van Hemert Martijn J, van den Worm Sjoerd H E, Knoops Kèvin, Mommaas A Mieke, Gorbalenya Alexander E, Snijder Eric J
Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
PLoS Pathog. 2008 May 2;4(5):e1000054. doi: 10.1371/journal.ppat.1000054.
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.
严重急性呼吸综合征冠状病毒（SARS-CoV）的复制和转录由一个复制/转录复合体（RTC）介导，该复合体的主要成分是病毒编码的非结构蛋白（nsps）。16种SARS-CoV的nsps由两种大型前体多蛋白自动加工产生。RTC被认为与SARS-CoV感染细胞胞质中特有的病毒诱导双膜结构相关。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，我们从感染细胞中分离出活性RTC，并利用它们开发了首个针对其体外活性的可靠检测方法。在这个体外系统中，RTC忠实地再现了基因组RNA和所有8种亚基因组mRNA的合成。体外主要合成正链RNA，RTC活性不需要蛋白质合成。所有RTC活性、酶促和假定的跨膜nsps以及病毒RNA都与重膜结构一起沉降。此外，沉淀的RTC需要添加一种细胞质宿主因子来恢复其体外活性。新合成的亚基因组RNA似乎被释放，而基因组RNA主要仍与含RTC的部分相关。RTC活性被去污剂处理破坏，表明膜起重要作用。RTC似乎受到膜的保护，因为新合成的病毒RNA和几个复制酶/转录酶亚基对蛋白酶和核酸酶具有抗性，仅在添加非离子去污剂后才变得易降解。我们的数据确立了SARS-CoV RNA合成对病毒诱导膜结构的重要功能依赖性。
