Proteome-Wide Overexpression of Host Proteins for Identification of Factors Affecting Tombusvirus RNA Replication: an Inhibitory Role of Protein Kinase C

Nawaz-ul-Rehman, Muhammad Shah; Martinez-Ochoa, N.; Pascal, Helene; Sasvari, Zsuzsanna; Herbst, Christin; Xu, Kai; Baker, Jannine; Sharma, M.; Herbst, Alan; Nagy, Peter D.

doi:10.1128/jvi.00019-12

Cited by 42 publications

(28 citation statements)

References 90 publications

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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.…”

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confidence: 99%

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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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“…The yeast Nedd4-type Rsp5p E3 ubiquitin ligase carrying WW domain was identified in several genome-wide screens previously (13,42,48). In addition to Rsp5p, several cellular WWdomain proteins, including Wwm1p, Prp40p, and plant AtDrh1, AtFCA, and AtPrp40c, bind to the tombusvirus replication proteins and inhibit their functions (64,65).…”

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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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“…Another group of cellular restriction factors is the WW motif-containing host proteins, such as Rsp5p Nedd4-like E3 ubiquitin ligase, which regulate the degradation of tombusviral p92 pol in yeast cells and inhibit the activity of VRC in vitro (45,46). Cellular kinases, such as Pkc1p, could also restrict TBSV replication in yeast (32). Taken altogether, studies of cellular restriction factors could help to unravel the full arsenal of the native cellintrinsic innate immune system in the host cell.…”

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confidence: 99%

“…We have performed several genome-wide screens of yeast genes by using different global proteomics approaches that have led to the identification of over 500 host genes/proteins putatively involved in TBSV replication or recombination (17,19,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). The abovementioned systematic screens have identified host stimulatory and restriction factors of TBSV replication.…”

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The Hop-Like Stress-Induced Protein 1 Cochaperone Is a Novel Cell-Intrinsic Restriction Factor for Mitochondrial Tombusvirus Replication

Lin

Nagy

2014

J Virol

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“…ecent genome-wide screens have revealed that replication of RNA viruses inside the infected cells depends on hundreds of factors, most of which have not been characterized (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). In addition, RNA viruses extensively rewire the cells by changing metabolic, secretory, and trafficking pathways, reorganize host membranes, and alter quality control mechanisms (17,18).…”

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The GEF1 Proton-Chloride Exchanger Affects Tombusvirus Replication via Regulation of Copper Metabolism in Yeast

Sasvari

Kovalev

Nagy

2013

J Virol

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Replication of plus-strand RNA viruses [(؉)RNA viruses] is performed by viral replicases, whose function is affected by many cellular factors in infected cells. In this paper, we demonstrate a surprising role for Gef1p proton-chloride exchanger in replication of Tomato bushy stunt virus (TBSV) model (؉)RNA virus. A genetic approach revealed that Gef1p, which is the only proton-chloride exchanger in Saccharomyces cerevisiae, is required for TBSV replication in the yeast model host. We also show that the in vitro activity of the purified tombusvirus replicase from gef1⌬ yeast was low and that the in vitro assembly of the viral replicase in a cell extract was inhibited by the cytosolic fraction obtained from gef1⌬ yeast. Altogether, our data reveal that Gef1p modulates TBSV replication via regulating Cu 2؉ metabolism in the cell. This conclusion is supported by several lines of evidence, including the direct inhibitory effect of Cu 2؉ ions on the in vitro assembly of the viral replicase, on the activity of the viral RNA-dependent RNA polymerase, and an inhibitory effect of deletion of CCC2 copper pump on TBSV replication in yeast, while altered iron metabolism did not reduce TBSV replication. In addition, applying a chloride channel blocker impeded TBSV replication in Nicotiana benthamiana protoplasts or in whole plants. Overall, blocking Gef1p function seems to inhibit TBSV replication through altering Cu 2؉ ion metabolism in the cytosol, which then inhibits the normal functions of the viral replicase.

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Proteome-Wide Overexpression of Host Proteins for Identification of Factors Affecting Tombusvirus RNA Replication: an Inhibitory Role of Protein Kinase C

Cited by 42 publications

References 90 publications

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

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

The Hop-Like Stress-Induced Protein 1 Cochaperone Is a Novel Cell-Intrinsic Restriction Factor for Mitochondrial Tombusvirus Replication

The GEF1 Proton-Chloride Exchanger Affects Tombusvirus Replication via Regulation of Copper Metabolism in Yeast

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