Hidalgo Paloma, Anzures Lourdes, Hernández-Mendoza Armando, Guerrero Adán, Wood Christopher D, Valdés Margarita, Dobner Thomas, Gonzalez Ramón A
Centro de Investigación en Dinámica Celular, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos (UAEM), Cuernavaca, Morelos, México Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, México.
Centro de Investigación en Dinámica Celular, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos (UAEM), Cuernavaca, Morelos, México.
J Virol. 2016 Jan 13;90(7):3411-27. doi: 10.1128/JVI.00033-16.
Adenovirus (Ad) replication compartments (RC) are nuclear microenvironments where the viral genome is replicated and a coordinated program of late gene expression is established. These virus-induced nuclear sites seem to behave as central hubs for the regulation of virus-host cell interactions, since proteins that promote efficient viral replication as well as factors that participate in the antiviral response are coopted and concentrated there. To gain further insight into the activities of viral RC, here we report, for the first time, the morphology, composition, and activities of RC isolated from Ad-infected cells. Morphological analyses of isolated RC particles by superresolution microscopy showed that they were indistinguishable from RC within infected cells and that they displayed a dynamic compartmentalization. Furthermore, the RC-containing fractions (RCf) proved to be functional, as they directed de novo synthesis of viral DNA and RNA as well as RNA splicing, activities that are associated with RC in vivo. A detailed analysis of the production of viral late mRNA from RCf at different times postinfection revealed that viral mRNA splicing occurs in RC and that the synthesis, posttranscriptional processing, and release from RC to the nucleoplasm of individual viral late transcripts are spatiotemporally separate events. The results presented here demonstrate that RCf are a powerful system for detailed study into RC structure, composition, and activities and, as a result, the determination of the molecular mechanisms that induce the formation of these viral sites of adenoviruses and other nuclear-replicating viruses.
RC may represent molecular hubs where many aspects of virus-host cell interaction are controlled. Here, we show by superresolution microscopy that RCf have morphologies similar to those of RC within Ad-infected cells and that they appear to be compartmentalized, as nucleolin and DBP display different localization in the periphery of these viral sites. RCf proved to be functional, as they direct de novo synthesis of viral DNA and mRNA, allowing the detailed study of the regulation of viral genome replication and expression. Furthermore, we show that the synthesis and splicing of individual viral late mRNA occurs in RC and that they are subject to different temporal patterns of regulation, from their synthesis to their splicing and release from RC to the nucleoplasm. Hence, RCf represent a novel system to study molecular mechanisms that are orchestrated in viral RC to take control of the infected cell and promote an efficient viral replication cycle.
腺病毒（Ad）复制区室（RC）是细胞核微环境，病毒基因组在此进行复制，并建立晚期基因表达的协调程序。这些病毒诱导的核位点似乎充当调节病毒与宿主细胞相互作用的中心枢纽，因为促进病毒高效复制的蛋白质以及参与抗病毒反应的因子都被招募并集中于此。为了进一步深入了解病毒RC的活性，我们首次在此报告从感染Ad的细胞中分离出的RC的形态、组成和活性。通过超分辨率显微镜对分离出的RC颗粒进行形态学分析表明，它们与感染细胞内的RC没有区别，并且呈现出动态区室化。此外，含RC的组分（RCf）被证明具有功能，因为它们指导病毒DNA和RNA的从头合成以及RNA剪接，这些活性在体内与RC相关。对感染后不同时间RCf产生病毒晚期mRNA的详细分析表明，病毒mRNA剪接发生在RC中，并且单个病毒晚期转录本从合成、转录后加工到从RC释放到核质是时空上分开的事件。此处呈现的结果表明，RCf是用于详细研究RC结构、组成和活性的强大系统，因此可用于确定诱导腺病毒和其他核复制病毒这些病毒位点形成的分子机制。
RC可能代表控制病毒与宿主细胞相互作用诸多方面的分子枢纽。在此，我们通过超分辨率显微镜显示，RCf具有与Ad感染细胞内RC相似的形态，并且它们似乎是区室化的，因为核仁素和DBP在这些病毒位点的周边显示出不同的定位。RCf被证明具有功能，因为它们指导病毒DNA和mRNA的从头合成，从而允许对病毒基因组复制和表达的调控进行详细研究。此外，我们表明单个病毒晚期mRNA的合成和剪接发生在RC中，并且它们从合成到剪接以及从RC释放到核质受到不同的时间调控模式。因此，RCf代表了一种新系统，用于研究在病毒RC中精心编排以控制感染细胞并促进高效病毒复制周期的分子机制。
