SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro.
SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro.
复制标题
SARS-Coronavirus复制/转录复合物受到膜保护，需要在体外活动的宿主因子。
DOI：
10.1371/journal.ppat.1000054
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发表时间：
2008-05-02
期刊：
影响因子：
6.7
通讯作者：
Snijder, Eric J.
中科院分区：
文献类型：
--
作者：
van Hemert, Martijn J.;van den Worm, Sjoerd H. E.;Knoops, Kevin;Mommaas, A. Mieke;Gorbalenya, Alexander E.;Snijder, Eric J.
SARS 冠状病毒 (SARS-CoV) 的复制和转录是由复制/转录复合体 (RTC) 介导的，其中病毒编码的非结构蛋白 (nsps) 是主要成分。 16 个 SARS-CoV nsps 是通过两个大的前体多蛋白的自动加工产生的。 RTC被认为与SARS-CoV感染细胞的细胞质中病毒诱导的特征性双膜结构有关。为了研究这些结构与病毒 RNA 合成之间的联系，并剖析 RTC 的组织和功能，我们从受感染的细胞中分离出活性 RTC，并利用它们开发了第一个对其体外活性的稳健检测。 RTC 在该体外系统中忠实地再现了基因组 RNA 和所有八个亚基因组 mRNA 的合成。主要合成正链RNA，体外RTC活性不需要蛋白质合成。所有 RTC 活性、酶促和推定的跨膜 nsps 以及病毒 RNA 均与重膜结构共同沉积。此外，沉淀的 RTC 需要添加细胞质宿主因子才能重建其体外活性。新合成的亚基因组 RNA 似乎被释放，而基因组 RNA 仍然主要与含有 RTC 的部分相关。 RTC 活性被洗涤剂处理破坏，表明膜的重要作用。 RTC 似乎受到膜的保护，因为新合成的病毒 RNA 和几个复制酶/转录酶亚基具有蛋白酶和核酸酶抗性，只有在添加非离子去污剂后才容易被降解。我们的数据确定了 SARS-CoV RNA 合成对病毒诱导的膜结构的重要功能依赖性。 SARS 冠状病毒（SARS-CoV）会在受感染宿主细胞的细胞质中复制，引起危及生命的严重急性呼吸综合征。 SARS-CoV 生命周期中关键的早期步骤是形成复制/转录复合体 (RTC)，驱动病毒基因组复制和亚基因组 mRNA 合成。病毒编码的酶形成了该 RTC 的核心，人们认为它与源自修饰的宿主细胞膜的病毒诱导的特征性膜结构有关。为了研究这些膜结构与 SARS-CoV RNA 合成之间的联系，并表征 RTC 的组成和功能，我们分离了这些复合物并开发了第一个体外测定法来研究它们的活性。 SARS-CoV 基因组 RNA 和所有八种亚基因组 mRNA 均在此体外反应中合成。通过离心，可以从细胞质中分离出 RTC 活性以及膜结构、病毒酶和 RNA。这些分离的 RTC 的活性依赖于细胞质宿主因子。 RTC 活性被去垢剂处理破坏，这表明膜在保护复合物免受蛋白酶和核酸酶消化方面发挥着关键作用。我们的数据建立了病毒 RNA 合成和细胞内膜之间的功能联系，并表明宿主因素在 SARS-CoV RNA 合成中发挥着至关重要的作用。
SARS-coronavirus (SARS-CoV) replication and transcription are mediated by a replication/transcription complex (RTC) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity. The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures. The SARS-coronavirus (SARS-CoV), which causes the life-threatening severe acute respiratory syndrome, replicates in the cytoplasm of infected host cells. A critical early step in the SARS-CoV life cycle is the formation of a replication/transcription complex (RTC) that drives viral genome replication and subgenomic mRNA synthesis. Virus-encoded enzymes form the core of this RTC, which is believed to be associated with characteristic virus-induced membrane structures derived from modified host cell membranes. To investigate the connection between these membrane structures and SARS-CoV RNA synthesis, and to characterize RTC composition and function, we isolated these complexes and developed the first in vitro assay to study their activity. SARS-CoV genomic RNA and all eight subgenomic mRNAs were synthesized in this in vitro reaction. By centrifugation, RTC activity could be isolated from the cytoplasm, together with membrane structures, viral enzymes, and RNA. The activity of these isolated RTCs was dependent on a cytoplasmic host factor. RTC activity was destroyed by detergent treatment, suggesting a critical role for membranes that appeared to protect the complex against protease and nuclease digestion. Our data establish a functional connection between viral RNA synthesis and intracellular membranes and show that host factors play a crucial role in SARS-CoV RNA synthesis.
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