Authentic Replication and Recombination of Tomato Bushy Stunt Virus RNA in a Cell-Free Extract from Yeast

Self Cite

pol replication proteins. We find that overexpression of Rsp5p inhibits TBSV replication in Saccharomyces cerevisiae yeast, while downregulation of Rsp5p leads to increased TBSV accumulation. The inhibition is caused by Rsp5p-guided degradation of p92 pol , while the negative effect on the p33 level is less pronounced. Interestingly, recombinant Rsp5p also inhibits TBSV RNA replication in a cell-free replication assay, likely due to its ability to bind to p33 and p92 pol . We show that the WW domain of Rsp5p, which is involved in protein interactions, is responsible for inhibition of TBSV replication, whereas the HECT domain, involved in protein ubiquitination, is not necessary for Rsp5p-mediated inhibition of viral replication. Overall, our data suggest that direct binding between Rsp5p and p92 pol reduces the stability of p92 pol , with consequent inhibition of TBSV replicase activity.Various interactions with their host cells are critical for plus-stranded (ϩ)RNA viruses as they attempt to utilize the host translation machinery to produce viral proteins, gain access to the resources of the host cells, co-opt host proteins, and subvert host membranes (1, 17). Additional levels of interaction between virus and host reflect antiviral responses which may involve innate immunity, as well as other antiviral processes and factors. On-going research with several model viruses is striving to map all the interactions between viruses and hosts and characterize the functions of the co-opted host factors. In this regard, recent research has led to the identification of a large number of host proteins which affect the replication of various (ϩ)RNA viruses and minus-stranded RNA viruses (4,5,9,11,22,35,39). The roles and functions of most of the host proteins identified as being involved in RNA virus replication, however, are currently unknown.Tombusviruses, such as Tomato bushy stunt virus (TBSV), are among the most advanced model systems in relation to the identification of host factors affecting (ϩ)RNA virus replication. The TBSV genome codes for only five proteins, two of which are the replication proteins translated directly from the genomic RNA (45). One of these replication proteins is the abundant p33 replication cofactor; the other is the RNA-dependent RNA polymerase (RdRp) p92pol . Due to the overlapping expression strategy, p33 is identical with the N-terminal portion of the larger p92 pol protein (Fig. 1A). Both replication proteins contain an RNA-binding motif (arginine-proline-rich motif), phosphorylation sites that affect RNA binding by the p33 protein, a p33-p33/p92 interaction domain, and two transmembrane domains (Fig. 1A) (18,19,32,36,37). Three short stretches of amino acids in p33 and p92 pol are involved in binding to the Pex19p host protein that facilitates the transportation of p33 and p92 pol from the cytosol to the cytosolic surface of the peroxisomes, the site of replicase complex formation and viral RNA replication (25). The essential nature of the above-named domains for obtaining functional rep...

Section: Methodsmentioning

confidence: 99%

The Nedd4-Type Rsp5p Ubiquitin Ligase Inhibits Tombusvirus Replication by Regulating Degradation of the p92 Replication Protein and Decreasing the Activity of the Tombusvirus Replicase

Barajas

Nagy

2009

Self Cite

“…5. The TBSV replication assay was performed for 3 h at 25°C, followed by phenol-chloroform extraction and denaturing PAGE as described earlier (60,61).…”

Section: Methodsmentioning

confidence: 99%

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

Sasvari

Kovalev

Nagy

2013

Self Cite

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.

“…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%

“…The presented data indicate that binding of the WW domain from Rsp5p to p33 and p92 pol (that contains p33 sequence due to the overlapping expression strategy) blocks interaction of the replication proteins with (i) the viral RNA, (ii) the oligomerization with other p33 and p92 pol molecules, and (iii) a set of stimulatory (susceptibility) host factors subverted for TBSV replication. All of these interactions are needed for the assembly of new VRCs and the activation of p92 pol , which is initially inactive in the cytosol after translation (30,37,39,56). Based on these features involving many of the VRC components, the WW-domain proteins seem to exhibit a complex mechanism as CIRFs.…”

Section: Rsp5p and Ww-domain Proteins Act As Cirfs Against Tombusvirumentioning

confidence: 99%

See 1 more Smart Citation

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

Barajas

Kovalev

Qin

et al. 2015

Self Cite

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...