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.Positive-strand RNA viruses are a large class of viral pathogens causing numerous clinically and economically important diseases of humans, animals, and plants. Although such viruses encompass substantial variation in morphology, genetic organization, host range, and other properties, they all share fundamental similarities in their basic replication mechanisms. For example, genome replication by positive-strand RNA viruses is universally associated with intracellular membranes, which usually are induced by viral replication proteins to form invaginations or vesicles (51). In the early steps of positive-strand RNA virus replication, the viral genomic RNAs first serve as templates for translating these replication proteins and often other viral proteins. Once such proteins induce formation of the membraneassociated replication complexes, the incoming viral genomic RNA must be recruited away from translation to serve as a template for RNA replication. This genomic RNA transition from translation to RNA replication is a crucial step in early infection and must be tightly regulated to effectively balance translation and replication (44). Nevertheless, the mechanisms of...

Selective Repression of Translation by the Brome Mosaic Virus 1a RNA Replication Protein

“…These PK mutants had similar stabilities to that of wildtype 1a (36). The two single amino acid substitutions, R136A and K691A, in the methyltransferase and helicase domains, respectively, were previously characterized for RNA replication, replicase assembly, and other 1a-associated activities (3,36,48). We first determined whether 1a mutations could affect the abundance of the BMV RNAs.…”

“…Since 1a acts on RNA1, this could work as a regulatory loop to modulate the degree of inhibition. The 1a protein is thus the ultimate multitasking protein in BMV infection, with demonstrated roles in replication, RNA modification, replicase assembly, rapid repair of truncations in the BMV 3Ј UTRs, and translation (2,7,16,21,23,48). Since 1a repression of viral RNA replication can be partially reversed by overexpressing 2a (Fig.…”

“…The N-terminal domain contains activities for 7-methyl-guanosine methyltransferase and covalent GTP binding required for viral RNA capping (2,23). The C-terminal domain contains all of the motifs of the DEAD box RNA helicase domain, with ATPase and GTPase activities (24,48). 1a is the primary viral protein determinant for the subcellular localization of the BMV RNA replication complex (9,39,40).…”

An unrecognized function for COPII components in recruiting the viral replication protein BMV 1a to the perinuclear ER

“…BMV 2a pol contains a central RNA-dependent RNA polymerase (RdRp) domain and serves as a replicase. BMV 1a contains an N-terminal RNAcapping domain (Ahola and Ahlquist, 1999;Kong et al, 1999) and a C-terminal NTPase or helicase-like domain (Wang et al, 2005). In yeast, expression of 1a and 2a pol , along with other host factors, supports the replication of BMV genomic RNAs.…”

Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

“…BMV encodes two replication proteins, 1a and 2a pol . 2a pol serves as the replicase, whereas 1a has an N-terminal methyltransferase domain (3,4) and a C-terminal ATPase/helicase-like domain (5). Together, 1a and 2a pol are necessary and sufficient for BMV replication.…”