Cdc34p Ubiquitin-Conjugating Enzyme Is a Component of the Tombusvirus Replicase Complex and Ubiquitinates p33 Replication Protein

Li, Zhenghe; Barajas, Daniel; Panavas, Tadas; Herbst, D. Alan; Nagy, Peter D.

doi:10.1128/jvi.00702-08

Cited by 121 publications

(218 citation statements)

References 69 publications

(109 reference statements)

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“…Information from genome-wide genetic or proteomic screens (Kushner et al, 2003;Panavas et al, 2005;Serviene et al, 2006;Li et al, 2008;Gancarz et al, 2011;Lozano-Durán et al, 2011b) supports the idea that ubiquitination of viral proteins is important for viral multiplication. Direct demonstration of Ub conjugation of viral proteins has also been reported in a few cases (described further below; Table II).…”

Section: Viral Protein Targets: Is Ub Friend or Foe?mentioning

confidence: 83%

“…Cdc34p was found to interact directly with the TBSV p33 replication protein, leading to its monoubiquitination and biubiquitination (Li et al, 2008). Ubiquitination of p33 seems to have no effect on its metabolic stability (Barajas and Nagy, 2010).…”

Section: Monoubiquitination Of Viral Replication Proteinsmentioning

confidence: 99%

“…In the case of the tombusvirus Tomato bushy stunt virus (TBSV), Cdc34p E2 Ub-conjugating enzyme was described as a novel component of the viral replication complex and as critical for efficient replicase activity (Li et al, 2008). Cdc34p was found to interact directly with the TBSV p33 replication protein, leading to its monoubiquitination and biubiquitination (Li et al, 2008).…”

Section: Monoubiquitination Of Viral Replication Proteinsmentioning

confidence: 99%

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Ubiquitin and Plant Viruses, Let’s Play Together!

2012

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Section: Viral Protein Targets: Is Ub Friend or Foe?mentioning

confidence: 83%

Section: Monoubiquitination Of Viral Replication Proteinsmentioning

confidence: 99%

Section: Monoubiquitination Of Viral Replication Proteinsmentioning

confidence: 99%

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Ubiquitin and Plant Viruses, Let’s Play Together!

2012

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“…Saccharomyces cerevisiae strain BY4741 (MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0) and the single-gene deletion strain ⌬pex19, as well as the ERG25/THC (BY4741; URA3::CMV-tTA) strain with the regulatable TET promoter from the Hughes collection, were obtained from Open Biosystems (Huntville, AL). The following yeast expression plasmids have been generated before: pGAD-His92 (41), pGBK-His33/DI-72 (16), pGBK-His33/CUP1 (15), pGAD-His92CUP1 (25), and pYC/DI72 (37).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Sterol Biosynthesis Reduces Tombusvirus Replication in Yeast and Plants

Sharma

Sasvari

Nagy

2010

J Virol

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The replication of plus-strand RNA viruses depends on subcellular membranes. Recent genome-wide screens have revealed that the sterol biosynthesis genes ERG25 and ERG4 affected the replication of Tomato bushy stunt virus (TBSV) in a yeast model host. To further our understanding of the role of sterols in TBSV replication, we demonstrate that the downregulation of ERG25 or the inhibition of the activity of Erg25p with an inhibitor (6-amino-2-n-pentylthiobenzothiazole; APB) leads to a 3-to 5-fold reduction in TBSV replication in yeast. In addition, the sterol biosynthesis inhibitor lovastatin reduced TBSV replication by 4-fold, confirming the importance of sterols in viral replication. We also show reduced stability for the p92 pol viral replication protein as well as a decrease in the in vitro activity of the tombusvirus replicase when isolated from APB-treated yeast. Moreover, APB treatment inhibits TBSV RNA accumulation in plant protoplasts and in Nicotiana benthamiana leaves. The inhibitory effect of APB on TBSV replication can be complemented by exogenous stigmasterol, the main plant sterol, suggesting that sterols are required for TBSV replication. The silencing of SMO1 and SMO2 genes, which are orthologs of ERG25, in N. benthamiana reduced TBSV RNA accumulation but had a lesser inhibitory effect on the unrelated Tobacco mosaic virus, suggesting that various viruses show different levels of dependence on sterol biosynthesis for their replication.Plus-stranded RNA [(ϩ)RNA] viruses usurp various intracellular/organellar membranes for their replication. These cellular membranes are thought to facilitate the building of viral factories, promote a high concentration of membrane-bound viral proteins, and provide protection against cellular nucleases and proteases (1,12,35,44). The membrane lipids and proteins may serve as scaffolds for targeting the viral replication proteins or for the assembly of the viral replicase complex. The subcellular membrane also may provide critical lipid or protein cofactors to activate/modulate the function of the viral replicase. Indeed, the formation of spherules, consisting of lipid membranes bended inward and viral replication proteins as well as recruited host proteins, has been demonstrated for several (ϩ)RNA viruses (20,30,48). These virus-induced spherules serve as sites of viral replication. Importantly, (ϩ)RNA viruses also induce membrane proliferation that requires new lipid biosynthesis. Therefore, it is not surprising that several genome-wide screens for the identification of host factors affecting (ϩ)RNA virus replication unraveled lipid biosynthesis/metabolism genes (8,23,38,50). However, in spite of these intensive efforts, understanding the roles of various lipids and lipid biosynthesis enzymes and pathways in (ϩ)RNA virus replication is limited.Tomato bushy stunt virus (TBSV) is among the most advanced model systems regarding the identification of host factors affecting (ϩ)RNA virus replication (32). Among the five proteins encoded by the TBSV genome, only the p33 re...

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“…The host proteins interacted with the viral replication proteins and viral RNA or affected TBSV replication and recombination when deleted/downregulated or overexpressed in host cells (11,13,(40)(41)(42)(43)(44)(45)(46)(47)(48). Cataloging of the host factors affecting TBSV replication is one of the most complete among pathogens at a single cell level, thus facilitating mechanistic studies.…”

mentioning

confidence: 99%

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

Barajas

Kovalev

Qin

et al. 2015

J Virol

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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...

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Cdc34p Ubiquitin-Conjugating Enzyme Is a Component of the Tombusvirus Replicase Complex and Ubiquitinates p33 Replication Protein

Cited by 121 publications

References 69 publications

Ubiquitin and Plant Viruses, Let’s Play Together!

Ubiquitin and Plant Viruses, Let’s Play Together!

Inhibition of Sterol Biosynthesis Reduces Tombusvirus Replication in Yeast and Plants

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

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