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Peter D. Nagy - One of the best experts on this subject based on the ideXlab platform.
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Genome-Wide Screens for Identification of Host Factors in Viral Replication
Methods of Molecular Biology, 2020Co-Authors: Tadas Panavas, Elena Serviene, Judit Pogany, Peter D. NagyAbstract:: The central step in virus infection cycle is replication, which depends on viral and host factors. Model hosts, such as yeast, can be very valuable to identify host factors and study the functional interactions of host factors with viral proteins and/or the virus nucleic acids. The advantages of using yeast include the availability of (i) single gene-deletion library, (ii) the essential gene library (yTHC), (iii) the controllable small or large-scale expression of viral proteins and nucleic acids, and (iv) the rapid growth of yeast strains. Here, we describe procedures, which facilitate high-throughput analysis of Tombusvirus replication in yeast.
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co opting the fermentation pathway for Tombusvirus replication compartmentalization of cellular metabolic pathways for rapid atp generation
PLOS Pathogens, 2019Co-Authors: Melissa Molho, Shengjie Zhang, Longshen Wang, Peter D. NagyAbstract:The viral replication proteins of plus-stranded RNA viruses orchestrate the biogenesis of the large viral replication compartments, including the numerous viral replicase complexes, which represent the sites of viral RNA replication. The formation and operation of these virus-driven structures require subversion of numerous cellular proteins, membrane deformation, membrane proliferation, changes in lipid composition of the hijacked cellular membranes and intensive viral RNA synthesis. These virus-driven processes require plentiful ATP and molecular building blocks produced at the sites of replication or delivered there. To obtain the necessary resources from the infected cells, tomato bushy stunt virus (TBSV) rewires cellular metabolic pathways by co-opting aerobic glycolytic enzymes to produce ATP molecules within the replication compartment and enhance virus production. However, aerobic glycolysis requires the replenishing of the NAD+ pool. In this paper, we demonstrate the efficient recruitment of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes into the viral replication compartment. Depletion of Pdc1 in combination with deletion of the homologous PDC5 in yeast or knockdown of Pdc1 and Adh1 in plants reduced the efficiency of Tombusvirus replication. Complementation approach revealed that the enzymatically functional Pdc1 is required to support Tombusvirus replication. Measurements with an ATP biosensor revealed that both Pdc1 and Adh1 enzymes are required for efficient generation of ATP within the viral replication compartment. In vitro reconstitution experiments with the viral replicase show the pro-viral function of Pdc1 during the assembly of the viral replicase and the activation of the viral p92 RdRp, both of which require the co-opted ATP-driven Hsp70 protein chaperone. We propose that compartmentalization of the co-opted fermentation pathway in the tombusviral replication compartment benefits the virus by allowing for the rapid production of ATP locally, including replenishing of the regulatory NAD+ pool by the fermentation pathway. The compartmentalized production of NAD+ and ATP facilitates their efficient use by the co-opted ATP-dependent host factors to support robust Tombusvirus replication. We propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for Tombusviruses to replicate rapidly to speed ahead of antiviral responses of the hosts and to outcompete other pathogenic viruses. We also show the dependence of turnip crinkle virus, bamboo mosaic virus, tobacco mosaic virus and the insect-infecting Flock House virus on the fermentation pathway, suggesting that a broad range of viruses might induce this pathway to support rapid replication.
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Tombusvirus rna replication depends on the tor pathway in yeast and plants
Virology, 2018Co-Authors: Junichi Inaba, Peter D. NagyAbstract:Abstract Similar to other (+)RNA viruses, tomato bushy stunt virus (TBSV) utilizes metabolites, lipids, membranes, and co-opted host factors during replication. The coordination of cell metabolism and growth with environmental cues is performed by the target of rapamycin (TOR) kinase in eukaryotic cells. In this paper, we find that TBSV replication partially inhibits TOR activity, likely due to recruitment of glycolytic enzymes to the viral replication compartment, which results in reduced ATP levels in the cytosol. Complete inhibition of TOR activity with rapamycin in yeast or AZD8055 inhibitor in plants reduces Tombusvirus replication. We find that high glucose concentration, which stimulates TOR activity, enhanced Tombusvirus replication in yeast. Depletion of yeast Sch9 or plant S6K1 kinase, a downstream effector of TOR, also inhibited Tombusvirus replication in yeast and plant or the assembly of the viral replicase in vitro. Altogether, the TOR pathway is crucial for TBSV to replicate efficiently in hosts.
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assembly hub function of er localized snare proteins in biogenesis of Tombusvirus replication compartment
PLOS Pathogens, 2018Co-Authors: Zsuzsanna Sasvari, Nikolay Kovalev, Paulina Alatriste Gonzalez, Kai Xu, Peter D. NagyAbstract:Positive-strand RNA viruses assemble numerous membrane-bound viral replicase complexes within large replication compartments to support their replication in infected cells. Yet the detailed mechanism of how given subcellular compartments are subverted by viruses is incompletely understood. Although, Tomato bushy stunt virus (TBSV) uses peroxisomal membranes for replication, in this paper, we show evidence that the ER-resident SNARE (soluble NSF attachment protein receptor) proteins play critical roles in the formation of active replicase complexes in yeast model host and in plants. Depletion of the syntaxin 18-like Ufe1 and Use1, which are components of the ER SNARE complex in the ERAS (ER arrival site) subdomain, in yeast resulted in greatly reduced Tombusvirus accumulation. Over-expression of a dominant-negative mutant of either the yeast Ufe1 or the orthologous plant Syp81 syntaxin greatly interferes with Tombusvirus replication in yeast and plants, thus further supporting the role of this host protein in Tombusvirus replication. Moreover, Tombusvirus RNA replication was low in cell-free extracts from yeast with repressed Ufe1 or Use1 expression. We also present evidence for the mislocalization of the tombusviral p33 replication protein to the ER membrane in Ufe1p-depleted yeast cells. The viral p33 replication protein interacts with both Ufe1p and Use1p and co-opts them into the TBSV replication compartment in yeast and plant cells. The co-opted Ufe1 affects the virus-driven membrane contact site formation, sterol-enrichment at replication sites, recruitment of several pro-viral host factors and subversion of the Rab5-positive PE-rich endosomes needed for robust TBSV replication. In summary, we demonstrate a critical role for Ufe1 and Use1 SNARE proteins in TBSV replication and propose that the pro-viral functions of Ufe1 and Use1 are to serve as assembly hubs for the formation of the extensive TBSV replication compartments in cells. Altogether, these findings point clearly at the ERAS subdomain of ER as a critical site for the biogenesis of the TBSV replication compartment.
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Tombusvirus host interactions co opted evolutionarily conserved host factors take center court
Annual Review of Virology, 2016Co-Authors: Peter D. NagyAbstract:Plant positive-strand (+)RNA viruses are intracellular infectious agents that reorganize subcellular membranes and rewire the cellular metabolism of host cells to achieve viral replication in elaborate replication compartments. This review describes the viral replication process based on Tombusviruses, highlighting common strategies with other plant and animal viruses. Overall, the works on Tomato bushy stunt virus (TBSV) have revealed intriguing and complex functions of co-opted cellular translation factors, heat shock proteins, DEAD-box helicases, lipid transfer proteins, and membrane-deforming proteins in virus replication. The emerging picture is that many of the co-opted host factors are from highly expressed and conserved protein families. By hijacking host proteins, phospholipids, sterols, and the actin network, TBSV exerts supremacy over the host cell to support viral replication in large replication compartments. Altogether, these advances in our understanding of Tombusvirus-host interactions are...
Daniel Barajas - One of the best experts on this subject based on the ideXlab platform.
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role of viral rna and co opted cellular escrt i and escrt iii factors in formation of Tombusvirus spherules harboring the Tombusvirus replicase
Journal of Virology, 2016Co-Authors: Nikolay Kovalev, Daniel Barajas, Judit Pogany, Isabel Fernandez De Castro Martin, Kunj B Pathak, Cristina Risco, Peter D. NagyAbstract: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.
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the proteasomal rpn11 metalloprotease suppresses Tombusvirus rna recombination and promotes viral replication via facilitating assembly of the viral replicase complex
Journal of Virology, 2015Co-Authors: Reddisiva K Prasanth, Daniel Barajas, Peter D. NagyAbstract:RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in Tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a “matchmaker” that brings the viral p92pol replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress Tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the Tombusvirus VRCs. IMPORTANCE RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, Tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in Tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for Tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then Tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for Tombusviruses by regulating viral replication and genetic recombination.
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Novel mechanism of regulation of Tomato bushy stunt virus replication by cellular WW-domain proteins
Journal of Virology, 2014Co-Authors: Daniel Barajas, Nikolay Kovalev, Peter D. NagyAbstract:Replication of (+)RNA viruses depends on several co-opted host proteins but is also under the control of cell-intrinsic restriction factors (CIRFs). By using Tombusviruses, small model viruses of plants, we dissect the mechanism of inhibition of viral replication by cellular WW-domain-containing proteins, which act as CIRFs. By using fusion proteins between the WW domain and the p33 replication protein, we show that the WW domain inhibits the ability of p33 to bind to the viral RNA and to other p33 and p92 replication proteins leading to inhibition of viral replication in yeast and in a cell extract. Overexpression of WW-domain protein in yeast also leads to reduction of several co-opted host factors in the viral replicase complex (VRC). These host proteins, such as eEF1A, Cdc34 E2 ubiquitin-conjugating enzyme, and ESCRT proteins (Bro1p and Vps4p), are known to be involved in VRC assembly. Simultaneous coexpression of proviral cellular factors with WW-domain protein partly neutralizes the inhibitory effect of the WW-domain protein. We propose that cellular WW-domain proteins act as CIRFs and also as regulators of Tombusvirus replication by inhibiting the assembly of new membrane-bound VRCs at the late stage of infection. We suggest that Tombusviruses could sense the status of the infected cells via the availability of cellular susceptibility factors versus WW-domain proteins for binding to p33 replication protein that ultimately controls the formation of new VRCs. This regulatory mechanism might explain how Tombusviruses could adjust the efficiency of RNA replication to the limiting resources of the host cells during infections. IMPORTANCE Replication of positive-stranded RNA viruses, which are major pathogens of plants, animals, and humans, is inhibited by several cell-intrinsic restriction factors (CIRFs) in infected cells. We define here the inhibitory roles of the cellular Rsp5 ubiquitin ligase and its WW domain in plant-infecting Tombusvirus replication in yeast cells and in vitro using purified components. The WW domain of Rsp5 binds to the viral RNA-binding sites of p33 and p92 replication proteins and blocks the ability of these viral proteins to use the viral RNA for replication. The WW domain also interferes with the interaction (oligomerization) of p33 and p92 that is needed for the assembly of the viral replicase. Moreover, WW domain also inhibits the subversion of several cellular proteins into the viral replicase, which otherwise play proviral roles in replication. Altogether, Rsp5 is a CIRF against a Tombusvirus, and it possibly has a regulatory function during viral replication in infected cells.
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host factors with regulatory roles in Tombusvirus replication
Current Opinion in Virology, 2012Co-Authors: Peter D. Nagy, Daniel Barajas, Judit PoganyAbstract: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 p92pol 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.
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similar roles for yeast dbp2 and arabidopsis rh20 dead box rna helicases to ded1 helicase in Tombusvirus plus strand synthesis
Virology, 2012Co-Authors: Nikolay Kovalev, Daniel Barajas, Peter D. NagyAbstract:Abstract 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.
Judit Pogany - One of the best experts on this subject based on the ideXlab platform.
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Genome-Wide Screens for Identification of Host Factors in Viral Replication
Methods of Molecular Biology, 2020Co-Authors: Tadas Panavas, Elena Serviene, Judit Pogany, Peter D. NagyAbstract:: The central step in virus infection cycle is replication, which depends on viral and host factors. Model hosts, such as yeast, can be very valuable to identify host factors and study the functional interactions of host factors with viral proteins and/or the virus nucleic acids. The advantages of using yeast include the availability of (i) single gene-deletion library, (ii) the essential gene library (yTHC), (iii) the controllable small or large-scale expression of viral proteins and nucleic acids, and (iv) the rapid growth of yeast strains. Here, we describe procedures, which facilitate high-throughput analysis of Tombusvirus replication in yeast.
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role of viral rna and co opted cellular escrt i and escrt iii factors in formation of Tombusvirus spherules harboring the Tombusvirus replicase
Journal of Virology, 2016Co-Authors: Nikolay Kovalev, Daniel Barajas, Judit Pogany, Isabel Fernandez De Castro Martin, Kunj B Pathak, Cristina Risco, Peter D. NagyAbstract: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.
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template role of double stranded rna in Tombusvirus replication
Journal of Virology, 2014Co-Authors: Nikolay Kovalev, Judit Pogany, Peter D. NagyAbstract: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.
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host factors with regulatory roles in Tombusvirus replication
Current Opinion in Virology, 2012Co-Authors: Peter D. Nagy, Daniel Barajas, Judit PoganyAbstract: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 p92pol 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.
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defining the roles of cis acting rna elements in Tombusvirus replicase assembly in vitro
Journal of Virology, 2012Co-Authors: Kunj B Pathak, K A White, Judit Pogany, Kai Xu, Peter D. NagyAbstract:ABSTRACT 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.
Andrew K White - One of the best experts on this subject based on the ideXlab platform.
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Tombusvirus like viruses tombusviridae
2020Co-Authors: Andrew K WhiteAbstract:Abstract Tombusvirus is the type genus of the family Tombusviridae and is closely related to two other genera in this family, Aureusvirus and Zeavirus. Collectively, members of this trio are distributed worldwide, infect wild and cultivated hosts, and elicit mild to severe symptoms. All possess plus-sense, single-stranded RNA genomes that are packaged into ~30 nm spherical capsids. Decades of research have generated a large amount of data on this group and allowed for the development of molecular models to describe steps in the infectious process. This article updates these findings and other fundamental viral features, as well as biotechnological applications.
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Tombusvirus polymerase structure and function
Virus Research, 2017Co-Authors: Chaminda D Gunawardene, Logan W Donaldson, Andrew K WhiteAbstract:Abstract Tombusviruses are small icosahedral viruses that possess plus-sense RNA genomes ∼4.8 kb in length. The type member of the genus, tomato bushy stunt virus (TBSV), encodes a 92 kDa (p92) RNA-dependent RNA polymerase (RdRp) that is responsible for viral genome replication and subgenomic (sg) mRNA transcription. Several functionally relevant regions in p92 have been identified and characterized, including transmembrane domains, RNA-binding segments, membrane targeting signals, and oligomerization domains. Moreover, conserved Tombusvirus-specific motifs in the C-proximal region of the RdRp have been shown to modulate viral genome replication, sg mRNA transcription, and trans-replication of subviral replicons. Interestingly, p92 is initially non-functional, and requires an accessory viral protein, p33, as well as viral RNA, host proteins, and intracellular membranes to become active. These and other host factors, through a well-orchestrated process guided by the viral replication proteins, mediate the assembly of membrane-associated virus replicase complexes (VRCs). Here, we describe what is currently known about the structure and function of the Tombusvirus RdRp and how it utilizes host components to build VRCs that synthesize viral RNAs.
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conserved motifs in a Tombusvirus polymerase modulate genome replication subgenomic transcription and amplification of defective interfering rnas
Journal of Virology, 2015Co-Authors: Chaminda D Gunawardene, Karolina Jaluba, Andrew K WhiteAbstract:The replication of plus-strand RNA virus genomes is mediated by virally encoded RNA-dependent RNA polymerases (RdRps). We have investigated the role of the C-proximal region in the RdRp of tomato bushy stunt virus (TBSV) in mediating viral RNA synthesis. TBSV is the prototype species in the genus Tombusvirus, family Tombusviridae, and its RdRp is responsible for replicating the viral genome, transcribing two subgenomic mRNAs, and supporting replication of defective interfering RNAs. Comparative sequence analysis of the RdRps of tombusvirids identified three highly conserved motifs in their C-proximal regions, and these sequences were subsequently targeted for mutational analysis in TBSV. The results revealed that these motifs are important for (i) synthesizing viral genomic RNA and subgenomic mRNAs, (ii) facilitating plus- and/or minus-strand synthesis, and (iii) modulating trans-replication of a defective interfering RNA. These motifs were also found to be conserved in other plant viruses as well as in a fungal and insect virus. The collective findings are discussed in relation to viral RNA synthesis and taxonomy. IMPORTANCE Little is currently known about the structure and function of the viral polymerases that replicate the genomes of RNA plant viruses. Tombusviruses, the prototype of the tombusvirids, have been used as model plus-strand RNA plant viruses for understanding many of the steps in the infectious process; however, their polymerases remain poorly characterized. To help address this issue, the function of the C-terminal region of the polymerase of a Tombusvirus was investigated. Three conserved motifs were identified and targeted for mutational analysis. The results revealed that these polymerase motifs are important for determining what type of viral RNA is produced, facilitating different steps in viral RNA production, and amplifying subgenomic RNA replicons. Accordingly, the C-terminal region of the Tombusvirus polymerase is needed for a variety of fundamental activities. Furthermore, as these motifs are also present in distantly related viruses, the significance of these results extends beyond tombusvirids.
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Tombusvirus recruitment of host translational machinery via the 3 utr
RNA, 2010Co-Authors: Beth L Nicholson, Baodong Wu, Irina Chevtchenko, Andrew K WhiteAbstract:RNA viruses recruit the host translational machinery by different mechanisms that depend partly on the structure of their genomes. In this regard, the plus-strand RNA genomes of several different pathogenic plant viruses do not contain traditional translation-stimulating elements, i.e., a 59-cap structure and a 39-poly(A) tail, and instead rely on a 39-cap-independent translational enhancer (39CITE) located in their 39 untranslated regions (UTRs) for efficient synthesis of viral proteins. We investigated the structure and function of the I-shaped class of 39CITE in Tombusviruses—also present in aureusviruses and carmoviruses—using biochemical and molecular approaches and we determined that it adopts a complex higher-order RNA structure that facilitates translation by binding simultaneously to both eukaryotic initiation factor (eIF) 4F and the 59 UTR of the viral genome. The specificity of 39CITE binding to eIF4F is mediated, at least in part, through a direct interaction with its eIF4E subunit, whereas its association with the viral 59 UTR relies on complementary RNA–RNA base-pairing. We show for the first time that this tripartite 59 UTR/39CITE/eIF4F complex forms in vitro in a translationally relevant environment and is required for recruitment of ribosomes to the 59 end of the viral RNA genome by a mechanism that shares some fundamental features with cap-dependent translation. Notably, our results demonstrate that the 39CITE facilitates the initiation step of translation and validate a molecular model that has been proposed to explain how several different classes of 39CITE function. Moreover, the virus–host interplay defined in this study provides insights into natural host resistance mechanisms that have been linked to 39CITE activity.
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context influenced cap independent translation of Tombusvirus mrnas in vitro
Virology, 2008Co-Authors: Beth L Nicholson, Andrew K WhiteAbstract:Tomato bushy stunt virus (TBSV) possesses a positive-strand RNA genome that is not 5′-capped or 3′-polyadenylated. Previous analysis revealed that the TBSV genome contains a 3′-cap-independent translational enhancer (3′CITE) in its 3′-untranslated region (3′UTR) that facilitates translation of viral mRNAs in vivo. A long-range 5′–3′ RNA–RNA interaction between the 3′CITE and the 5′UTR of viral mRNAs is necessary for function, and this RNA bridge has been proposed to mediate delivery of translation-related factors bound to the 3′CITE to the 5′-end of the message. Although fully functional when assayed in plant protoplasts, the TBSV 3′CITE was previously found to be unable to activate translation in vitro in wheat germ extract (wge). In the current report we have determined that (i) another Tombusvirus, Carnation Italian ringspot virus (CIRV), contains a TBSV-like 3′CITE that is active in wge; (ii) the CIRV 3′CITE functions in vitro in a manner analogous to the TBSV 3′CITE in vivo; (iii) the TBSV 3′CITE is able to competitively inhibit CIRV 3′CITE-dependent translation in wge and (iv) the TBSV 3′CITE can enhance translation in wge when present in short viral messages. These results reveal the contrasting activities of different TBSV-like 3′CITEs in vitro and shed light on the nature of the defect in TBSV.
Jozsef Burgyan - One of the best experts on this subject based on the ideXlab platform.
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identification of sequence elements of Tombusvirus associated defective interfering rnas required for symptom modulation
Archives of Virology, 2006Co-Authors: Csaba Hornyik, Zoltan Havelda, Jozsef BurgyanAbstract:Defective interfering (DI) RNAs of Tombusviruses are short, non-coding, symptom-modulating RNAs originating from the viral genome. The presence of homologous DI RNA in virus infection attenuates the otherwise lethal viral symptoms. Nicotiana benthamiana plants infected with tomato bushy stunt Tombusvirus pepper isolate (TBSV-P) show severe symptoms, which culminate in the death of the plant. In contrast, plants co-inoculated with TBSV-P and TBSV-P-derived DI RNA display attenuated symptoms. However, co-inoculation of TBSV-P with heterologous DI RNA, originating from Carnation Italian ringspot Tombusvirus results in development of apical necrotic symptoms. To localize the symptom-determining factors on DI RNA genome, chimeras of protective and non-protective DI RNAs have been constructed. All chimeras were biologically active and accumulated to a high level in the presence of helper virus. We identified a 5′ proximal sequence element of the DI RNA as the most important symptom determinant region. However, our results demonstrated that the symptom modulating ability of this region is also influenced by the sequence composition of whole DI RNAs.
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defective interfering rna hinders the activity of a Tombusvirus encoded posttranscriptional gene silencing suppressor
Journal of Virology, 2005Co-Authors: Zoltan Havelda, Csaba Hornyik, Anna Valoczi, Jozsef BurgyanAbstract:Defective interfering (DI) RNAs are subviral replicons originating from the viral genome and are associated with many plant RNA viruses and nearly all animal RNA viruses. The presence of DI RNAs in Tombusvirus-infected plants reduces the accumulation of helper virus RNA and results in the development of attenuated symptoms similar to those caused by Tombusviruses defective in p19, the posttranscriptional gene silencing (PTGS) suppressor. In situ analysis of infected plants containing DI RNAs revealed that the extent of virus infection was spatially restricted as was found for p19-defective Tombusvirus. Previously, p19 was shown to suppress PTGS by sequestering the small interfering RNAs (siRNAs), which act as the specificity determinant for PTGS. Our results demonstrate that DI RNAs dramatically elevate the level of virus-specific siRNAs in viral infections, resulting in the saturation of p19 and the accumulation of unbound siRNAs. Moreover, we showed that, at low temperature, where PTGS is inhibited, DI RNAs are not able to efficiently interfere with virus accumulation and protect the plants. These data show that the activation of PTGS plays a pivotal role in DI RNA-mediated interference. Our data also support a role for 21-nucleotide siRNAs in PTGS signaling.
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in situ characterization of cymbidium ringspot Tombusvirus infection induced posttranscriptional gene silencing in nicotiana benthamiana
Journal of Virology, 2003Co-Authors: Zoltan Havelda, Csaba Hornyik, Aniello Crescenzi, Jozsef BurgyanAbstract:In plants, posttranscriptional gene silencing (PTGS) is an ancient and effective defense mechanism against viral infection. A number of viruses encode proteins that suppress virus-activated PTGS. The p19 protein of Tombusviruses is a potent PTGS suppressor which interferes with the onset of PTGS-generated systemic signaling and is not required for viral replication or for viral movement in Nicotiana benthamiana. This unique feature of p19 suppressor allowed us to analyze the mechanism of PTGS-based host defense and its viral suppression without interfering with other viral functions. In contrast to the necrotic symptoms caused by wild-type Tombusvirus, the infection of p19-defective mutant virus results in the development of a typical PTGS-associated recovery phenotype in N. benthamiana. In this report we show the effect of PTGS on the viral infection process for N. benthamiana infected with either wild-type Cymbidium Ringspot Tombusvirus (CymRSV) or a p19-defective mutant (Cym19stop). In situ analyses of different virus-derived products revealed that PTGS is not able to reduce accumulation of virus in primary infected cells regardless of the presence of p19 PTGS suppressor. We also showed that both CymRSV and Cym19stop viruses move systemically in the vasculature, with similar efficiencies. However, in contrast to the uniform accumulation of CymRSV throughout systemically infected leaves, the presence of Cym19stop virus was confined to and around the vascular bundles. These results suggest that the role of p19 is to prevent the onset of mobile signal-induced systemic PTGS ahead of the viral infection front, leading to generalized infection.
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the 5 terminal region of a Tombusvirus genome determines the origin of multivesicular bodies
Journal of General Virology, 1996Co-Authors: Jozsef Burgyan, Luisa Rubino, Marcello RussoAbstract:Multivesicular bodies (MVB) are membranous cytoplasmic inclusions that are invariably associated with Tombusvirus infections regardless of the virus species, the host, or the tissue examined. MVB are virus-induced structures since they are absent from tissues of healthy plants and are always present both in infected plants and protoplasts. MVB derive from peroxisomes in cells infected by a number of Tombusviruses including cymbidium ringspot virus (CymRSV) and from mitochondria in cells infected by another Tombusvirus, carnation Italian ringspot virus (CIRV). By using common restriction sites in full-length infectious clones, hybrid clones of these two viruses were constructed. In addition, a mutant of CIRV was prepared in which the protein encoded by the first open reading frame was shortened by 22 amino acids. All mutant transcripts were viable and infected Nicotiana benthamiana plants. Infected leaf tissue samples were collected, processed for thin sectioning, and observed in the electron microscope. The origin of MVB was shown to be under the control of the 5′ region of the viral genome. A sequence as short as about 600 nucleotides in ORF 1 contained the determinants for formation of MVB from peroxisomes or mitochondria.
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localization of cis acting sequences essential for cymbidium ringspot Tombusvirus defective interfering rna replication
Journal of General Virology, 1995Co-Authors: Zoltan Havelda, Tamas Dalmay, Jozsef BurgyanAbstract:The smallest defective interfering RNA (DI-2) of cymbidium ringspot Tombusvirus (CyRSV) was used to identify the cis-acting sequences necessary for its replication by making a series of deletions throughout the 404 nt long molecule and testing the biological activity of mutants. Deletion or substitution of the conserved sequence blocks (A, B and C) always yielded inactive molecules. The deletion of only a few nucleotides could be tolerated beyond the natural deletion sites in blocks A and B. However, either half of block C1 (34 nt) and the first 25 nt of C2 (102 nt) could be deleted without loss of infectivity. It was also demonstrated that either one of the two halves of block C1 was specifically required for replication. We suggest that the last 77 nt of the viral genome and either half of block C1 represent the complementary strand promoter sequence recognized by the viral replicase.
