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Journal of Virology, November 2005, p. 13747-13758, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13747-13758.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Brome Mosaic Virus 1a Nucleoside Triphosphatase/Helicase Domain Plays Crucial Roles in Recruiting RNA Replication Templates

Xiaofeng Wang,1,2 Wai-Ming Lee,1,{ddagger} Tokiko Watanabe,1 Michael Schwartz,1,2,{dagger} Michael Janda,1,2 and Paul Ahlquist1,2*

Institute for Molecular Virology,1 Howard Hughes Medical Institute, University of Wisconsin, Madison, Wisconsin 537062

Received 23 December 2004/ Accepted 4 August 2005

Positive-strand RNA virus RNA replication is invariably membrane associated and frequently involves viral proteins with nucleoside triphosphatase (NTPase)/helicase motifs or activities. Brome mosaic virus (BMV) encodes two RNA replication factors: 1a has a C-terminal NTPase/helicase-like domain, and 2apol has a central polymerase domain. 1a accumulates on endoplasmic reticulum membranes, recruits 2apol, and induces 50- to 70-nm membrane invaginations (spherules) serving as RNA replication compartments. 1a also recruits BMV replication templates such as genomic RNA3. In the absence of 2apol, 1a dramatically stabilizes RNA3 by transferring RNA3 to a membrane-associated, nuclease-resistant state that appears to correspond to the interior of the 1a-induced spherules. Prior results show that the 1a NTPase/helicase-like domain contributes to RNA recruitment. Here, we tested mutations in the conserved helicase motifs of 1a to further define the roles of this domain in RNA template recruitment. All 1a helicase mutations tested showed normal 1a accumulation, localization to perinuclear endoplasmic reticulum membranes, and recruitment of 2apol. Most 1a helicase mutants also supported normal spherule formation. Nevertheless, these mutations severely inhibited RNA replication and 1a-induced stabilization of RNA3 in vivo. For such 1a mutants, the membrane-associated RNA3 pool was both reduced and highly susceptible to added nuclease. Thus, 1a recruitment of viral RNA templates to a membrane-associated, nuclease-resistant state requires additional functions beyond forming spherules and recruiting RNA to membranes, and these functions depend on the 1a helicase motifs. The possibility that, similar to some double-stranded RNA viruses, the 1a NTPase/helicase-like domain may be involved in importing viral RNAs into a preformed replication compartment is discussed.

* Corresponding author. Mailing address: Institute for Molecular Virology, University of Wisconsin-Madison, 1525 Linden Dr., Madison,WI 53706-1576. Phone: (608) 263-5916. Fax: (608) 265-9214. E-mail: ahlquist{at}wisc.edu.

{ddagger} Present address: Department of Pediatrics, University of Wisconsin, Madison, WI 53792.

{dagger} Present address: Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235.

Journal of Virology, November 2005, p. 13747-13758, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13747-13758.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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