title: Bromovirus RNA replication compartment formation requires concerted action of 1a's self-interacting RNA capping and helicase domains.
abstract: All positive-strand RNA viruses replicate their genomes in association with rearranged intracellular membranes such as single- or double-membrane vesicles. Brome mosaic virus (BMV) RNA synthesis occurs in vesicular endoplasmic reticulum (ER) membrane invaginations, each induced by many copies of viral replication protein 1a, which has N-terminal RNA capping and C-terminal helicase domains. Although the capping domain is responsible for 1a membrane association and ER targeting, neither this domain nor the helicase domain was sufficient to induce replication vesicle formation. Moreover, despite their potential for mutual interaction, the capping and helicase domains showed no complementation when coexpressed in trans. Cross-linking showed that the capping and helicase domains each form trimers and larger multimers in vivo, and the capping domain formed extended, stacked, hexagonal lattices in vivo. Furthermore, coexpressing the capping domain blocked the ability of full-length 1a to form replication vesicles and replicate RNA and recruited full-length 1a into mixed hexagonal lattices with the capping domain. Thus, BMV replication vesicle formation and RNA replication depend on the direct linkage and concerted action of 1a's self-interacting capping and helicase domains. In particular, the capping domain's strong dominant-negative effects showed that the ability of full-length 1a to form replication vesicles was highly sensitive to disruption by non-productively titrating lattice-forming self-interactions of the capping domain. These and other findings shed light on the roles and interactions of 1a domains in replication compartment formation and support prior results suggesting that 1a induces replication vesicles by forming a capsid-like interior shell.
authors.affiliation: Institute for Molecular Virologya and Howard Hughes Medical Institute, Madison, Madison, Wisconsin 53706, USA.
authors.forename: Arturo
authors.initials: A
authors.lastname: Diaz
authors.forename: Andreas
authors.initials: A
authors.lastname: Gallei
authors.forename: Paul
authors.initials: P
authors.lastname: Ahlquist
mesh_terms: D017795:Bromovirus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D002467:Cell Nucleus / Q000821:virology; D004721:Endoplasmic Reticulum / Q000821:virology; D015967:Gene Expression Regulation, Viral; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012315:RNA Caps / Q000235:genetics* / Q000378:metabolism; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000821:virology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D012315:RNA Caps; D012367:RNA, Viral; D014764:Viral Proteins; D020365:RNA Helicases
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