Host transcription factor Rpb11p affects tombusvirus replication and recombination via regulating the accumulation of viral replication proteins

Previous genome-wide screens identified over 100 host genes whose deletion/down-regulation affected tombusvirus replication and 32 host genes that affected tombusvirus RNA recombination in yeast, a model host for replication of Tomato bushy stunt virus (TBSV). Down-regulation of several of the identified host genes affected the accumulation levels of p33 and p92(pol) replication proteins, raising the possibility that these host factors could be involved in the regulation of the amount of viral replication proteins and, thus, they are indirectly involved in TBSV replication and recombination. To test this model, we developed a tightly regulated expression system for recombinant p33 and p92(pol) replication proteins in yeast. We demonstrate that high accumulation level of p33 facilitated efficient viral RNA replication, while the effect of p33 level on RNA recombination was less pronounced. On the other hand, high level of p92(pol) accumulation promoted TBSV RNA recombination more efficiently than RNA replication. As predicted, Rpb11p, which is part of the polII complex, affected the accumulation levels of p33 and p92(pol) as well as altered RNA replication and recombination. An in vitro assay with the tombusvirus replicase further supported that Rpb11p affects TBSV replication and recombination only indirectly, via regulating p33 and p92(pol) levels. In contrast, the mechanism by which Rpt4p endopeptidase/ATPase and Mps1p threonine/tyrosine kinase affect TBSV recombination is different from that proposed for Rpb11p. We propose a model that the concentration (molecular crowding) of replication proteins within the viral replicase is a factor affecting viral replication and recombination.     

Phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus adjacent to the RNA binding site affects viral RNA replication

Replication of the nonsegmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. In this paper, we demonstrate that p33 is phosphorylated in vivo and in vitro by a membrane-bound plant kinase. Phosphorylation of p33 was also demonstrated in vitro by using purified protein kinase C. The related p28 replication protein of Turnip crinkle virus was also found to be phosphorylated in vivo, suggesting that posttranslational modification of replication proteins is a general feature among members of the large Tombusviridae family. Based on in vitro studies with purified recombinant p33, we show evidence for phosphorylation of threonine and serine residues adjacent to the essential RNA-binding site in p33. Phosphorylation-mimicking aspartic acid mutations rendered p33 nonfunctional in plant protoplasts and in yeast, a model host. Comparable mutations within the prereadthrough portion of p92 did not abolish replication. The nonphosphorylation-mimicking alanine mutants of CNV were able to replicate in plant protoplasts and in yeast, albeit with reduced efficiency when compared to the wild type. These alanine mutants also showed altered subgenomic RNA synthesis and a reduction in the ratio between plus- and minus-strand RNAs produced during CNV infection. These findings suggest that phosphorylation of threonine/serine residues adjacent to the essential RNA-binding site in the auxiliary p33 protein likely plays a role in viral RNA replication and subgenomic RNA synthesis during tombusvirus infections.     

Interaction between the replicase proteins of Tomato bushy stunt virus in vitro and in vivo

Tomato bushy stunt virus (TBSV), a tombusvirus with a non-segmented, plus-stranded RNA genome, codes for p33 and p92 replicase proteins. The sequence of p33 overlaps with the N-terminal domain of p92, which also contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its non-overlapping C-terminal portion. In this research, we demonstrate in vitro interactions between p33:p33 and p33:p92 using surface plasmon resonance analysis with purified recombinant p33 and p92. The sequence in p33 involved in the above protein-protein interactions was mapped to the C-terminal region, which also contains an RNA-binding site. Using the yeast two-hybrid assay, we confirmed that two short regions within p33 could promote p33:p33 and p33:p92 interactions in vivo. Mutations in either p33 or p92 within the short regions involved in p33:p33 and p33:p92 interactions decreased the replication of a TBSV defective interfering RNA in yeast, a model host, supporting the significance of these protein interactions in tombusvirus replication.     

Translation elongation factor 1A is a component of the tombusvirus replicase complex and affects the stability of the p33 replication co-factor

Host RNA-binding proteins are likely to play multiple, integral roles during replication of plus-strand RNA viruses. To identify host proteins that bind to viral RNAs, we took a global approach based on the yeast proteome microarray, which contains 4080 purified yeast proteins. The biotin-labeled RNA probes included two distantly related RNA viruses, namely Tomato bushy stunt virus (TBSV) and Brome mosaic virus (BMV). Altogether, we have identified 57 yeast proteins that bound to TBSV RNA and/or BMV RNA. Among the identified host proteins, eleven bound to TBSV RNA and seven bound to BMV RNA with high selectivity, whereas the remaining 39 host proteins bound to both viral RNAs. The interaction between the TBSV replicon RNA and five of the identified host proteins was confirmed via gel-mobility shift and co-purification experiments from yeast. Over-expression of the host proteins in yeast, a model host for TBSV, revealed 4 host proteins that enhanced TBSV replication as well as 14 proteins that inhibited replication. Detailed analysis of one of the identified yeast proteins binding to TBSV RNA, namely translation elongation factor eEF1A, revealed that it is present in the highly purified tombusvirus replicase complex. We also demonstrate binding of eEF1A to the p33 replication protein and a known cis-acting element at the 3′ end of TBSV RNA. Using a functional mutant of eEF1A, we provide evidence on the involvement of eEF1A in TBSV replication.     

Kinetics and functional studies on interaction between the replicase proteins of Tomato Bushy Stunt Virus: requirement of p33:p92 interaction for replicase assembly

The assembly of the functional replicase complex via protein:protein and RNA:protein interactions among the viral-coded proteins, host factors and the viral RNA on cellular membranes is a key step in the replication process of plus-stranded RNA viruses. In this work, we have characterized essential interactions between p33:p33 and p33:p92 replication proteins of Tomato bushy stunt virus (TBSV), a tombusvirus with a non-segmented, plus-stranded RNA genome. Surface plasmon resonance (SPR) measurements with purified recombinant p33 and p92 demonstrate that p33 interacts with p92 in vitro and that the interaction requires the S1 subdomain, whereas the S2 subdomain plays lesser function. Kinetic SPR analyses showed that binding of S1 subdomain to the C-terminal half of p33 takes place with moderate binding affinity in the nanomolar range whereas S2 subdomain binds to p33 with micromolar affinity. Using mutated p33 and p92 proteins, we identified critical amino acid residues within the p33:p92 interaction domain that play essential role in replication and the assembly of the tombusviral replicase. In addition, we show that interaction takes place between replication proteins of TBSV and the closely related Cucumber necrosis virus but not between TBSV and the more distantly related Turnip crinkle virus, suggesting that selective protein interactions might prevent the assembly of chimeric replicases carrying replication proteins from different viruses during mixed infections.     

The role of the p33:p33/p92 interaction domain in RNA replication and intracellular localization of p33 and p92 proteins of Cucumber necrosis tombusvirus

Replication of plus-stranded RNA viruses is performed by the viral replicase complex, which, together with the viral RNA, must be targeted to intracellular membranes, where replication takes place in membraneous vesicles/spherules. Tombusviruses code for two overlapping replication proteins, the p33 auxiliary protein and the p92 polymerase. Using replication-competent fluorescent protein-tagged p33 of Cucumber necrosis virus (CNV), we determined that two domains affected p33 targeting to peroxisomal membranes in yeast: an N-proximal hydrophobic trans-membrane sequence and the C-proximal p33:p33/p92 interaction domain. On the contrary, only the deletion of the p33:p33/p92 interaction domain, but not the trans-membrane sequence, altered the intracellular targeting of p92 protein in the presence of wt p33 and DI-72(+) RNA. Moreover, unlike p33, p92 lacking the trans-membrane sequence was still functional in supporting the replication of a replicon RNA in yeast, whereas the p33:p33/p92 interaction domain in both p33 and p92 was essential for replication. In addition, p33 was also shown to facilitate the recruitment of the viral RNA to peroxisomal membranes and that p33 is colocalized with (+) and (-)-stranded viral RNAs. Also, FRET and pull-down analyses confirmed that p33 interacts with other p33 molecules in yeast cells. Based on these data, we propose that p33 facilitates the formation of multimolecular complexes, including p33, p92, viral RNA, and unidentified host factors, which are then targeted to the peroxisomal membranes, the sites of CNV replication.     

Replication of Tomato bushy stunt virus RNA in a plant in vitro system

An ideal system to investigate individual determinants of the replication process of (+)-strand RNA viruses is a cell-free extract that supports viral protein and RNA synthesis in a synchronized manner. Here, we applied a translation/replication system based on cytoplasmic extracts of Nicotiana tabacum cells to Tomato bushy stunt virus (TBSV) RNA. In vitro translated TBSV proteins p33 and p92 form viral replicase, which, in the same reaction, accomplishes the entire replication cycle on exogenous TBSV DI or full-length RNA. Tests of mutant TBSV RNAs confirmed the template specificity of the in vitro replication reaction. Complementation experiments ascertained the significance of an earlier identified TBSV host factor. Interestingly, formation of the viral replicase occurs also in the absence of concurrent protein synthesis demonstrating that translation and RNA replication are not functionally linked in this system. Our studies with cell-free extracts of a plant host thus confirmed earlier findings and enabled novel insights into the TBSV RNA replication process.     

Exploiting alternative subcellular location for replication: tombusvirus replication switches to the endoplasmic reticulum in the absence of peroxisomes

Plus-strand RNA virus replication takes place on distinct membranous surfaces in infected cells via the assembly of viral replicase complexes involving multiple viral and host proteins. One group of tombusviruses, such as Tomato bushy stunt virus (TBSV), replicate on the surfaces of peroxisomal membranes in plant and yeast cells. Surprisingly, previous genome-wide screen performed in yeast demonstrated that a TBSV replicon RNA replicated as efficiently in yeast defective in peroxisome biogenesis as in the wt yeast (Panavas et al., Proc Natl Acad Sci U S A, 2005). To further test how the lack of peroxisomes could affect tombusvirus replication, we used yeast cells missing either PEX3 or PEX19 genes, which are absolutely essential for peroxisome biogenesis. Confocal microscopy-based approach revealed that the wild-type tombusvirus p33 replication protein accumulated in the endoplasmic reticulum (ER) in pex3Delta or pex19Delta yeast, suggesting that tombusvirus replication could take place on the surface of ER membrane. The activities of the isolated tombusvirus replicase preparations from wt, pex3Delta or pex19Delta yeasts were comparable, demonstrating that the assembly of the replicase was as efficient in the ER as in the authentic subcellular environments. The generation/accumulation of tombusvirus recombinants was similar in wt, pex3Delta and pex19Delta yeasts, suggesting that the rate of mistakes occurring during tombusvirus replication is comparable in the presence or absence of peroxisomes. Overall, this work demonstrates that a tombusvirus, relying on the wt replication proteins, can efficiently replicate on an alternative intracellular membrane. This suggests that RNA viruses might have remarkable flexibility for using various host membranes for their replication.     

Expression of the Arabidopsis Xrn4p 5'-3' exoribonuclease facilitates degradation of tombusvirus RNA and promotes rapid emergence of viral variants in plants

Rapid RNA virus evolution is a major problem due to the devastating diseases caused by human, animal and plant-pathogenic RNA viruses. A previous genome-wide screen for host factors affecting recombination in Tomato bushy stunt tombusvirus (TBSV), a small monopartite plant virus, identified Xrn1p 5'-3' exoribonuclease of yeast, a model host, whose absence led to increased appearance of recombinants [Serviene, E., Shapka, N., Cheng, C.P., Panavas, T., Phuangrat, B., Baker, J., Nagy, P.D., (2005). Genome-wide screen identifies host genes affecting viral RNA recombination. Proc. Natl. Acad. Sci. U. S. A. 102 (30), 10545-10550]. In this paper, we tested if over-expression of Xrn1p in yeast or expression of the analogous Xrn4p cytoplasmic 5'-3' exoribonuclease, which has similar function in RNA degradation in Arabidopsis as Xrn1p in yeast, in Nicotiana benthamiana could affect the accumulation of tombusvirus RNA. We show that over-expression of Xrn1p led to almost complete degradation of TBSV RNA replicons in yeast, suggesting that Xrn1p is involved in TBSV degradation. Infection of N. benthamiana expressing AtXrn4p with Cucumber necrosis tombusvirus (CNV) led to enhanced viral RNA degradation, suggesting that the yeast and the plant cytoplasmic 5'-3' exoribonuclease play similar roles. We also observed rapid emergence of novel CNV genomic RNA variants formed via deletions of 5' terminal sequences in N. benthamiana expressing AtXrn4p. Three of the newly emerging 5' truncated CNV variants were infectious in N. benthamiana protoplasts, whereas one CNV variant caused novel symptoms and moved systemically in N. benthamiana plants. Altogether, this paper establishes that a single plant gene can contribute to the emergence of novel viral variants.     

The host Pex19p plays a role in peroxisomal localization of tombusvirus replication proteins

Replication of Tomato bushy stunt virus (TBSV) RNA takes place on the cytosolic membrane surface of peroxisomes in plants and in yeast, a model host. To identify the host proteins involved in assisting the peroxisomal localization of the tombusvirus p33 replication protein, we tested if p33 could bind directly to yeast proteins involved in peroxisomal transport in vitro. This work has led to the demonstration of Pex19p-p33 interaction via pull-down and co-purification experiments. Pex19p was also detected in the tombusvirus replicase after protein cross-linking, suggesting that Pex19p transiently binds to the replicase as could be expected from a transporter. To validate the importance of Pex19p-p33 interaction in TBSV replication in yeast, we re-targeted Pex19p to the mitochondria, which resulted in the re-distribution of a large fraction of p33 to the mitochondria. The expression of the mitochondrial-targeted Pex19p inhibited TBSV RNA accumulation by 2-4-fold in vivo and reduced the in vitro activity of the tombusvirus replicase by 80%. These data support the model that Pex19p is a cellular transporter for localization of p33 replication protein to the host peroxisomal membranes.     

De novo generation of defective interfering RNAs of tomato bushy stunt virus by high multiplicity passage

Defective interfering (DI) RNAs were generated de novo in each of 12 independent isolates of tomato bushy stunt virus (TBSV) upon serial passage at high multiplicities of infection (m.o.i.) in plants, but not in any of 4 additional isolates after 11 serial passages at low m.o.i. The DI RNAs were detected in RNA isolated from virus particles and in 2.3 M LiCl-soluble RNA fractions isolated from inoculated leaves. Symptom attenuation leading to persistent infections was closely correlated with the passage in which DIs first developed. Comparisons of nucleotide sequences of 10 cDNA clones from 2 DI RNA populations and with a previously characterized TBSV DI RNA revealed the same four regions of sequence from the TBSV genome were strictly conserved in each of the DI RNAs: the virus 5' leader sequence of 168 bases; a region of approximately 200-250 bases from the viral polymerase gene; approximately 70 bases from the 3' terminus of the viral p19 and p22 genes; and approximately 130 bases from the 3' terminal noncoding region. Conservation of the sequence motif present in all of the DIs suggests that there might be a common mechanism of DI formation as well as selection pressure to maintain sequences essential for replication and encapsidation.     

Ubiquitination of tombusvirus p33 replication protein plays a role in virus replication and binding to the host Vps23p ESCRT protein

Post-translational modifications of viral replication proteins could be widespread phenomena during the replication of plus-stranded RNA viruses. In this article, we identify two lysines in the tombusvirus p33 replication co-factor involved in ubiquitination and show that the same lysines are also important for the p33 to interact with the host Vps23p ESCRT-I factor. We find that the interaction of p33 with Vps23p is also affected by a “late-domain”-like sequence in p33. The combined mutations of the two lysines and the late-domain-like sequences in p33 reduced replication of a replicon RNA of Tomato bushy stunt virus in yeast model host, in plant protoplasts, and plant leaves, suggesting that p33-Vps23p ESCRT protein interaction affects tombusvirus replication. Using ubiquitin-mimicking p33 chimeras, we demonstrate that high level of p33 ubiquitination is inhibitory for TBSV replication. These findings argue that optimal level of p33 ubiquitination plays a regulatory role during tombusvirus infections.     

An inhibitory function of WW domain-containing host proteins in RNA virus replication

To identify new genes affecting Tomato bushy stunt virus (TBSV) replication in yeast model host, we are studying protein families, whose members have been identified during previous high throughput screening. In this paper, we have characterized the WW domain-containing protein family from yeast and plants. We find that, in addition to Rsp5 E3 ubiquitin ligase, yeast Wwm1 and Prp40 and three Arabidopsis WW domain-containing proteins are strong inhibitors of TBSV replication. The tombusvirus replicase complex isolated from yeast with down-regulated Wwm1 protein level was more active. Accumulation of viral p92^pol was reduced when Wwm1 was over-expressed, suggesting that the stability of p92^pol might be reduced, as observed with Rsp5. Moreover, replication of two insect RNA viruses is also inhibited by Wwm1 and Rsp5, suggesting that WW domain-containing proteins might have broad regulatory effects on RNA viruses. Thus, artificial antiviral proteins with WW domains could be useful antiviral strategy.     

Similar roles for yeast Dbp2 and Arabidopsis RH20 DEAD-box RNA helicases to Ded1 helicase in tombusvirus plus-strand synthesis

Recruited host factors aid replication of plus-strand RNA viruses. In this paper, we show that Dbp2 DEAD-box helicase of yeast, which is a homolog of human p68 DEAD-box helicase, directly affects replication of Tomato bushy stunt virus (TBSV). We demonstrate that Dbp2 binds to the 3′-end of the viral minus-stranded RNA and enhances plus-strand synthesis by the viral replicase in a yeast-based cell-free TBSV replication assay. In vitro data with wt and an ATPase-deficient Dbp2 mutant indicate that Dbp2 unwinds local secondary structures at the 3′-end of the TBSV (−)RNA. We also show that Dbp2 complements the replication deficiency of TBSV in yeast containing reduced amount of Ded1 DEAD-box helicase, another host factor involved in TBSV replication, suggesting that Dbp2 and Ded1 helicases play redundant roles in TBSV replication. We also show that the orthologous AtRH20 DEAD-box helicase from Arabidopsis can increase tombusvirus replication in vitro and in yeast.     

Defective-interfering RNAs and elevated temperatures inhibit replication of tomato bushy stunt virus in inoculated protoplasts

Tomato bushy stunt virus (TBSV) genomic RNA and one of its defective interfering (DI) RNAs were inoculated in various combinations to protoplasts of Nicotiana benthamiana. Ethidium bromide staining of electrophoretically separated RNAs from infected protoplasts, incorporation of [3H]uridine into TBSV and DI RNAs, and Northern hybridization at different times after inoculation clearly demonstrated reduced accumulation of genomic RNA in the presence of DI RNA. Accumulation of genomic RNA was very rapid between 3 and 9 hr postinfection. The presence of equimolar amounts of genomic and DI RNA in the inoculum resulted in a 65% suppression of genomic RNA accumulation. Suppression of genomic RNA was mediated by a reduction in the rate at which genomic RNA accumulated. Analysis of protoplasts inoculated with increasing ratios of DI:genomic RNA suggested that DI RNA-mediated suppression of genomic RNA synthesis results from competition for factors essential for viral replication. Incubation of protoplasts at different temperatures also had a profound effect on replication of both genomic and DI RNAs. Both replicated well at 27 degrees but were barely detectable at 32 degrees. Suppression of genomic RNA synthesis by DI RNA was similar at all temperatures tested. Thus, this study suggests that DI suppression of TBSV symptoms in whole plants and symptom attenuation at elevated temperatures are primarily the result of reduced viral replication.     

The overlapping RNA-binding domains of p33 and p92 replicase proteins are essential for tombusvirus replication

Two of the five viral-coded proteins of tombusviruses, which are small, nonsegmented, plus-stranded RNA viruses of plants, are required for replication in infected cells. These replicase proteins, namely, p33 and p92, of cucumber necrosis virus are expressed directly from the genomic RNA via a readthrough mechanism. Their overlapping domains contain an arginine/proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). Site-directed mutagenesis of p33 expressed in Escherichia coli, followed by a gel shift assay, defined two of the four arginines as required for efficient RNA binding in vitro. In vivo testing of 19 RPR motif mutants revealed that the RPR motif, and therefore the ability to bind RNA, is important for the replication of tombusviruses and their associated defective interfering (DI) RNAs. Mutation within the RPR motif also affected the ratio of subgenomic versus genomic RNAs in infected cells. To test whether the RPR motif is essential for the function of either p33 or p92 in replication, we used a two-component system developed by, J. Virol. 5845-5851), in which p92 was expressed from the genomic RNA of a tombusvirus, while p33 was expressed from a DI RNA. The protoplast experiments with the two-component system revealed that the RPR motif is essential for the replication function of both proteins. Interestingly, mutations within the RPR motif of p33 and p92 had different effects on RNA replication, suggesting different roles for the RNA-binding motifs of these proteins in tombusvirus replication.     

A temperature sensitive mutant of heat shock protein 70 reveals an essential role during the early steps of tombusvirus replication

By co-opting host proteins for their replication, plus-stranded RNA viruses can support robust replication and suppress host anti-viral responses. Tomato bushy stunt virus (TBSV) recruit the cellular heat shock protein 70 (Hsp70), an abundant cytosolic chaperone, into the replicase complex. By taking advantage of yeast model host, we demonstrate that the four-member SSA subfamily of HSP70 genes is essential for TBSV replication. The constitutively expressed SSA1 and SSA2, which are resident proteins in the viral replicase, can be complemented by the heat-inducible SSA3 and/or SSA4 for TBSV replication. Using a yeast strain carrying a temperature sensitive ssa1^ts, but lacking functional SSA2/3/4, we show that inactivation of Ssa1p^ts led to a defect in membrane localization of the viral replication proteins, resulting in cytosolic distribution of the viral proteins and lack of replicase activity. An in vitro replicase assembly assay with Ssa1p^ts revealed that functional Ssa1p is required during the replicase assembly process, but not during minus- or plus-strand synthesis. Temperature shift experiments from nonpermissive to permissive in ssa1^ts yeast revealed that the re-activated Ssa1p^ts could promote efficient TBSV replication in the absence of other SSA genes. We also demonstrate that the purified recombinant Ssa3p can facilitate the in vitro assembly of the TBSV replicase on yeast membranes, demonstrating that Ssa3p can fully complement the function of Ssa1p. Taken together, the cytosolic SSA subfamily of Hsp70 proteins play essential and multiple roles in TBSV replication.     

3'-Terminal RNA secondary structures are important for accumulation of tomato bushy stunt virus DI RNAs

The plus-strand RNA genome of tomato bushy stunt virus (TBSV) contains a 351-nucleotide (nt)-long 3'-untranslated region. We investigated the role of the 3'-proximal 130 nt of this sequence in viral RNA accumulation within the context of a TBSV defective interfering (DI) RNA. Sequence comparisons between different tombusviruses revealed that the 3' portion of the 130-nt sequence is highly conserved and deletion analysis confirmed that this segment is required for accumulation of DI RNAs in protoplasts. Computer-aided sequence analysis and in vitro solution structure probing indicated that the conserved sequence consists of three stem-loop (SL) structures (5'-SL3-SL2-SL1-3'). The existence of SLs 1 and 3 was also supported by comparative secondary structure analysis of sequenced tombusvirus genomes. Formation of the stem regions in all three SLs was found to be very important, and modification of the terminal loop sequences of SL1 and SL2, but not SL3, decreased DI RNA accumulation in vivo. For SL3, alterations to an internal loop resulted in significantly reduced DI RNA levels. Collectively, these data indicate that all three SLs are functionally relevant and contribute substantially to DI RNA accumulation. In addition, secondary structure analysis of other tombusvirus replicons and related virus genera revealed that a TBSV satellite RNA and members of the closely related genus Aureusvirus (family Tombusviridae) share fundamental elements of this general structural arrangement. Thus, this secondary structure model appears to extend beyond tombusvirus genomes. These conserved 3'-terminal RNA elements likely function in vivo by promoting and/or regulating minus-strand synthesis.     

Silencing of Nicotiana benthamiana Xrn4p exoribonuclease promotes tombusvirus RNA accumulation and recombination

The cytosolic 5′-to-3′ exoribonuclease Xrn1p plays a major role in recombination and degradation of Tomato bushy stunt tombusvirus (TBSV) replicon (rep)RNA in yeast, a model host (Serviene, E., Shapka, N., Cheng, C.P., Panavas, T., Phuangrat, B., Baker, J., and Nagy, P.D., 2005. Genome-wide screen identifies host genes affecting viral RNA recombination. Proc. Natl. Acad. Sci. U. S. A. 102(30), 10545-10550.). To test if the plant cytosolic 5′-to-3′ exoribonuclease Xrn4p, similar to the yeast Xrn1p, could also affect TBSV recombination, in this paper, we silenced XRN4 in Nicotiana benthamiana, an experimental host. The accumulation of tombusvirus genomic RNA and repRNA increased by 50% and 220%, respectively, in XRN4-silenced N. benthamiana. We also observed up to 125-fold increase in the emergence of new recombinants and partly degraded viral RNAs in the silenced plants. Using a cell-free assay based on a yeast extract, which supports authentic replication and recombination of TBSV, we demonstrate that the purified recombinant Xrn1p efficiently inhibited the accumulation of recombinants and partly degraded viral RNAs. Altogether, the data from a plant host and cell-free system confirm a central role for the plant cytosolic 5′-to-3′ exoribonuclease in TBSV replication, recombination and viral RNA degradation.