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J Virol, August 1998, p. 6689-6698, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

ORF1a-Encoded Replicase Subunits Are Involved in the Membrane Association of the Arterivirus Replication Complex

Yvonne van der Meer,1 Hans van Tol,1 Jacomine Krijnse Locker,2 and Eric J. Snijder1,*

Department of Virology, Leiden University Medical Center, Leiden, The Netherlands,1 and Cell Biology Program, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany2

Received 17 March 1998/Accepted 27 April 1998

Among the functions of the replicase of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are important viral enzyme activities such as proteases and the putative RNA polymerase and RNA helicase functions. The replicase is expressed in the form of two polyproteins (open reading frame 1a [ORF1a] and ORF1ab), which are processed into 12 nonstructural proteins by three viral proteases. In immunofluorescence assays, the majority of these cleavage products localized to the perinuclear region of the cell. A dense granular and vesicular staining was observed, which strongly suggested membrane association. By using confocal microscopy and double-label immunofluorescence, the distribution of the EAV replicase was shown to overlap with that of PDI, a resident protein of the endoplasmic reticulum and intermediate compartment. An in situ labeling of nascent viral RNA with bromo-UTP demonstrated that the membrane-bound complex in which the replicase subunits accumulate is indeed the site of viral RNA synthesis. A number of ORF1a-encoded hydrophobic domains were postulated to be involved in the membrane association of the arterivirus replication complex. By using various biochemical methods (Triton X-114 extraction, membrane purification, and sodium carbonate treatment), replicase subunits containing these domains were shown to behave as integral membrane proteins and to be membrane associated in infected cells. Thus, contribution to the formation of a membrane-bound scaffold for the viral replication-transcription complex appears to be an important novel function for the arterivirus ORF1a replicase polyprotein.

* Corresponding author. Mailing address: Department of Virology, Leiden University Medical Center, AZL P4-26, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Phone: 31 71 5261657. Fax: 31 71 5266761. E-mail: Snijder{at}Virology.AZL.NL.

J Virol, August 1998, p. 6689-6698, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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