews David A, Yamamoto Masato, Curiel David T
Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
J Mol Biol. 2010 Jan 8;395(1):55-78. doi: 10.1016/j.jmb.2009.10.034. Epub 2009 Oct 21.
Oncolytic adenoviruses represent a promising therapeutic medicine for human cancer therapy, but successful translation into human clinical trials requires careful evaluation of their viral characteristics. While the function of adenovirus proteins has been analyzed in detail, the dynamics of adenovirus infection remain largely unknown due to technological constraints that prevent adequate tracking of adenovirus particles after infection. Fluorescence labeling of adenoviral particles is one new strategy designed to directly analyze the dynamic processes of viral infection in virus-host cell interactions. We hypothesized that the double labeling of an adenovirus with fluorescent proteins would allow us to properly analyze intracellular viruses and the fate of viral proteins in a live analysis of an adenovirus as compared to single labeling. Thus, we generated a fluorescently labeled adenovirus with both a red fluorescent minor capsid protein IX (pIX) [pIX monomeric red fluorescent protein 1 (mRFP1)] and a green fluorescent minor core protein V (pV) [pV enhanced green fluorescent protein (EGFP)], resulting in Ad5-IX-mRFP1-E3-V-EGFP. The fluorescent signals for pIX-mRFP1 and pV-EGFP were detected within 10 min in living cells. However, a growth curve analysis of Ad5-IX-mRFP1-E3-V-EGFP showed an approximately 150-fold reduced production of the viral progeny at 48 h postinfection as compared to adenovirus type 5. Interestingly, pIX-mRFP1 and pV-EGFP were initially localized in the cytoplasm and nucleolus, respectively, at 18 h postinfection. These proteins were observed in the nucleus during the late stage of infection, and relocalization of the proteins was observed in an adenoviral-replication-dependent manner. These results indicate that simultaneous detection of adenoviruses using dual-fluorescent proteins is suitable for real-time analysis, including identification of infected cells and monitoring of viral spread, which will be required for a complete evaluation of oncolytic adenoviruses.
溶瘤腺病毒是一种很有前景的人类癌症治疗药物，但要成功转化为人体临床试验，需要仔细评估其病毒特性。虽然腺病毒蛋白的功能已得到详细分析，但由于技术限制，腺病毒感染的动态过程在很大程度上仍不清楚，这些技术限制使得在感染后无法充分追踪腺病毒颗粒。腺病毒颗粒的荧光标记是一种旨在直接分析病毒 - 宿主细胞相互作用中病毒感染动态过程的新策略。我们假设，与单标记相比，用荧光蛋白对腺病毒进行双重标记将使我们能够在对腺病毒的实时分析中正确分析细胞内病毒和病毒蛋白的命运。因此，我们构建了一种荧光标记的腺病毒，其带有红色荧光的次要衣壳蛋白IX（pIX）[pIX单体红色荧光蛋白1（mRFP1）]和绿色荧光的次要核心蛋白V（pV）[pV增强型绿色荧光蛋白（EGFP）]，得到Ad5 - IX - mRFP1 - E3 - V - EGFP。在活细胞中，10分钟内即可检测到pIX - mRFP1和pV - EGFP的荧光信号。然而，与5型腺病毒相比，Ad5 - IX - mRFP1 - E3 - V - EGFP的生长曲线分析显示，感染后48小时病毒子代产量降低了约150倍。有趣的是，在感染后18小时，pIX - mRFP1和pV - EGFP最初分别定位于细胞质和核仁。在感染后期，这些蛋白在细胞核中被观察到，并且蛋白的重新定位是以腺病毒复制依赖的方式被观察到的。这些结果表明，使用双荧光蛋白同时检测腺病毒适用于实时分析，包括识别感染细胞和监测病毒传播，这对于全面评估溶瘤腺病毒是必需的。
