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Journal of Virology, December 2001, p. 12370-12381, Vol. 75, No. 24
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.24.12370-12381.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Identification of Sequences in Brome Mosaic Virus Replicase Protein 1a That Mediate Association with Endoplasmic Reticulum Membranes

Johan A. den Boon,1 Jianbo Chen,1 and Paul Ahlquist1,2,*

Institute for Molecular Virology1 and Howard Hughes Medical Institute,2 University of Wisconsin-Madison, Madison, Wisconsin 53706

Received 23 May 2001/Accepted 6 September 2001

RNA replication of all positive-strand RNA viruses is closely associated with intracellular membranes. Brome mosaic virus (BMV) RNA replication occurs on the perinuclear region of the endoplasmic reticulum (ER), both in its natural plant host and in the yeast Saccharomyces cerevisiae. The only viral component in the BMV RNA replication complex that localizes independently to the ER is 1a, a multifunctional protein with an N-terminal RNA capping domain and a C-terminal helicase-like domain. The other viral replication components, the RNA polymerase-like protein 2a and the RNA template, depend on 1a for recruitment to the ER. We show here that, in membrane extracts, 1a is fully susceptible to proteolytic digestion in the absence of detergent and thus, a finding consistent with its roles in RNA replication, is wholly or predominantly on the cytoplasmic face of the ER with no detectable lumenal protrusions. Nevertheless, 1a association with membranes is resistant to high-salt and high-pH treatments that release most peripheral membrane proteins. Membrane flotation gradient analysis of 1a deletion variants and 1a segments fused to green fluorescent protein (GFP) showed that sequences in the N-terminal RNA capping module of 1a mediate membrane association. In particular, a region C-terminal to the core methyltransferase homology was sufficient for high-affinity ER membrane association. Confocal immunofluorescence microscopy showed that even though these determinants mediate ER localization, they fail to localize GFP to the narrow region of the perinuclear ER, where full-length 1a normally resides. Instead, they mediate a more globular or convoluted distribution of ER markers. Thus, additional sequences in 1a that are distinct from the primary membrane association determinants contribute to 1a's normal subcellular distribution, possibly through effects on 1a conformation, orientation, or multimerization on the membrane.

* Corresponding author. Mailing address: Institute for Molecular Virology and Howard Hughes Medical Institute, University of Wisconsin---Madison, 1525 Linden Dr., Madison, WI 53706. Phone: (608) 263-5916. Fax: (608) 265-9214. E-mail: ahlquist{at}facstaff.wisc.edu.

Journal of Virology, December 2001, p. 12370-12381, Vol. 75, No. 24
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.24.12370-12381.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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