Targeting conserved co-opted host factors to block virus replication: Using allosteric inhibitors of the cytosolic Hsp70s to interfere with tomato bushy stunt virus replication.

To further our understanding of the pro-viral roles of the host cytosolic heat shock protein 70 (Hsp70) family, we chose the conserved Arabidopsis thaliana Hsp70-2 and the unique Erd2 (early response to dehydration 2), which contain Hsp70 domains. Based on in vitro studies with purified components, we show that AtHsp70-2 and AtErd2 perform pro-viral functions equivalent to that of the yeast Ssa1 Hsp70. These functions include activation of the tombusvirus RdRp, and stimulation of replicase assembly. Yeast-based complementation studies demonstrate that AtHsp70-2 or AtErd2 are present in the purified tombusvirus replicase. RNA silencing and over-expression studies in Nicotiana benthamiana suggest that both Hsp70-2 and Erd2 are co-opted by tomato bushy stunt virus (TBSV). Moreover, we used allosteric inhibitors of Hsp70s to inhibit replication of TBSV and related plant viruses in plants. Altogether, interfering with the functions of the co-opted Hsp70s could be an effective antiviral approach against tombusviruses in plants.

Reconstitution of an RNA Virus Replicase in Artificial Giant Unilamellar Vesicles Supports Full Replication and Provides Protection for the Double-Stranded RNA Replication Intermediate.

Positive-strand RNA [(+)RNA] viruses are important pathogens of humans, animals, and plants and replicate inside host cells by coopting numerous host factors and subcellular membranes. To gain insights into the assembly of viral replicase complexes (VRCs) and dissect the roles of various lipids and coopted host factors, we have reconstituted Tomato bushy stunt virus (TBSV) replicase using artificial giant unilamellar vesicles (GUVs). We demonstrate that reconstitution of VRCs on GUVs with endoplasmic reticulum (ER)-like phospholipid composition results in a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)RNA viruses. TBSV VRCs assembled on GUVs provide significant protection of the double-stranded RNA (dsRNA) replication intermediate against the dsRNA-specific RNase III. The lipid compositions of GUVs have pronounced effects on in vitro TBSV replication, including (-) and (+)RNA synthesis. The GUV-based assay has led to the discovery of the critical role of phosphatidylserine in TBSV replication and a novel role for phosphatidylethanolamine in asymmetrical (+)RNA synthesis. The GUV-based assay also showed stimulatory effects by phosphatidylinositol-3-phosphate [PI(3)P] and ergosterol on TBSV replication. We demonstrate that eEF1A and Hsp70 coopted replicase assembly factors, Vps34 phosphatidylinositol 3-kinase (PI3K) and the membrane-bending ESCRT factors, are required for reconstitution of the active TBSV VRCs in GUVs, further supporting that the novel GUV-based in vitro approach recapitulates critical steps and involves essential coopted cellular factors of the TBSV replication process. Taken together, this novel GUV assay will be highly suitable to dissect the functions of viral and cellular factors in TBSV replication.IMPORTANCE Understanding the mechanism of replication of positive-strand RNA viruses, which are major pathogens of plants, animals, and humans, can lead to new targets for antiviral interventions. These viruses subvert intracellular membranes for virus replication and coopt numerous host proteins, whose functions during virus replication are not yet completely defined. To dissect the roles of various host factors in Tomato bushy stunt virus (TBSV) replication, we have developed an artificial giant unilamellar vesicle (GUV)-based replication assay. The GUV-based in vitro approach recapitulates critical steps of the TBSV replication process. GUV-based reconstitution of the TBSV replicase revealed the need for a complex mixture of phospholipids, especially phosphatidylserine and phosphatidylethanolamine, in TBSV replication. The GUV-based approach will be useful to dissect the functions of essential coopted cellular factors.

Interviral Recombination between Plant, Insect, and Fungal RNA Viruses: Role of the Intracellular Ca2+/Mn2+ Pump.

Recombination is one of the driving forces of viral evolution. RNA recombination events among similar RNA viruses are frequent, although RNA recombination could also take place among unrelated viruses. In this paper, we have established efficient interviral recombination systems based on yeast and plants. We show that diverse RNA viruses, including the plant viruses tomato bushy stunt virus, carnation Italian ringspot virus, and turnip crinkle virus-associated RNA; the insect plus-strand RNA [(+)RNA] viruses Flock House virus and Nodamura virus; and the double-stranded L-A virus of yeast, are involved in interviral recombination events. Most interviral recombinants are minus-strand recombinant RNAs, and the junction sites are not randomly distributed, but there are certain hot spot regions. Formation of interviral recombinants in yeast and plants is accelerated by depletion of the cellular SERCA-like Pmr1 ATPase-driven Ca2+/Mn2+ pump, regulating intracellular Ca2+ and Mn2+ influx into the Golgi apparatus from the cytosol. The interviral recombinants are generated by a template-switching mechanism during RNA replication by the viral replicase. Replication studies revealed that a group of interviral recombinants is replication competent in cell-free extracts, in yeast, and in the plant Nicotiana benthamiana We propose that there are major differences among the viral replicases to generate and maintain interviral recombinants. Altogether, the obtained data promote the model that host factors greatly contribute to the formation of recombinants among related and unrelated viruses. This is the first time that a host factor's role in affecting interviral recombination is established.IMPORTANCE Viruses with RNA genomes are abundant, and their genomic sequences show astonishing variation. Genetic recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis, and adaptation to their hosts. We utilized a previously identified intracellular Ca2+/Mn2+ pump-deficient yeast to search for interviral recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral recombinants. Altogether, interviral RNA recombinants were observed between plant and insect viruses, and between a fungal double-stranded RNA (dsRNA) virus and an insect virus, in the yeast host. In addition, interviral recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca2+/Mn2+ pump was depleted. These findings underline the crucial role of the host in promoting RNA recombination among unrelated viruses.

Cell-Free and Cell-Based Approaches to Explore the Roles of Host Membranes and Lipids in the Formation of Viral Replication Compartment Induced by Tombusviruses.

Plant positive strand RNA viruses are intracellular infectious agents that take advantage of cellular lipids and membranes to support replication and protect viral RNA from degradation by host antiviral responses. In this review, we discuss how Tomato bushy stunt virus (TBSV) co-opts lipid transfer proteins and modulates lipid metabolism and transport to facilitate the assembly of the membrane-bound viral replicase complexes within intricate replication compartments. Identification and characterization of the proviral roles of specific lipids and proteins involved in lipid metabolism based on results from yeast (Saccharomyces cerevisiae) model host and cell-free approaches are discussed. The review also highlights the advantage of using liposomes with chemically defined composition to identify specific lipids required for TBSV replication. Remarkably, all the known steps in TBSV replication are dependent on cellular lipids and co-opted membranes.

Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase.

UNLABELLED: Plus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs). Tomato bushy stunt virus (TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule)-like structures in the peroxisomal membrane with tight openings toward the cytosol. In this study, using a yeast (Saccharomyces cerevisiae) vps23Δ bro1Δ double-deletion mutant, we showed that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell extracts. Dual expression of dominant-negative versions of Arabidopsis homologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression in Nicotiana benthamiana leaves. We also demonstrated the critical role of Snf7p (CHMP4), Vps20p, and Vps24p ESCRT-III proteins in tombusvirus replication in yeast and in vitro. Electron microscopic imaging of vps23Δ yeast revealed the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also showed that the length of the viral RNA affects the sizes of spherules formed in N. benthamiana cells. The 4.8-kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ∼600-nucleotide (nt)-long defective interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a "measuring string" during VRC assembly and spherule formation. IMPORTANCE: Plant positive-strand RNA viruses, similarly to animal positive-strand RNA viruses, replicate in membrane-bound viral replicase complexes in the cytoplasm of infected cells. Identification of cellular and viral factors affecting the formation of the membrane-bound viral replication complex is a major frontier in current virology research. In this study, we dissected the functions of co-opted cellular ESCRT-I (endosomal sorting complexes required for transport I) and ESCRT-III proteins and the viral RNA in tombusvirus replicase complex formation using in vitro, yeast-based, and plant-based approaches. Electron microscopic imaging revealed the lack of tombusvirus-induced spherule-like structures in ESCRT-I or ESCRT-III deletion yeasts replicating TBSV RNA, demonstrating the requirement for these co-opted cellular factors in tombusvirus replicase formation. The work could be of broad interest in virology and beyond.

Activation of Tomato Bushy Stunt Virus RNA-Dependent RNA Polymerase by Cellular Heat Shock Protein 70 Is Enhanced by Phospholipids In Vitro.

UNLABELLED: Similar to other positive-strand RNA viruses, tombusviruses are replicated by the membrane-bound viral replicase complex (VRC). The VRC consists of the p92 virus-coded RNA-dependent RNA polymerase (RdRp), the viral p33 RNA chaperone, and several co-opted host proteins. In order to become a functional RdRp after its translation, the p92 replication protein should be incorporated into the VRC, followed by its activation. We have previously shown in a cell-free yeast extract-based assay that the activation of the Tomato bushy stunt virus (TBSV) RdRp requires a soluble host factor(s). In this article, we identify the cellular heat shock protein 70 (Hsp70) as the co-opted host factor required for the activation of an N-terminally truncated recombinant TBSV RdRp. In addition, small-molecule-based blocking of Hsp70 function inhibits RNA synthesis by the tombusvirus RdRp in vitro. Furthermore, we show that neutral phospholipids, namely, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), enhance RdRp activation in vitro. In contrast, phosphatidylglycerol (PG) shows a strong and dominant inhibitory effect on in vitro RdRp activation. We also demonstrate that PE and PC stimulate RdRp-viral plus-strand RNA [(+)RNA] interaction, while PG inhibits the binding of the viral RNA to the RdRp. Based on the stimulatory versus inhibitory roles of various phospholipids in tombusvirus RdRp activation, we propose that the lipid composition of targeted subcellular membranes might be utilized by tombusviruses to regulate new VRC assembly during the course of infection. IMPORTANCE: The virus-coded RNA-dependent RNA polymerase (RdRp), which is responsible for synthesizing the viral RNA progeny in infected cells of several positive-strand RNA viruses, is initially inactive. This strategy is likely to avoid viral RNA synthesis in the cytosol that would rapidly lead to induction of RNA-triggered cellular antiviral responses. During the assembly of the membrane-bound replicase complex, the viral RdRp becomes activated through an incompletely understood process that makes the RdRp capable of RNA synthesis. By using TBSV RdRp, we show that the co-opted cellular Hsp70 chaperone and neutral phospholipids facilitate RdRp activation in vitro. In contrast, phosphatidylglycerol (PG) has a dominant inhibitory effect on in vitro RdRp activation and RdRp-viral RNA interaction, suggesting that the membranous microdomain surrounding the RdRp greatly affects its ability for RNA synthesis. Thus, the activation of the viral RdRp likely depends on multiple host components in infected cells.

Noncanonical role for the host Vps4 AAA+ ATPase ESCRT protein in the formation of Tomato bushy stunt virus replicase.

Assembling of the membrane-bound viral replicase complexes (VRCs) consisting of viral- and host-encoded proteins is a key step during the replication of positive-stranded RNA viruses in the infected cells. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the involvement of eleven cellular ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. The ESCRT proteins are involved in endosomal sorting of cellular membrane proteins by forming multiprotein complexes, deforming membranes away from the cytosol and, ultimately, pinching off vesicles into the lumen of the endosomes. In this paper, we show an unexpected key role for the conserved Vps4p AAA+ ATPase, whose canonical function is to disassemble the ESCRT complexes and recycle them from the membranes back to the cytosol. We find that the tombusvirus p33 replication protein interacts with Vps4p and three ESCRT-III proteins. Interestingly, Vps4p is recruited to become a permanent component of the VRCs as shown by co-purification assays and immuno-EM. Vps4p is co-localized with the viral dsRNA and contacts the viral (+)RNA in the intracellular membrane. Deletion of Vps4p in yeast leads to the formation of crescent-like membrane structures instead of the characteristic spherule and vesicle-like structures. The in vitro assembled tombusvirus replicase based on cell-free extracts (CFE) from vps4Δ yeast is highly nuclease sensitive, in contrast with the nuclease insensitive replicase in wt CFE. These data suggest that the role of Vps4p and the ESCRT machinery is to aid building the membrane-bound VRCs, which become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA. Other (+)RNA viruses of plants and animals might also subvert Vps4p and the ESCRT machinery for formation of VRCs, which require membrane deformation and spherule formation.

Template role of double-stranded RNA in tombusvirus replication.

UNLABELLED: Replication of plus-strand RNA [(+)RNA] viruses of plants is a relatively simple process that involves complementary minus-strand RNA [(-)RNA] synthesis and subsequent (+)RNA synthesis. However, the actual replicative form of the (-)RNA template in the case of plant (+)RNA viruses is not yet established unambiguously. In this paper, using a cell-free replication assay supporting a full cycle of viral replication, we show that replication of Tomato bushy stunt virus (TBSV) leads to the formation of double-stranded RNA (dsRNA). Using RNase digestion, DNAzyme, and RNA mobility shift assays, we demonstrate the absence of naked (-)RNA templates during replication. Time course experiments showed the rapid appearance of dsRNA earlier than the bulk production of new (+)RNAs, suggesting an active role for dsRNA in replication. Radioactive nucleotide chase experiments showed that the mechanism of TBSV replication involves the use of dsRNA templates in strand displacement reactions, where the newly synthesized plus strand replaces the original (+)RNA in the dsRNA. We propose that the use of dsRNA as a template for (+)RNA synthesis by the viral replicase is facilitated by recruited host DEAD box helicases and the viral p33 RNA chaperone protein. Altogether, this replication strategy allows TBSV to separate minus- and plus-strand syntheses in time and regulate asymmetrical RNA replication that leads to abundant (+)RNA progeny. IMPORTANCE: Positive-stranded RNA viruses of plants use their RNAs as the templates for replication. First, the minus strand is synthesized by the viral replicase complex (VRC), which then serves as a template for new plus-strand synthesis. To characterize the nature of the (-)RNA in the membrane-bound viral replicase, we performed complete RNA replication of Tomato bushy stunt virus (TBSV) in yeast cell-free extracts and in plant extracts. The experiments demonstrated that the TBSV (-)RNA is present as a double-stranded RNA that serves as the template for TBSV replication. During the production of new plus strands, the viral replicase displaces the old plus strand in the dsRNA template, leading to asymmetrical RNA synthesis. The presented data are in agreement with the model that the dsRNA is present in nuclease-resistant membranous VRCs. This strategy likely allows TBSV to protect the replicating viral RNA from degradation as well as to evade the early detection of viral dsRNAs by the host surveillance system.

How yeast can be used as a genetic platform to explore virus-host interactions: from 'omics' to functional studies.

The yeast Saccharomyces cerevisiae is an advanced model organism that has emerged as an effective host to gain insights into the intricate interactions of viruses with host cells. RNA viruses have limited coding potential and need to coopt numerous host cellular factors to facilitate their replication. To identify the host factors subverted by viruses, high-throughput genomics and global proteomics approaches have been performed with plant viruses such as brome mosaic virus (BMV) and tomato bushy stunt virus (TBSV). Accordingly, several hundred susceptibility and restriction factors for BMV and TBSV have been identified using yeast as a model host. Amazingly, host factors affecting viral genetic recombination and evolution have also been identified in genome-wide screens in yeast. The roles of many yeast host factors involved in various steps of the viral replication process have been validated by exploiting the orthologous genes in plant hosts. This Opinion summarizes the advantages of using simple viruses and yeast model host to advance our general understanding of virus-host interactions. The knowledge gained on host factors could lead to novel specific or broad-range resistance and antiviral tools against viruses.

Characterization of dominant-negative and temperature-sensitive mutants of tombusvirus replication proteins affecting replicase assembly.

The assembly of the viral replicase complex (VRC) on subcellular membranes is a key step in the replication process of plus-stranded RNA viruses. In this work, we have identified lethal and temperature sensitive (ts) point mutations within the essential p33:p33/p92 interaction domain of p33 and p92 replication proteins of Cucumber necrosis virus, a tombusvirus. Mutations within the p33:p33/p92 interaction domain also affected viral RNA recombination in yeast model host. An in vitro approach based on yeast cell free extract demonstrated that several p33 and p92 mutants behaved as dominant-negative during VRC assembly, and they showed reduced binding to the viral (+)RNA and affected activation of the p92 RdRp protein, while they did not directly influence (-) or (+)-strand synthesis. Overall, the presented data provide direct evidence that the p33:p33/p92 interaction domains in p33 and p92 are needed for the early stage of virus replication and also influence viral recombination.

Host factors with regulatory roles in tombusvirus replication.

Similar to animal viruses, the abundant plant positive-strand RNA viruses replicate in infected cells by exploiting the vast resources of the host. This review focuses on virus-host interactions during tombusvirus replication. The multifunctional tombusvirus p33 replication protein not only interacts with itself, the viral p92(pol) polymerase, and viral RNA, but also with approximately 100 cellular proteins and subcellular membranes. Several negative regulatory host proteins, such as cyclophilins and WW motif containing proteins, also bind to p33 and interfere with p33's functions. To explain how p33 can perform multiple functions, we propose that a variety of interactions involving p33 result in the commitment of p33 molecules to specific tasks. This facilitates tight spatial and temporal organization of viral replication in infected cells.

p33-Independent activation of a truncated p92 RNA-dependent RNA polymerase of Tomato bushy stunt virus in yeast cell-free extract.

Plus-stranded RNA viruses replicate in membrane-bound structures containing the viral replicase complex (VRC). A key component of the VRC is the virally encoded RNA-dependent RNA polymerase (RdRp), which should be activated and incorporated into the VRC after its translation. To study the activation of the RdRp of Tomato bushy stunt virus (TBSV), a small tombusvirus of plants, we used N-terminal truncated recombinant RdRp, which supported RNA synthesis in a cell-free yeast extract-based assay. The truncated RdRp required a cis-acting RNA replication element and soluble host factors, while unlike the full-length TBSV RdRp, the truncated RdRp did not need the viral p33 replication cofactor or cellular membranes for RNA synthesis. Interestingly, the truncated RdRp used 3'-terminal extension for initiation and terminated prematurely at an internal cis-acting element. However, the truncated RdRp could perform de novo initiation on a TBSV plus-strand RNA template in the presence of the p33 replication cofactor, cellular membranes, and soluble host proteins. Altogether, the data obtained with the truncated RdRp indicate that this RdRp still requires activation, but with the participation of fewer components than with the full-length RdRp, making it suitable for future studies on dissection of the RdRp activation mechanism.

A Co-Opted DEAD-Box RNA helicase enhances tombusvirus plus-strand synthesis.

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.

The TPR domain in the host Cyp40-like cyclophilin binds to the viral replication protein and inhibits the assembly of the tombusviral replicase.

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.

Defining the roles of cis-acting RNA elements in tombusvirus replicase assembly in vitro.

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 dependence of viral RNA replication on co-opted host factors.

Positive-sense RNA ((+)RNA) viruses such as hepatitis C virus exploit host cells by subverting host proteins, remodelling subcellular membranes, co-opting and modulating protein and ribonucleoprotein complexes, and altering cellular metabolic pathways during infection. To facilitate RNA replication, (+)RNA viruses interact with numerous host molecules through protein-protein, RNA-protein and protein-lipid interactions. These interactions lead to the formation of viral replication complexes, which produce new viral RNA progeny in host cells. This Review presents the recent progress that has been made in understanding the role of co-opted host proteins and membranes during (+)RNA virus replication, and discusses common themes employed by different viruses.

Non-template functions of the viral RNA in plant RNA virus replication.

In addition to the essential function of the viral RNA as a template during replication of positive-stranded (+)RNA viruses, the RNA also has crucial non-template functions. These functions during replication include the recruitment of the viral RNA to the site of replication and assembly of the functional viral replicase complex. The RNA recruitment elements are specifically recognized by the viral replication protein, but also affected by host factors such as elongation factor 1A or P-body proteins. The cis-elements for replicase assembly can partially overlap with RNA recruitment elements and they may provide a platform for the assembly of the replicase complex consisting of viral and host proteins. This review focuses on our current knowledge obtained with tombusviruses and other plant viruses. Altogether, understanding of the non-template functions of the viral RNA during viral replication provides new insights into virus-host interactions.

Translation elongation factor 1A facilitates the assembly of the tombusvirus replicase and stimulates minus-strand synthesis.

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.

A high-throughput approach for studying virus replication in yeast.

Viruses are intracellular pathogens that are dependent on viral and host factors for multiplication. Model hosts, such as yeast, can be very valuable in identifying host factors involved in viral replication. Yeast is also useful for studies on functional interactions of host factors with viral proteins and/or virus nucleic acids. The advantages of using yeast include the availability of a single gene-deletion library and the essential gene library (yTHC); the controllable small- or large-scale expression of viral proteins and nucleic acids; and the rapid growth of yeast strains. Procedures that facilitate high-throughput analysis of host factors and plant and animal RNA virus replication in yeast, with a plant virus (tombusvirus; TBSV) and an animal virus (nodavirus; FHV) as examples, are described.

Global genomics and proteomics approaches to identify host factors as targets to induce resistance against Tomato bushy stunt virus.

The success of RNA viruses as pathogens of plants, animals, and humans depends on their ability to reprogram the host cell metabolism to support the viral infection cycle and to suppress host defense mechanisms. Plus-strand (+)RNA viruses have limited coding potential necessitating that they co-opt an unknown number of host factors to facilitate their replication in host cells. Global genomics and proteomics approaches performed with Tomato bushy stunt virus (TBSV) and yeast (Saccharomyces cerevisiae) as a model host have led to the identification of 250 host factors affecting TBSV RNA replication and recombination or bound to the viral replicase, replication proteins, or the viral RNA. The roles of a dozen host factors involved in various steps of the replication process have been validated in yeast as well as a plant host. Altogether, the large number of host factors identified and the great variety of cellular functions performed by these factors indicate the existence of a truly complex interaction between TBSV and the host cell. This review summarizes the advantages of using a simple plant virus and yeast as a model host to advance our understanding of virus-host interactions at the molecular and cellular levels. The knowledge of host factors gained can potentially be used to inhibit virus replication via gene silencing, expression of dominant negative mutants, or design of specific chemical inhibitors leading to novel specific or broad-range resistance and antiviral tools against (+)RNA plant viruses.