To identify host genes affecting replication of Tomato bushy stunt virus (TBSV), a small model positive-stranded RNA virus, we overexpressed 5,500 yeast proteins individually in Saccharomyces cerevisiae, which supports TBSV replication. In total, we identified 141 host proteins, and overexpression of 40 of those increased and the remainder decreased the accumulation of a TBSV replicon RNA. Interestingly, 36 yeast proteins were identified previously by various screens, greatly strengthening the relevance of these host proteins in TBSV replication. To validate the results from the screen, we studied the effect of protein kinase C1 (Pkc1), a conserved host kinase involved in many cellular processes, which inhibited TBSV replication when overexpressed. Using a temperature-sensitive mutant of Pkc1p revealed a high level of TBSV replication at a semipermissive temperature, further supporting the idea that Pkc1p is an inhibitor of TBSV RNA replication. A direct inhibitory effect of Pkc1p was shown in a cell-free yeast extract-based TBSV replication assay, in which Pkc1p likely phosphorylates viral replication proteins, decreasing their abilities to bind to the viral RNA. We also show that cercosporamide, a specific inhibitor of Pkc-like kinases, leads to increased TBSV replication in yeast, in plant single cells, and in whole plants, suggesting that Pkc-related pathways are potent inhibitors of TBSV in several hosts.

Plus-strand (+)RNA viruses co-opt host RNA-binding proteins (RBPs) to perform many functions during viral replication. A few host RBPs have been identified that affect the recruitment of viral (+)RNAs for replication. Other subverted host RBPs help the assembly of the membrane-bound replicase complexes, regulate the activity of the replicase and control minus- or plus-strand RNA synthesis. Host RBPs also affect the stability of viral RNAs, which have to escape cellular RNA degradation pathways. While many host RBPs seem to have specialized functions, others participate in multiple events during infection. Several conserved RBPs, such as eEF1A, hnRNP proteins and the Lsm 1–7 complex, are co-opted by evolutionarily diverse (+)RNA viruses, underscoring some common themes in virus-host interactions. On the other hand, viruses also hijack unique RBPs, suggesting that (+)RNA viruses could utilize different RBPs to perform similar functions. Moreover, different (+) RNA viruses have adapted distinctive strategies for co-opting unique RBPs. Altogether, a deeper understanding of the functions of the host RBPs subverted for viral replication will help development of novel antiviral strategies and give new insights into host RNA biology.

Replication of plus-strand RNA viruses depends on lipids present in cellular membranes. Recent genome-wide screens have revealed that eight phospholipid biosynthesis genes affected the replication of Tomato bushy stunt virus (TBSV) in yeast model host. To test the importance of phospholipids in TBSV replication, we studied one of the identified genes, namely INO2, which forms a heterodimer with Ino4, and is a transcription activator involved in regulation of phospholipid biosynthesis. Deletion of INO2, or double deletion of INO2/INO4, reduced TBSV replication and inhibited the activity of the viral replicase complex. In addition, the stability of the viral replication protein is decreased as well as the localization pattern of the viral protein changed dramatically in ino2Δ ino4Δ yeast. Over-expression of Opi1, a repressor of Ino2 and phospholipid biosynthesis, also inhibited TBSV RNA accumulation. In contrast, over-expression of Ino2 stimulated TBSV RNA accumulation. We also observed an inhibitory effect on Flock house virus (FHV) replication and the reduced stability of the FHV replication protein in ino2Δ ino4Δ yeast. These data are consistent with the important role of phospholipids in RNA virus replication.

In addition to its central role as a template for replication and translation, the viral plus-strand RNA genome also has nontemplate functions, such as recruitment to the site of replication and assembly of the viral replicase, activities that are mediated by cis-acting RNA elements within viral genomes. Two noncontiguous RNA elements, RII(+)-SL (located internally in the tombusvirus genome) and RIV (located at the 3′-terminus), are involved in template recruitment into replication and replicase assembly; however, the importance of each of these RNA elements for these two distinct functions is not fully elucidated. We used an in vitro replicase assembly assay based on yeast cell extract and purified recombinant tombusvirus replication proteins to show that RII(+)-SL, in addition to its known requirement for recruitment of the plus-strand RNA into replication, is also necessary for assembly of an active viral replicase complex. Additional studies using a novel two-component RNA system revealed that the recruitment function of RII(+)-SL can be provided in trans by a separate RNA and that the replication silencer element, located within RIV, defines the template that is used for initiation of minus-strand synthesis. Collectively, this work has revealed new functions for tombusvirus cis-acting RNA elements and provided insights into the pioneering round of minus-strand synthesis.

The replication of plus-strand RNA viruses depends on many cellular factors. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an abundant metabolic enzyme that is recruited to the replicase complex of Tomato bushy stunt virus (TBSV) and affects asymmetric viral RNA synthesis. To further our understanding on the role of GAPDH in TBSV replication, we used an in vitro TBSV replication assay based on recombinant p33 and p92pol viral replication proteins and cell-free yeast extract. We found that the addition of purified recombinant GAPDH to the cell extract prepared from GAPDH-depleted yeast results in increased plus-strand RNA synthesis and asymmetric production of viral RNAs. Our data also demonstrate that GAPDH interacts with p92pol viral replication protein, which may facilitate the recruitment of GAPDH into the viral replicase complex in the yeast model host. In addition, we have identified a dominant negative mutant of GAPDH, which inhibits RNA synthesis and RNA recruitment in vitro. Moreover, this mutant also exhibits strong suppression of tombusvirus accumulation in yeast and in virus-infected Nicotiana benthamiana. Overall, the obtained data support the model that the co-opted GAPDH plays a direct role in TBSV replication by stimulating plus-strand synthesis by the viral replicase.

Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV). To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3′-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3′-end of the TBSV (−)RNA, rendering the RNA compatible for initiation of (+)-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is another host factor for TBSV, play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (−)RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV), a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells.

Author Summary

Subverted host factors play a role in plus-strand RNA virus replication. Small RNA viruses do not code for their own helicases and they might recruit host RNA helicases to aid their replication in infected cells. In this paper, the authors show that the Ded1p DEAD-box helicase, which is an essential translation factor in yeast, is recruited by Tomato bushy stunt virus (TBSV) into its replicase complex. They also show that Ded1p binds to the viral (−)RNA and promotes (+)-strand TBSV synthesis when added to a yeast-based cell-free extract depleted for Ded1p. An ATPase defective Ded1p mutant failed to promote TBSV replication in vitro, suggesting that the helicase activity of Ded1p is essential for its function during TBSV replication. In addition, the authors also show that another host protein, which also binds to the (−)RNA, namely glyceraldehyde-3-phosphate dehydrogenase (GAPDH), further enhances TBSV (+)RNA when added together with Ded1p to yeast-based cell-free extract. In summary, the authors show that the major functions of Ded1p and GAPDH host proteins are to promote TBSV replication via selectively enhancing (+)-strand synthesis.

Replication of plus-stranded RNA viruses is greatly affected by numerous host-coded proteins acting either as susceptibility or resistance factors. Previous genome-wide screens and global proteomics approaches with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of cyclophilins, which are a large family of host prolyl isomerases, in TBSV replication. In this paper, we identified those members of the large cyclophilin family that interacted with the viral replication proteins and inhibited TBSV replication. Further characterization of the most effective cyclophilin, the Cyp40-like Cpr7p, revealed that it strongly inhibits many steps during TBSV replication in a cell-free replication assay. These steps include viral RNA recruitment inhibited via binding of Cpr7p to the RNA-binding region of the viral replication protein; the assembly of the viral replicase complex and viral RNA synthesis. Since the TPR (tetratricopeptide repeats) domain, but not the catalytic domain of Cpr7p is needed for the inhibitory effect on TBSV replication, it seems that the chaperone activity of Cpr7p provides the negative regulatory function. We also show that three Cyp40-like proteins from plants can inhibit TBSV replication in vitro and Cpr7p is also effective against Nodamura virus, an insect pathogen. Overall, the current work revealed a role for Cyp40-like proteins and their TPR domains as regulators of RNA virus replication.

Author Summary

Replication of plus-stranded RNA viruses, which are important pathogens of humans, animals and plants, can be inhibited by host-coded proteins. In this paper, the authors show that the Cyp40-like Cpr7p prolyl isomerase of yeast can effectively inhibit tombusvirus replication. This inhibition is due to binding of the TPR (tetratricopeptide repeats) domain of Cpr7p to the RNA-binding region of the tombusvirus replication proteins that leads to inhibition of RNA binding by the viral replication proteins, interference with the assembly of the viral replicase and blocking viral RNA synthesis. Cpr7p is also effective against the distantly-related alfanodaviruses of insects. Overall, this work reveals a role for a Cyp40-like protein as a regulator of RNA virus replication. This function of Cyp40 during RNA virus infection seems to be conserved between yeast and plants.

Host factors are recruited into viral replicase complexes to aid replication of plus-strand RNA viruses. In this paper, we show that deletion of eukaryotic translation elongation factor 1Bgamma (eEF1Bγ) reduces Tomato bushy stunt virus (TBSV) replication in yeast host. Also, knock down of eEF1Bγ level in plant host decreases TBSV accumulation. eEF1Bγ binds to the viral RNA and is one of the resident host proteins in the tombusvirus replicase complex. Additional in vitro assays with whole cell extracts prepared from yeast strains lacking eEF1Bγ demonstrated its role in minus-strand synthesis by opening of the structured 3′ end of the viral RNA and reducing the possibility of re-utilization of (+)-strand templates for repeated (-)-strand synthesis within the replicase. We also show that eEF1Bγ plays a synergistic role with eukaryotic translation elongation factor 1A in tombusvirus replication, possibly via stimulation of the proper positioning of the viral RNA-dependent RNA polymerase over the promoter region in the viral RNA template.These roles for translation factors during TBSV replication are separate from their canonical roles in host and viral protein translation.

Author Summary

RNA viruses recruit numerous host proteins to facilitate their replication and spread. Among the identified host proteins are RNA-binding proteins (RBPs), such as ribosomal proteins, translation factors and RNA-modifying enzymes. In this paper, the authors show that deletion of eukaryotic translation elongation factor 1Bgamma (eEF1Bγ) reduces Tomato bushy stunt virus (TBSV) replication in a yeast model host. Knock down of eEF1Bγ level in plant host also decreases TBSV accumulation. Moreover, the authors demonstrate that eEF1Bγ binds to the viral RNA and is present in the tombusvirus replicase complex. Functional studies revealed that eEF1Bγ promotes minus-strand synthesis by serving as an RNA chaperone. The authors also show that eEF1Bγ and eukaryotic translation elongation factor 1A, another host factor, function together to promote tombusvirus replication.

RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

Summary

Post-translational modifications of viral replication proteins could be widespread phenomena during the replication of plus-stranded RNA viruses. In this paper, 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.

Previous genome-wide screens identified >100 host genes affecting tombusvirus replication using yeast model host. One of those factors was Nsr1p (nucleolin), which is an abundant RNA binding shuttle protein involved in rRNA maturation and ribosome assembly. We find that over-expression of Nsr1p in yeast or in Nicotiana benthamiana inhibited the accumulation of tombusvirus RNA by ~10-fold. Regulated over-expression of Nsr1p revealed that Nsr1p should be present at the beginning of viral replication for efficient inhibition, suggesting that Nsr1p inhibits an early step in the replication process. In vitro experiments revealed that Nsr1p binds preferably to the 3' UTR in the viral RNA. The purified recombinant Nsr1p inhibited the in vitro replication of the viral RNA in a yeast cell-free assay when pre-incubated with the viral RNA before the assay. These data support the model that Nsr1p/nucleolin inhibits tombusvirus replication by interfering with the recruitment of the viral RNA for replication.

Summary

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 tombusvirus (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 ssa1ts, but lacking functional SSA2/3/4, we show that inactivation of Ssa1pts 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 Ssa1pts 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 ssa1ts yeast revealed that the re-activated Ssa1pts 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.

Replication of plus-strand RNA viruses depends on host factors that are recruited into viral replicase complexes. Previous studies showed that eukaryotic translation elongation factor (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. Using a random library of eEF1A mutants, we identified one mutant that decreased and three mutants that increased Tomato bushy stunt virus (TBSV) replication in a yeast model host. Additional in vitro assays with whole cell extracts prepared from yeast strains expressing the eEF1A mutants demonstrated several functions for eEF1A in TBSV replication: facilitating the recruitment of the viral RNA template into the replicase complex; the assembly of the viral replicase complex; and enhancement of the minus-strand synthesis by promoting the initiation step. These roles for eEF1A are separate from its canonical role in host and viral protein translation, emphasizing critical functions for this abundant cellular protein during TBSV replication.

Author Summary

Plus-stranded RNA viruses are important pathogens of plants, animals and humans. They replicate in the infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. In this paper, we show that the eukaryotic translation elongation factor (eEF1A), which is one of the resident host proteins in the highly purified tombusvirus replicase complex, is important for Tomato bushy stunt virus (TBSV) replication in a yeast model host. Based on a random library of eEF1A mutants, we identified eEF1A mutants that either decreased or increased TBSV replication. In vitro studies revealed that eEF1A facilitated the recruitment of the viral RNA template for replication and the assembly of the viral replicase complex, as well as eEF1A enhanced viral RNA synthesis in vitro. Altogether, this study demonstrates that eEF1A has several functions during TBSV replication.

Positive-stranded RNA (+RNA) viruses exploit host cell machinery by subverting host proteins and membranes and altering cellular pathways during infection. To achieve robust replication, some +RNA viruses, such as poliovirus (PV), build special intracellular compartments, called viral replication organelles. A recent work from the Altan-Bonnett laboratory [1] gave new insights into the formation of poliovirus replication organelles, which are unique subcellular structures containing many individual replication complexes as a result of dynamic cellular membrane remodeling.

Viruses are masters of evolution due to high frequency mutations and genetic recombination. In spite of the significance of viral RNA recombination that promotes the emergence of drug-resistant virus strains, the role of host and environmental factors in RNA recombination is poorly understood. Here we report that the host Met22p/Hal2p bisphosphate-3′-nucleotidase regulates the frequency of viral RNA recombination and the efficiency of viral replication. Based on Tomato bushy stunt virus (TBSV) and yeast as a model host, we demonstrate that deletion of MET22 in yeast or knockdown of AHL, SAL1 and FRY1 nucleotidases/phosphatases in plants leads to increased TBSV recombination and replication. Using a cell-free TBSV recombination/replication assay, we show that the substrate of the above nucleotidases, namely 3′-phosphoadenosine-5′-phosphate pAp, inhibits the activity of the Xrn1p 5′-3′ ribonuclease, a known suppressor of TBSV recombination. Inhibition of the activity of the nucleotidases by LiCl and NaCl also leads to increased TBSV recombination, demonstrating that environmental factors could also affect viral RNA recombination. Thus, host factors in combination with environmental factors likely affect virus evolution and adaptation.

Author Summary

Viral RNA recombination plays a major role in virus evolution. Yet, we know little about the roles of host and environmental factors in viral RNA recombination. In this work, using TBSV and yeast as a model host, we show that MET22 nucleotidase suppresses viral RNA recombination. In vitro experiments with a cell-free extract from yeast revealed that the substrate of Met22p bisphosphate-3′-nucleotidase, namely pAp, could promote TBSV RNA recombination via inhibiting the activity of the Xrn1p 5′-3′ ribonuclease, a known suppressor of viral RNA recombination. Altogether, we demonstrate that the Met22/Xrn1 pathway and environmental factors affecting this pathway, namely salt-stress caused by LiCl and NaCl, play a role in viral RNA recombination. The authors also provide evidence that a similar pathway affects TBSV recombination in plants as well. The most pronounced increase in accumulation of TBSV RNA recombinants was seen in the triple-nucleotidase gene silenced plants treated with LiCl, suggesting that the combined effect of genetic and environmental factors could be critical in regulation of the rate of viral RNA recombination.

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 p92pol 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.

Recent in vitro proteomics screens revealed that many host proteins could interact with the replication proteins of Tomato bushy stunt virus (TBSV), which is a small, plus-stranded RNA virus (Z. Li, D. Barajas, T. Panavas, D. A. Herbst, and P. D. Nagy, J. Virol. 82:6911-6926, 2008). To further our understanding of the roles of host factors in TBSV replication, we have tested the effect of Rsp5p, which is a member of the Nedd4 family of E3 ubiquitin ligases. The full-length Rsp5p, via its WW domain, is shown to interact with p33 and the central portion of p92pol 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 p92pol, 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 p92pol. 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 p92pol reduces the stability of p92pol, with consequent inhibition of TBSV replicase activity.

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 were 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 that 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.

Plus-stranded RNA viruses replicate in infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of seven ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. In this paper, we show that the expression of dominant negative Vps23p, Vps24p, Snf7p, and Vps4p ESCRT factors inhibited virus replication in the plant host, suggesting that tombusviruses co-opt selected ESCRT proteins for the assembly of the viral replicase complex. We also show that TBSV p33 replication protein interacts with Vps23p ESCRT-I and Bro1p accessory ESCRT factors. The interaction with p33 leads to the recruitment of Vps23p to the peroxisomes, the sites of TBSV replication. The viral replicase showed reduced activity and the minus-stranded viral RNA in the replicase became more accessible to ribonuclease when derived from vps23Δ or vps24Δ yeast, suggesting that the protection of the viral RNA is compromised within the replicase complex assembled in the absence of ESCRT proteins. The recruitment of ESCRT proteins is needed for the precise assembly of the replicase complex, which might help the virus evade recognition by the host defense surveillance system and/or prevent viral RNA destruction by the gene silencing machinery.

Author Summary

Plus-stranded RNA viruses, which are important pathogens of humans, animals and plants, replicate in infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. In this paper, we show that a group of host factors called ESCRT proteins (endosomal sorting complexes required for transport) play important roles in tombusvirus replication. The expression of dominant negative mutants of ESCRT factors inhibited virus replication in the plant host, suggesting that tombusviruses co-opt selected ESCRT proteins for the assembly of the viral replicase complex. In addition, we show direct interaction between the viral p33 replication protein and Vps23p ESCRT-I and Bro1p accessory ESCRT factors. The interaction with p33 leads to the recruitment of Vps23p to the peroxisomes, the sites of tombusvirus replication. We also showed that the viral RNA within the viral replicase complex became more sensitive to ribonuclease in the absence of ESCRT factors, suggesting that the protection of the viral RNA is compromised within the replicase complex assembled in the absence of ESCRT proteins. Intriguingly, the host ESCRT factors also affect the budding of several enveloped viruses, intracellular transport of proteins and cytokinesis. Overall, this work demonstrates that a plus-stranded RNA virus uses the endosomal sorting pathway in a unique way.

Defective interfering (DI) RNAs are subviral RNAs produced during multiplication of RNA viruses by the error-prone viral replicase. DI-RNAs are parasitic RNAs that are derived from and associated with the parent virus, taking advantage of viral-coded protein factors for their multiplication. Recent advances in the field of DI RNA biology has led to a greater understanding about generation and evolution of DI-RNAs as well as the mechanism of symptom attenuation. Moreover, DI-RNAs are versatile tools in the hands of virologists and are used as less complex surrogate templates to understand the biology of their helper viruses. The ease of their genetic manipulation has resulted in rapid discoveries on cis-acting RNA replication elements required for replication and recombination. DI-RNAs have been further exploited to discover host factors that modulate Tomato bushy stunt virus replication, as well as viral RNA recombination. This review discusses the current models on generation and evolution of DI-RNAs, the roles of viral and host factors in DI-RNA replication, and the mechanisms of disease attenuation.

Background

Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV), a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ) and quinacrine (QC), which are active against prion-based diseases.

Methodology/Principal Findings

Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i) inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii) reduction of minus-strand synthesis by the tombusvirus replicase; and (iii) inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication.

Conclusion/Significance

Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses.

Plus-stranded RNA viruses coopt host proteins to promote their robust replication in infected hosts. Tomato bushy stunt tombusvirus (TBSV) is a model virus that can replicate a small replicon RNA in Saccharomyces cerevisiae and in plants. The tombusvirus replicase complex contains heat shock protein 70 (Hsp70), an abundant cytosolic chaperone, which is required for TBSV replication. To dissect the function of Hsp70 in TBSV replication, in this paper we use an Hsp70 mutant (ssa1 ssa2) yeast strain that supports a low level of TBSV replication. Using confocal laser microscopy and cellular fractionation experiments, we find that the localization of the viral replication proteins changes to the cytosol in the mutant cells from the peroxisomal membranes in wild-type cells. An in vitro membrane insertion assay shows that Hsp70 promotes the integration of the viral replication proteins into subcellular membranes. This step seems to be critical for the assembly of the viral replicase complex. Using a gene-silencing approach and quercetin as a chemical inhibitor to downregulate Hsp70 levels, we also confirm the significance of cytosolic Hsp70 in the replication of TBSV and other plant viruses in a plant host. Taken together, our results suggest that cytosolic Hsp70 plays multiple roles in TBSV replication, such as affecting the subcellular localization and membrane insertion of the viral replication proteins as well as the assembly of the viral replicase.

Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA–RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA–RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA–based interaction spanning ∼3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.

Author Summary

Plus-strand (i.e. messenger-sensed) RNA viruses are responsible for significant diseases in plants and animals. The single-stranded RNA genomes of these viruses serve as templates for translation of viral proteins and perform other essential functions that generally involve local RNA structures, such as RNA hairpins. Interestingly, plant tombusviruses utilize a number of long-range intra-genomic RNA–RNA interactions to regulate important events during infection of their hosts, i.e. viral translation and transcription. Here, we report that an additional essential tombusvirus process, viral RNA replication, also requires a long-range RNA–RNA interaction. Our analyses indicate a role for this RNA–based interaction in the assembly of the viral replicase, which is responsible for executing viral RNA synthesis. This information was used to generate a comprehensive higher-order RNA structural model for functional long-range interactions in the genome of this eukaryotic RNA virus. The model highlights a critical role for global RNA structure in multiple viral processes that are necessary for successful infection of hosts.

Rice dwarf virus (RDV) replicates in and is transmitted by a leafhopper vector in a persistent-propagative manner. Previous cytopathologic and genetic data revealed that tubular structures, constructed by the nonstructural viral protein Pns10, contain viral particles and are directly involved in the intercellular spread of RDV among cultured leafhopper cells. Here, we demonstrated that RDV exploited these virus-containing tubules to move along actin-based microvilli of the epithelial cells and muscle fibers of visceral muscle tissues in the alimentary canal, facilitating the spread of virus in the body of its insect vector leafhoppers. In cultured leafhopper cells, the knockdown of Pns10 expression due to RNA interference (RNAi) induced by synthesized dsRNA from Pns10 gene strongly inhibited tubule formation and prevented the spread of virus among insect vector cells. RNAi induced after ingestion of dsRNA from Pns10 gene strongly inhibited formation of tubules, preventing intercellular spread and transmission of the virus by the leafhopper. All these results, for the first time, show that a persistent-propagative virus exploits virus-containing tubules composed of a nonstructural viral protein to traffic along actin-based cellular protrusions, facilitating the intercellular spread of the virus in the vector insect. The RNAi strategy and the insect vector cell culture provide useful tools to investigate the molecular mechanisms enabling efficient transmission of persistent-propagative plant viruses by vector insects.

Author Summary

Numerous plant viruses that seriously damage agricultural crops are transmitted by insects. However, the mechanisms enabling virus transmission by vector insects have been poorly understood, in part, due to the lack of useful tools. A persistent-propagative plant virus replicates and encodes nonstructural proteins to form various cytopathological structures in their two types of hosts: plants and vector insects. Here, we took advantage of unique biological tools, including insect vector cell culture and RNA interference (RNAi) induced by synthesized dsRNA, to investigate the molecular mechanisms facilitating the efficient spread of Rice dwarf virus (RDV), a persistent-propagative plant virus, among cells and organs of leafhopper vector. Our experimental evidence shows that RDV exploited virus-containing tubules composed of nonstructural viral protein Pns10 to traffic along actin-based cellular machinery, allowing efficient cell-to-cell spread of the virus in leafhopper vector. Consistently, and in support of a function of Pns10 tubules as a determinant for viral spread in vector insect, the introduction of dsRNA from Pns10 gene into cultured insect vector cells or intact insect strongly inhibited such tubule formation, preventing efficient viral intercellular spread in the leafhopper in vitro and in vivo and subsequent transmission by the vector, without significant effect on viral multiplication in leafhopper cells.

To identify host proteins interacting with Tomato bushy stunt virus (TBSV) replication proteins in a genome-wide scale, we have used a yeast (Saccharomyces cerevisiae) proteome microarray carrying 4,088 purified proteins. This approach led to the identification of 58 yeast proteins that interacted with p33 replication protein. The identified host proteins included protein chaperones, ubiquitin-associated proteins, translation factors, RNA-modifying enzymes, and other proteins with yet-unknown functions. We confirmed that 19 of the identified host proteins bound to p33 in vitro or in a split-ubiquitin-based two-hybrid assay. Further analysis of Cdc34p E2 ubiquitin-conjugating enzyme, which is one of the host proteins interacting with p33, revealed that Cdc34p is a novel component of the purified viral replicase. Downregulation of Cdc34p expression in yeast, which supports replication of a TBSV replicon RNA (repRNA), reduced repRNA accumulation and the activity of the tombusvirus replicase by up to fivefold. Overexpression of wild-type Cdc34p, but not that of an E2-defective mutant of Cdc34p, increased repRNA accumulation, suggesting a significant role for the ubiquitin-conjugating enzyme function of Cdc34p in TBSV replication. Also, Cdc34p was able to ubiquitinate p33 in vitro. In addition, we have shown that p33 becomes ubiquitinated in vivo. We propose that ubiquitination of p33 likely alters its function or affects the recruitment of host factors during TBSV replication.