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Journal of Virology, March 1999, p. 2016-2026, Vol. 73, No. 3
Division of Electron Microscopy, Department
of Biology, University of Oslo, Oslo, Norway,1
and
Department of Virology, Leiden University Medical
Center, Leiden, The Netherlands2
Received 13 October 1998/Accepted 1 December 1998
The replicase of equine arteritis virus (EAV; family
Arteriviridae, order Nidovirales) is expressed
in the form of two polyproteins (the open reading frame 1a [ORF1a]
and ORF1ab proteins). Three viral proteases cleave these precursors
into 12 nonstructural proteins, which direct both genome replication
and subgenomic mRNA transcription. Immunofluorescence assays showed
that most EAV replicase subunits localize to membranes in the
perinuclear region of the infected cell. Using replicase-specific
antibodies and cryoimmunoelectron microscopy, unusual double-membrane
vesicles (DMVs) were identified as the probable site of EAV RNA
synthesis. These DMVs were previously observed in cells infected with
different arteriviruses but were never implicated in viral RNA
synthesis. Extensive electron microscopic analysis showed that they
appear to be derived from paired endoplasmic reticulum membranes and that they are most likely formed by protrusion and detachment of
vesicular structures with a double membrane. Interestingly, very
similar membrane rearrangements were observed upon expression of
ORF1a-encoded replicase subunits nsp2 to nsp7 from an alphavirus-based expression vector. Apparently, the formation of a membrane-bound scaffold for the replication complex is a distinct step in the arterivirus life cycle, which is directed by the ORF1a protein and does
not depend on other viral proteins and/or EAV-specific RNA synthesis.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Open Reading Frame 1a-Encoded Subunits of the
Arterivirus Replicase Induce Endoplasmic Reticulum-Derived
Double-Membrane Vesicles Which Carry the Viral Replication
Complex
*
Corresponding author. Mailing address: Department of
Virology, Leiden University Medical Center, LUMC P4-26, PO Box 9600, 2300 RC Leiden, The Netherlands. Phone: 31 71 5261657. Fax: 31 71 5266761. E-mail: Snijder{at}Virology.AZL.NL.
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