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

Abstract

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.Positive strand RNA viruses form the largest group of animal viruses and include many important human pathogens, like poliovirus, hepatitis A and C virus, dengue virus, yellow fever virus, West Nile virus, and various human coronaviruses. Although these viruses differ in many aspects of their biology, including genome size, organization, and expression strategy, they are united by the fact that their RNA genome is replicated by cytoplasmic enzyme complexes. These complexes are associated with virus-induced membrane structures that are derived from host cell organelles (for reviews, see Refs. 1-3). Such membrane structures might function as scaffold for the replication machinery, provide a suitable microenvironment for viral RNA synthesis, serve to recruit membrane-bound host proteins, and/or provide protection against the host cell's antiviral responses (e.g. RNA degradation or responses triggered by the double-stranded RNA intermediates of viral RNA synthesis).Nidoviruses (corona-, roni-, and arteriviruses) have exceptionally large polycistronic RNA genomes and employ a unique transcription mechanism to produce a nested set of subgenomic (sg) 2 mRNAs. Therefore, among positive strand RNA viruses, nidovirus RNA synthesis is considered to be of unparalleled complexity (4, 5). In nidovirus-infected cells, newly synthesized viral R...

Nsp9 and Nsp10 Contribute to the Fatal Virulence of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Emerging in China

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The DEAD-box RNA helicase 5 positively regulates the replication of porcine reproductive and respiratory syndrome virus by interacting with viral Nsp9 in vitro

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