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Membrane-shaping host reticulon proteins play crucial roles in viral RNA replication compartment formation and function.

ABSTRACT: Positive-strand RNA viruses replicate their genomes on membranes with virus-induced rearrangements such as single- or double-membrane 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.

SUBMITTER: Diaz A

PROVIDER: S-EPMC2941330 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Membrane-shaping host reticulon proteins play crucial roles in viral RNA replication compartment formation and function.

Diaz Arturo A Wang Xiaofeng X Ahlquist Paul P

Proceedings of the National Academy of Sciences of the United States of America 20100830 37

Positive-strand RNA viruses replicate their genomes on membranes with virus-induced rearrangements such as single- or double-membrane 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 homolo ...[more]

PMID: 20805477

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Project description:Legionella pneumophila grows within cells ranging from environmental amoebae to human macrophages. In spite of this conserved strategy of pathogenesis, identification of host factors that restrict L. pneumophila intracellular replication has not been extended outside components of the mammalian innate immune response. We performed a double-stranded RNA (dsRNA) screen against more than 50% of the Drosophila melanogaster annotated open reading frames (ORFs) to identify host cell factors that restrict L. pneumophila The majority of analyzed dsRNAs that stimulated L. pneumophila intracellular replication were directed against host proteins involved in protein synthesis or cell cycle control. Consistent with disruption of the cell cycle stimulating intracellular replication, proteins involved in translation initiation also resulted in G1 arrest. Stimulation of replication was dependent on the stage of cell cycle arrest, as dsRNAs causing arrest during S phase had an inhibitory effect on intracellular replication. The inhibitory effects of S phase arrest could be recapitulated in a human cell line, indicating that cell cycle control of L. pneumophila replication is evolutionarily conserved. Synchronized HeLa cell populations in S phase and challenged with L. pneumophila failed to progress through the cell cycle and were depressed for supporting intracellular replication. Poor bacterial replication in S phase was associated with loss of the vacuole membrane barrier, resulting in exposure of bacteria to the cytosol and their eventual degradation. These results are consistent with the model that S phase is inhibitory for L. pneumophila intracellular survival as a consequence of failure to maintain the integrity of the membrane surrounding intracellular bacteria.IMPORTANCELegionella pneumophila has the ability to replicate within human macrophages and amoebal hosts. Here, we report that the host cell cycle influences L. pneumophila intracellular replication. Our data demonstrate that the G1 and G2/M phases of the host cell cycle are permissive for bacterial replication, while S phase is toxic for the bacterium. L. pneumophila replicates poorly within host cells present in S phase. The inability of L. pneumophila to replicate relies on its failure to control the integrity of its vacuole, leading to cytosolic exposure of the bacteria and eventual degradation. The data presented here argue that growth-arrested host cells that are encountered by L. pneumophila in either the environment or within human hosts are ideal targets for intracellular replication because their transit through S phase is blocked.

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Membrane-shaping host reticulon proteins play crucial roles in viral RNA replication compartment formation and function.

Publications

Membrane-shaping host reticulon proteins play crucial roles in viral RNA replication compartment formation and function.

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