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

Martijn J Van Hemert,Adriaan H De Wilde,Alexander E Gorbalenya,Eric J Snijder

doi:10.1074/jbc.m708136200

Martijn J Van Hemert, Adriaan H De Wilde + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m708136200

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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.

Highlights

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. 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)
For the coronaviruses mouse hepatitis virus (MHV) and SARS coronavirus and the arterivirus equine arteritis virus (EAV), immunoelectron microscopy revealed that both viral nonstructural proteins presumed to be part of the replication/transcription complex (RTC) and de novo made viral RNA are associated with these membranes [9, 12, 20, 23]
Metabolic labeling of EAV RNA synthesis with [3H]uridine revealed that it was maximal by 6 h postinfection, and RTCs were routinely isolated at this time point

Summary

Introduction

Including genome size, organization, and expression strategy, they are united by the fact that their RNA genome is replicated by cytoplasmic enzyme complexes. Pp1a and pp1ab undergo extensive autoproteolytic processing by three ORF1a-encoded proteases, which leads to the generation of 13 end products (nsps), named nsp to nsp (a recently described cleavage within nsp yields nsp7␣ and nsp7␤ [27]) Most of these replicase subunits appear to become associated with intracellular membranes in the perinuclear region of the infected cell [11,12,13], where they are thought to assemble into RTCs. The ORF1b-encoded subunits contain the core enzymatic activities that are involved in viral RNA synthesis, like the RNA-dependent RNA polymerase (RdRp) and RNA helicase [28, 29], whereas ORF1a encodes, in addition to the three protease domains, several putative transmembrane subunits. Viral RNA synthesis involves partially and fully double-stranded intermediates, known as replicative intermediates (RIs) and replicative forms (RFs), which are thought to be associated with plus and minus strand RNA synthesis, respectively [32, 33]

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