Many viruses that replicate in the cytoplasm compartmentalize their genome replication and transcription in organelle-like structures that enhance replication efficiency and protection from host defenses. In particular, recent studies with diverse positive-strand RNA viruses have further elucidated the ultrastructure of membrane-bounded RNA replication complexes and their close coordination with virion assembly and budding. The structure, function and assembly of some positive-strand RNA virus replication complexes have parallels and potential evolutionary links with the replicative cores of double-strand RNA virus and retrovirus virions, and more general similarities with the replication factories of cytoplasmic DNA viruses.

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Positive-strand RNA viruses replicate their genomes on membranes with virus-induced rearrangements such as single-or doublemembrane vesicles, but the mechanisms of such rearrangements, including the role of host proteins, are poorly understood. Brome mosaic virus (BMV) RNA synthesis occurs in ≈70 nm, negatively curved endoplasmic reticulum (ER) membrane invaginations induced by multifunctional BMV protein 1a. We show that BMV RNA replication is inhibited 80-90% by deleting the reticulon homology proteins (RHPs), a family of membrane-shaping proteins that normally induce and stabilize positively curved peripheral ER membrane tubules. In RHP-depleted cells, 1a localized normally to perinuclear ER membranes and recruited the BMV 2a pol polymerase. However, 1a failed to induce ER replication compartments or to recruit viral RNA templates. Partial RHP depletion allowed formation of functional replication vesicles but reduced their diameter by 30-50%. RHPs coimmunoprecipitated with 1a and 1a expression redirected >50% of RHPs from peripheral ER tubules to the interior of BMV-induced RNA replication compartments on perinuclear ER. Moreover, RHP-GFP fusions retained 1a interaction but shifted 1a-induced membrane rearrangements from normal vesicles to double membrane layers, a phenotype also induced by excess 1a-interacting 2a pol . Thus, RHPs interact with 1a, are incorporated into RNA replication compartments, and are required for multiple 1a functions in replication compartment formation and function. The results suggest possible RHP roles in the bodies and necks of replication vesicles.positive-strand RNA virus | reticulons | genomic RNA replication complex | endoplasmic reticulum vesicles A universal feature of (+)RNA viruses is that they replicate their RNA on intracellular membranes, usually in association with vesiculation or other membrane rearrangements (1-3). As a highly conserved feature of (+)RNA virus life cycles, such membrane involvement is a potentially valuable target for broadspectrum antiviral treatments.Brome mosaic virus (BMV) is a member of the alphavirus-like superfamily of human, animal, and plant viruses. BMV encodes two RNA replication proteins. BMV 1a contains an N-proximal domain with m 7 G methyltransferase and covalent m 7 GMPbinding activities required for viral RNA capping (4-6), and a Cterminal NTPase/RNA helicase-like domain (7). BMV 2a pol has a central RNA-dependent RNA polymerase-like domain and an N-terminal domain that interacts with the 1a helicase domain (8, 9). In both the yeast Saccharomyces cerevisiae and its natural plant hosts, BMV RNA is selectively encapsidated into progeny virions (10), and BMV RNA replication depends on 1a, 2a pol and specific cis-acting RNA signals (11), localizes to ER membranes (12, 13), generates a dramatic excess of positive-to negative-strand RNA (14), and efficiently directs subgenomic mRNA synthesis (14).BMV 1a localizes to perinuclear ER membranes and induces 60-to 75-nm vesicular invaginations or spherules. BMV 1a's high multiplicity in spherul...

Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2aPol) and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic α-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a–ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a–membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2aPol accumulation. The second class of helix A mutations not only maintains efficient 1a–membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2aPol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.