Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites

Barajas, Daniel; Xu, Kai; Martín, Isabel Fernández de Castro; Sasvari, Zsuzsanna; Brandizzí, Federica; Risco, Cristina; Nagy, Peter D.

doi:10.1371/journal.ppat.1004388

Cited by 102 publications

(151 citation statements)

References 97 publications

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“…The study of how intracellular pathogens manipulate lipid traffic and membrane biogenesis is an expanding research area (Sarowar et al, 2009;Mutsafi et al, 2013;Barajas et al, 2014b;Konan and Sanchez-Felipe, 2014;Nour and Modis, 2014;Paul et al, 2014). We show the importance of 3D imaging when combined with new methods for sensitive molecular mapping to better understand viral replication in cells.…”

Section: Discussionmentioning

confidence: 93%

Three dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex

Castro

Fernández

Barajas

et al. 2016

Journal of Cell Science

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Positive-strand RNA viruses, which can be devastating pathogens in humans, animals and plants, replicate their genomes on intracellular membranes. Here, we describe the three-dimensional ultrastructural organization of a tombusvirus replicase in yeast, a valuable model for exploring virus-host interactions. We visualized the intracellular distribution of a viral replicase protein using metal-tagging transmission electron microscopy, a highly sensitive nanotechnology whose full potential remains to be developed. These three-dimensional images show how viral replicase molecules are organized when they are incorporated into the active domains of the intracellular replication compartment. Our approach provides a means to study protein activation mechanisms in cells and to identify targets for new antiviral compounds.

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Section: Discussionmentioning

confidence: 93%

Three dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex

Castro

Fernández

Barajas

et al. 2016

Journal of Cell Science

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“…All the methods used here have previously been described (8,52,65). Leaves of N. benthamiana were agroinfiltrated with a construct expressing CNV 20kSTOP (for genomic RNA [gRNA] replication) or with a mixture of A. tumefaciens cultures separately expressing CNV p33 and p92 replication proteins, p19 protein (viral suppressor of gene silencing), and DI-72 RNA.…”

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confidence: 99%

Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase

Kovalev

Martín

Pogany

et al. 2016

J Virol

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Plus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs).One of the intriguing aspects of VRC formation is the need for extensive subcellular membrane deformations. Accordingly, (ϩ)RNA viruses replicate in various membranous structures that could be formed from various membranes, such as endoplasmic reticulum, mitochondria, endosome, chloroplast, peroxisome, and plasma membranes, or induced de novo (1,(3)(4)(5). Membrane deformations are possibly induced by co-opted cellular phospholipid kinases, local enrichment of sterols, and subverted membrane-bending proteins, such as ESCRT factors, reticulons, and amphiphysins (6-12).A major type of subcellular membrane deformation induced by some (ϩ)RNA viruses is represented by vesicle-like small invaginations with single narrow openings toward the cytosol (13,14). These structures, called spherules, contain the membranebound VRCs consisting of viral and co-opted cellular proteins in the infected cells (1)(2)(3)(15)(16)(17)(18). The membranous spherule structures sequester all the replication factors into a confined cytosolic area and likely protect the fragile viral (ϩ)RNA from degradation

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“…Hsp70, eEF1A, Cdc34p, and ESCRT proteins are involved in the assembly of the tombusviral VRC, while other subverted RNA-binding proteins (eEF1A, eEF1B␥, GAPDH, Ded1p, and RH2) facilitate viral RNA synthesis (1,3,18,31). Tombusvirus replication also depends on lipids, such as sterols and phospholipids, and oxysterol-binding proteins (ORPs) that help the formation of the sterol-and phospholipid-rich microenvironment needed for VRC formation (32)(33)(34).…”

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confidence: 99%

Activation of Tomato Bushy Stunt Virus RNA-Dependent RNA Polymerase by Cellular Heat Shock Protein 70 Is Enhanced by Phospholipids In Vitro

Pogany

Nagy

2015

J Virol

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Similar to other positive-strand RNA viruses, tombusviruses are replicated by the membrane-bound viral replicase complex (VRC). The VRC consists of the p92 virus-coded RNA-dependent RNA polymerase (RdRp), the viral p33 RNA chaperone, and several co-opted host proteins. In order to become a functional RdRp after its translation, the p92 replication protein should be incorporated into the VRC, followed by its activation. We have previously shown in a cell-free yeast extract-based assay that the activation of the Tomato bushy stunt virus (TBSV) RdRp requires a soluble host factor(s). In this article, we identify the cellular heat shock protein 70 (Hsp70) as the co-opted host factor required for the activation of an N-terminally truncated recombinant TBSV RdRp. In addition, small-molecule-based blocking of Hsp70 function inhibits RNA synthesis by the tombusvirus RdRp in vitro. Furthermore, we show that neutral phospholipids, namely, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), enhance RdRp activation in vitro. 1-7). Interestingly, the viral RdRp is assumed to be inactive in the cytosol to prevent the formation of a viral double-stranded RNA (dsRNA) intermediate that could trigger efficient gene silencing and RNAinduced innate immunity (8-10). Therefore, the VRC assembly, including the activation of the viral RdRp, could be a critical regulatory step during the infection cycle.The tombusvirus VRCs are housed in membranous spherule structures, which are membrane invaginations into peroxisomes or mitochondria in the cases of Tomato bushy stunt virus (TBSV) and Carnation Italian ringspot virus (CIRV), respectively (11-15). The tombusvirus VRCs contain the virus-coded p92 RdRp and p33 RNA chaperone and the dsRNA intermediate, as well as co-opted cellular proteins, such as the heat shock protein 70 (Hsp70) molecular chaperone (coded for by yeast [Saccharomyces cerevisiae] SSA1/2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH [coded for by TDH2/3 in yeast]), Cdc34p E2 ubiquitinconjugating enzyme, eukaryotic translation elongation factor 1A (eEF1A), eEF1B␥, the Vps4p ESCRT (endosomal sorting complex required for transport) protein, and DDX3-like Ded1p and eIF4AIII-like RH2 DEAD box helicases (16-30). Hsp70, eEF1A, Cdc34p, and ESCRT proteins are involved in the assembly of the tombusviral VRC, while other subverted RNA-binding proteins (eEF1A, eEF1B␥, GAPDH, Ded1p, and RH2) facilitate viral RNA synthesis (1,3,18,31). Tombusvirus replication also depends on lipids, such as sterols and phospholipids, and oxysterol-binding

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Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites

Cited by 102 publications

References 97 publications

Three dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex

Three dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex

Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase

Activation of Tomato Bushy Stunt Virus RNA-Dependent RNA Polymerase by Cellular Heat Shock Protein 70 Is Enhanced by Phospholipids In Vitro

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