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J. Biol. Chem., Vol. 283, Issue 24, 16525-16536, June 13, 2008

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The in Vitro RNA Synthesizing Activity of the Isolated Arterivirus Replication/Transcription Complex Is Dependent on a Host Factor^*

From the Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, LUMC E4-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands

The cytoplasmic replication of positive-stranded RNA viruses is associated with characteristic, virus-induced membrane structures that are derived from host cell organelles. We used the prototype arterivirus, equine arteritis virus (EAV), to gain insight into the structure and function of the replication/transcription complex (RTC) of nidoviruses. RTCs were isolated from EAV-infected cells, and their activity was studied using a newly developed in vitro assay for viral RNA synthesis, which reproduced the synthesis of both viral genome and subgenomic mRNAs. A detailed characterization of this system and its reaction products is described. RTCs isolated from cytoplasmic extracts by differential centrifugation were inactive unless supplemented with a cytosolic host protein factor, which, according to subsequent size fractionation analysis, has a molecular mass in the range of 59–70 kDa. This host factor was found to be present in a wide variety of eukaryotes. Several EAV replicase subunits cosedimented with newly made viral RNA in a heavy membrane fraction that contained all RNA-dependent RNA polymerase activity. This fraction contained the characteristic double membrane vesicles (DMVs) that were previously implicated in EAV RNA synthesis and could be immunolabeled for EAV nonstructural proteins (nsps). Replicase subunits directly involved in viral RNA synthesis (nsp9 and nsp10) or DMV formation (nsp2 and nsp3) exclusively cosedimented with the active RTC. Subgenomic mRNAs appeared to be released from the complex, whereas newly made genomic RNA remained more tightly associated. Taken together, our data strongly support a link between DMVs and the RNA-synthesizing machinery of arteriviruses.

Received for publication, October 1, 2007 , and in revised form, February 28, 2008.

^* This research was supported by Council for Chemical Sciences of the Netherlands Organization for Scientific Research Grant 700.52.306. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Molecular Virology Laboratory, Dept. of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, LUMC E4-P, P. O. Box 9600, 2300 RC Leiden, The Netherlands. Tel.: 31-71-5261657; Fax: 31-71-5266761; E-mail: e.j.snijder{at}lumc.nl.

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