title: Brome mosaic virus 1a nucleoside triphosphatase/helicase domain plays crucial roles in recruiting RNA replication templates.
abstract: 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 2a(pol) has a central polymerase domain. 1a accumulates on endoplasmic reticulum membranes, recruits 2a(pol), 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 2a(pol), 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 2a(pol). 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.
authors.affiliation: Institute for Molecular Virology, University of Wisconsin-Madison, 1525 Linden Dr., Madison,WI 53706-1576, USA.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Wang
authors.forename: Wai-Ming
authors.initials: WM
authors.lastname: Lee
authors.forename: Tokiko
authors.initials: T
authors.lastname: Watanabe
authors.forename: Michael
authors.initials: M
authors.lastname: Schwartz
authors.forename: Michael
authors.initials: M
authors.lastname: Janda
authors.forename: Paul
authors.initials: P
authors.lastname: Ahlquist
mesh_terms: D017795:Bromovirus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D043583:Nucleoside-Triphosphatase / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013698:Templates, Genetic
chemical_list: D012367:RNA, Viral; D043583:Nucleoside-Triphosphatase; D020365:RNA Helicases