Template Role of Double-Stranded RNA in Tombusvirus Replication

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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

Section: Reduced Tombusvirus Replication In Nicotiana Benthamianasupporting

confidence: 71%

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

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“…Tombusvirus replication leads to the production of dsRNA intermediates that are used to make an excess amount of new (ϩ)RNA progeny in infected cells (29). However, dsRNA is a strong inducer of RNA silencing, an innate antiviral response of the host cells (8)(9)(10).…”

Section: Discussionmentioning

confidence: 99%

“…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)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(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).…”

mentioning

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

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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

“…The TBSV repRNA can go through a single full cycle of replication (producing doublestranded RNA intermediate on added plus-stranded template and excess amount of new plus-stranded RNAs) in yeast CFE when purified recombinant p33 and p92 pol replication proteins are provided (Fig. 3B, lane 1) (37,56,73). When WW-domain protein or Rsp5p were added to the CFE assay, then TBSV replication was ϳ10% of the control assay containing purified GST protein (Fig.…”

Section: Inhibition Of the Rna-binding And Protein-interaction Functimentioning

confidence: 99%

Novel Mechanism of Regulation of Tomato Bushy Stunt Virus Replication by Cellular WW-Domain Proteins

Barajas

Kovalev

Qin

et al. 2015

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Replication of (؉)RNA viruses depends on several co-opted host proteins but is also under the control of cell-intrinsic restriction factors (CIRFs). By using tombusviruses, small model viruses of plants, we dissect the mechanism of inhibition of viral replication by cellular WW-domain-containing proteins, which act as CIRFs. By using fusion proteins between the WW domain and the p33 replication protein, we show that the WW domain inhibits the ability of p33 to bind to the viral RNA and to other p33 and p92 replication proteins leading to inhibition of viral replication in yeast and in a cell extract. Overexpression of WWdomain protein in yeast also leads to reduction of several co-opted host factors in the viral replicase complex (VRC). These host proteins, such as eEF1A, Cdc34 E2 ubiquitin-conjugating enzyme, and ESCRT proteins (Bro1p and Vps4p), are known to be involved in VRC assembly. Simultaneous coexpression of proviral cellular factors with WW-domain protein partly neutralizes the inhibitory effect of the WW-domain protein. We propose that cellular WW-domain proteins act as CIRFs and also as regulators of tombusvirus replication by inhibiting the assembly of new membrane-bound VRCs at the late stage of infection. We suggest that tombusviruses could sense the status of the infected cells via the availability of cellular susceptibility factors versus WWdomain proteins for binding to p33 replication protein that ultimately controls the formation of new VRCs. This regulatory mechanism might explain how tombusviruses could adjust the efficiency of RNA replication to the limiting resources of the host cells during infections. IMPORTANCEReplication of positive-stranded RNA viruses, which are major pathogens of plants, animals, and humans, is inhibited by several cell-intrinsic restriction factors (CIRFs) in infected cells. We define here the inhibitory roles of the cellular Rsp5 ubiquitin ligase and its WW domain in plant-infecting tombusvirus replication in yeast cells and in vitro using purified components. The WW domain of Rsp5 binds to the viral RNA-binding sites of p33 and p92 replication proteins and blocks the ability of these viral proteins to use the viral RNA for replication. The WW domain also interferes with the interaction (oligomerization) of p33 and p92 that is needed for the assembly of the viral replicase. Moreover, WW domain also inhibits the subversion of several cellular proteins into the viral replicase, which otherwise play proviral roles in replication. Altogether, Rsp5 is a CIRF against a tombusvirus, and it possibly has a regulatory function during viral replication in infected cells. P lus-stranded (ϩ)RNA viruses, which are widespread and emerging pathogens, replicate in the cytosol of infected cells by assembling membrane-bound viral replicase complexes (VRCs). The VRCs consist of the viral RNA and viral proteins, as well as co-opted host-coded proteins (1-8). Rapid progress has recently been made in understanding the functions of the viral replication proteins, including the v...