Source: UNIVERSITY OF KENTUCKY submitted to
ROLE OF PROMOTER AND ENHANCER ELEMENTS IN THE REPLICATION OF DEFECTIVE INTERFERING TOMBUSVIRUS RNA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0187118
Grant No.
(N/A)
Project No.
KY012012
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2000
Project End Date
Sep 30, 2005
Grant Year
(N/A)
Project Director
Nagy, P. D.
Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
PLANT PATHOLOGY
Non Technical Summary
RNA viruses of plants cause serious losses of many crops in the U.S. There are no effective measures to prevent the spread of plant viruses. The proposed research is to understand the replication of small parasitic RNAs that can interfere with RNA virus infections. This information may lead to development of novel virus control strategies.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21224991101100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1101 - Virology;
Goals / Objectives
Objective 1. To characterize the role of replication promoter elements in defective interfering (DI) RNA replication in tombusviruses in vitro. Objective 2. To characterize the role of replication enhancer elements in DI RNA replication and interference in tombusviruses in vitro. Objective 3. To construct super-competitive DI RNAs for control of helper virus infections.
Project Methods
Objective 1. Primary RNA sequences and secondary structures of RNA replication promoters, including promoters for minus and plus strand synthesis, will be delineated by using a recently developed in vitro assay with the partially purified tombusvirus RNA-dependent RNA polymerase. Enzymatic probing of RNA structure in combination with computer-based secondary structure prediction will be used to determine the secondary structure of the RNA promoters. Site-directed mutagenesis of RNA will be applied to depict the critical residues within the tombusvirus RNA promoters. Objective 2. An in vitro approach, similar to that described in Objective 1, will be applied to characterize replication enhancer elements. Objective 3. Based on the results of Objectives 1 and 2, super-competitive DI RNAs will be constructed using recombinant DNA technologies. RNA promoters and multiple copies of RNA enhancers with the highest activities will be combined in novel DI RNAs. The interference of these DI RNAs with helper virus infections will be tested in whole plants (Nicotiana benthamiana) and protoplasts.

Progress 10/01/00 to 09/30/05

Outputs
The PI and his associates have made progress in three main research areas with Tombusviruses: (1) Discovery of the role of the viral RNA template in assembly of the functional virus replicase complex; (2) Identification of a cis-acting RNA element involved in specific interaction with the viral replication proteins; (3) Dissection of the mechanism of stimulation of plus-strand synthesis by an RNA replication enhancer. (1) A highly active, template-dependent replicase complex for Cucumber necrosis virus (CNV) was obtained from Saccharomyces cerevisiae. The recombinant CNV replicase showed similar properties to the plant-derived CNV replicase. Isolation of functional replicase required the co-expression of the CNV p92 RNA-dependent RNA polymerase and the auxiliary p33 protein in yeast. Co-expression of a viral RNA template with the replicase proteins in yeast increased the activity of the purified CNV replicase by 40-fold, suggesting that the viral RNA might promote the assembly of the replicase complex and/or that the RNA increases the stability of the replicase. (2) The mechanism of template selection for genome replication in plus-strand RNA viruses is poorly understood. UsingTomato bushy stunt virus (TBSV), it was shown that recombinant p33 replicase protein binds specifically to an internal replication element (IRE) located within the p92 RNA-dependent RNA polymerase coding region of the viral genome. Specific binding of p33 to the IRE in vitro depends on the presence of a C . C mismatch within a conserved RNA helix. Importantly, there was a strong correlation between p33:IRE complex formation in vitro and viral replication in vivo, since mutations in the IRE that disrupted selective p33 binding in vitro also abolished TBSV RNA replication in both plant and in yeast cells. Based on these findings, it is proposed that the p33:IRE interaction provides a mechanism to selectively recruit viral RNAs into cognate viral replicase complexes. (3) Replication of RNA viruses is regulated by cis-acting RNA elements, including promoters, replication silencers and replication enhancers (REN). To dissect the function of an REN element involved in plus-strand RNA synthesis, we developed an in vitro trans-replication assay for tombusviruses. In this assay, two RNA strands were tethered together via short complementary regions with the REN present in the nontemplate RNA, whereas the promoter was located in the template RNA. We found that the template activity of the tombusvirus replicase preparation was stimulated in trans by the REN, suggesting that the REN is a functional enhancer when located in the vicinity of the promoter. This study revealed that the REN has dual function during RNA synthesis: (i) it binds to the viral replicase, and (ii) it interacts with the core plus-strand initiation promoter via a long-distance RNA-RNA interaction, which leads to stimulation of initiation of plus-strand RNA synthesis by the replicase in vitro. We propose that a REN is important for asymmetrical viral RNA replication that leads to more abundant plus-strand RNA progeny than the minus-strand intermediate, a hallmark of replication of plus-strand RNA viruses.

Impacts
Replication is the central step in the pathogenesis of plant viruses. Tombusviruses have become one of the premier systems to study plus-stranded RNA virus replication and recombination. The first purified recombinant Tombusvirus replicase showing high activity and template dependence was demonstrated. The research also identified a novel, regulatory, RNA elements, named a replication recruitment element and a replication enhancer element. In addition, an important role of the RNA in assembly of the viral replicase complex has been identified. These findings will greatly facilitate future studies on viral RNA replication and should enhance the means to limit damage caused by RNA viruses.

Publications

  • Panavas, T., Pogany, J., and Nagy, P. D. 2002. Internal initiation by the Cucumber necrosis virus RNA-dependent RNA polymerase is facilitated by promoter-like sequences. Virology, 296, 275-287.
  • Rajendran, K. S., Pogany, J., and Nagy, P. D. 2002. Comparison of Turnip crinkle virus RNA-dependent RNA polymerase preparations expressed in E. coli or derived from infected plants. Journal of Virology, 76, 1707-1717.
  • Nagy, P. D. and Pogany, J. 2000. Partial purification and characterization of Cucumber necrosis virus and Tomato bushy stunt virus RNA-dependent RNA polymerases: Similarities and differences in template usage between tombusvirus and carmovirus RNA-dependent RNA polymerases. Virology, 276: 279-288.
  • Panavas, T,, and Nagy, P.D. 2005. Mechanism of stimulation of plus-strand synthesis by an RNA replication enhancer in a tombusvirus. Journal of Virology 79:9777-85.
  • Stork, J., Panaviene, Z. and Nagy, P.D. 2005. Inhibition of in vitro RNA binding and replicase activity by phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus. Virology 343:79-92.
  • Shapka, N., Stork, J. and Nagy, P.D. 2005. Phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus adjacent to the RNA binding site affects viral RNA replication. Virology 343:65-78.
  • Panaviene, Z., Panavas, T., and Nagy, P.D. 2005. Role of an internal and two 3'-terminal RNA elements in assembly of tombusvirus replicase. Journal of Virology 79: 10608-18.
  • Panavas, T., and Nagy, P. D. 2003. Yeast as a model host to study replication and recombination of defective interfering RNA of Tomato bushy stunt virus. Virology 314: 315-325.
  • Pogany, J., Fabian, M., White, K.A., and Nagy, P. D. 2003. A replication silencer element in an RNA virus. EMBO Journal 22: 5602-5611.
  • Panavas, T., and Nagy, P. D. 2003. The RNA replication enhancer element of tombusviruses contains two interchangeable hairpins that are functional during plus-strand synthesis. Journal of Virology, 77, 258-269.
  • Panavas, T., Panaviene, Z., Pogany J., and Nagy, P. D. 2003. Generation of a novel cis-acting replication element by promoter duplication in tombusviruses. Virology, 310, 118-129.
  • Cheng, C.-P., Pogany, J., and Nagy, P. D. 2002. Mechanism of DI RNA formation in Tombusviruses: Dissecting the requirement for primer extension by the Tombusvirus RNA-dependent RNA polymerase in vitro. Virology, 304, 460-473.
  • Panavas, T., Pogany, J., and Nagy, P. D. 2002. Analysis of minimal promoter sequences for plus-strand synthesis by the Cucumber necrosis virus RNA-dependent RNA polymerase. Virology, 296, 263-274.


Progress 01/01/04 to 12/31/04

Outputs
The PI and his associates have made progress in two main research areas with Tombusviruses, which are small plant viruses: (1) discovery of the role of the viral RNA template in assembly of the functional virus replicase complex; (2) identification of a cis-acting RNA element involved in specific interaction with the viral replication proteins. The progress is outlined in detail below: (1) Purified recombinant viral replicases are useful to study the mechanism of viral RNA replication in vitro. A highly active, template-dependent replicase complex for Cucumber necrosis virus (CNV), which is a plus-stranded RNA virus, was obtained from Saccharomyces cerevisiae. The recombinant CNV replicase showed similar properties to the plant-derived CNV replicase (Nagy and Pogany, Virology, 2000) including, the ability (i) to initiate complementary RNA synthesis de novo on both plus- and minus-stranded templates, (ii) to generate shorter than full-length replicase products by internal initiation, and (iii) to perform primer extension from the 3' end of the template. Isolation of functional replicase required the co-expression of the CNV p92 RNA-dependent RNA polymerase and the auxiliary p33 protein in yeast. Moreover, co-expression of a viral RNA template with the replicase proteins in yeast increased the activity of the purified CNV replicase by 40-fold, suggesting that the viral RNA might promote the assembly of the replicase complex and/or that the RNA increases the stability of the replicase. (2) The mechanism of template selection for genome replication in plus-strand RNA viruses is poorly understood. Using the prototypical tombusvirus, Tomato bushy stunt virus (TBSV), it was shown that recombinant p33 replicase protein binds specifically to an internal replication element (IRE) located within the p92 RNA-dependent RNA polymerase coding region of the viral genome. Specific binding of p33 to the IRE in vitro depends on the presence of a C . C mismatch within a conserved RNA helix. Interestingly, the absence of the p33:p33 interaction domain in p33 prevented specific, but allowed non-specific, RNA binding, suggesting that a multimeric form of this protein is involved in the IRE-specific interaction. Further support for the selectivity of p33 binding in vitro was provided by the inability of the replicase proteins of the closely related Turnip crinkle virus and distantly related Hepatitis C virus to specifically recognize the TBSV IRE. Importantly, there was also a strong correlation between p33:IRE complex formation in vitro and viral replication in vivo, where mutations in the IRE that disrupted selective p33 binding in vitro also abolished TBSV RNA replication in both plant and in yeast cells. Based on these findings and the other known properties of p33 and the IRE, it is proposed that the p33:IRE interaction provides a mechanism to selectively recruit viral RNAs into cognate viral replicase complexes. Since all genera in Tombusviridae encode comparable replicase proteins, these results may be relevant to other members of this large virus family.

Impacts
Tombusviruses have become one of the premier systems to study plus-stranded RNA virus replication and recombination. The first purified recombinant Tombusvirus replicase showing high activity and template dependence was demonstrated. The research also identified a novel, regulatory, RNA element, named a replication recruitment element. In addition, an important role of the RNA in assembly of the viral replicase complex has been identified. These findings will greatly facilitate future studies on viral RNA replication and should enhance the means to limit damage caused by RNA viruses.

Publications

  • Pogany, J., White, K.A., and Nagy, P.D. 2004. Specific Binding of the Tombusvirus Replication Protein p33 to an Internal Replication Element in the Viral RNA is Essential for Replication. Journal of Virology (in press).
  • Monkewich, S., Lin, H-X, Fabian, M.R., Xu, W., Na, H., Ray, D., Chernysheva, O.A., Nagy, P.D., and White, K.A. 2004. p92 Polymerase Coding Region Contains an Internal RNA Element Required at an Early Step in Tombusvirus Genome Replication. Journal of Virology (in press).
  • Panaviene, Z. Panavas, T., Serva, S. and Nagy, P.D. 2004. Purification of the Cucumber necrosis virus replicase from yeast: The role of co-expressed viral RNA in stimulation of replicase activity. Journal of Virology, 78, 8254-8263.
  • Rajendran, K. S. and Nagy, P.D. 2004. Interaction between the replicase proteins of Tomato Bushy Stunt Virus in vitro and in vivo. Virology, 326: 250-261.
  • Boonrod, K., Galetzka, D., Nagy, P.D., Conrad, U. and Krczal, G. 2004. Single-chain antibodies against a plant viral RNA-dependent RNA polymerase confer virus resistance. Nature Biotechnolology, 22, 856-862.
  • Shapka, N. and Nagy, P.D. 2004. The AU-rich RNA recombination hot spot sequence of Brome mosaic virus is functional in tombusviruses: Implications for the mechanism of RNA recombination. Journal of Virology, 78, 2288-2300.
  • White, K.A., and Nagy, P.D. 2004. Advances in the Molecular Biology of Tombusviruses: Gene Expression, Genome Replication and Recombination. Progress in Nucleic Acid Research and Molecular Biology 78, 187-226.


Progress 01/01/03 to 12/31/03

Outputs
The PI and his associates have made progress in two main research areas with Tombusviruses, which are small plant viruses: (i) dissection of RNA replication signals in Tombusviruses based on purification of the viral replicase, which is the enzyme complex responsible for multiplying the viral genome, from infected plants; (ii) development of RNA recombination assays for Tombusviruses. In vitro analysis of template activities of a series of deletion mutants of plus and minus-stranded defective interfering (DI) RNAs revealed that a region, termed RIII(-), which is present in the minus-stranded RNA, contains a strong replication enhancer. The RIII(-) sequence enhanced RNA synthesis from the cPR promoter by ten-fold in the Cucumber necrosis virus RdRp assay. In addition to the RIII(-) enhancer, we also found that the 3 prime end of the minus-stranded DI RNA, adjacent to the 3 prime-terminal cPR promoter, contains a second replication enhancer element, termed the promoter proximal enhancer (PPE). The PPE element enhanced RNA synthesis by three-fold from the cPR promoter in the in vitro assay and it was also important for replication in vivo. The sequence of the PPE element is similar to the cPR promoter, suggesting that sequence duplication of the cPR promoter may have led to the generation of the PPE element. A novel RNA replication silencer element (RSE), which inhibited minus-strand RNA synthesis by seven-fold, was discovered by studying template-dependent RNA synthesis in vitro in a tombusvirus RdRp assay using DI-72(+) RNA, a model template. We found that RSE interacted with the 3 prime end of gPR (the minimal minus-strand initiation promoter) via forming a five base-pair pseudoknot-like structure. The interaction was confirmed by using: (i) RNA structure analysis; (ii) complementary mutations within the interacting sequences and; (iii) by inhibiting the interaction with the use of short complementary DNAs. We proposed that due to the RSE-gPR interaction, the 3 prime end sequence used for initiation is masked from recognition by the viral replicase. Similar RNA-RNA interactions may also regulate replication of other related viruses. The biological significance of RSE-gPR interaction was demonstrated in plant cell (protoplast) infection (this work was done in collaboration with Dr. A. White). These studies revealed that the interaction between RSE and gPR is essential for tombusvirus replication. Based on our findings, we propose that the replication silencer may serve important regulatory and/or structural functions during tombusvirus replication. We found that, in addition to initiating RNA synthesis de novo, the tombusvirus RdRp was capable of template-switching and primer extension in vitro. We also demonstrated that RII and RIII sequences promote efficient recombination in vivo.

Impacts
Based on the PI and others works in the last two years, Tombusviruses have become one of the premier systems to study plus-stranded RNA virus replication and recombination. The completed work identified novel regulatory RNA elements, such as replication enhancer and replication silencer.

Publications

  • Panavas, T., and Nagy, P. D. 2003. Yeast as a model host to study replication and recombination of defective interfering RNA of Tomato bushy stunt virus. Virology 314: 315-325.
  • Pogany, J., Fabian, M., White, K.A., and Nagy, P. D. 2003. A replication silencer element in an RNA virus. EMBO Journal 22: 5602-5611.
  • C.-P. Cheng, and Nagy, P. D. 2003. Mechanism of RNA recombination in Carmo- and Tombusviruses: Evidence for Template Switching by the RNA-dependent RNA polymerase in vitro. Journal of Virology 77: 12033-12047.
  • Panaviene, Z. and Nagy, P. D. 2003. Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo. Virology 317, 359-372.
  • Rajendran, K.S. and Nagy, P.D. 2003. Characterization of the RNA-binding domains in the replicase proteins of tomato bushy stunt virus. Journal of Virology 77, 9244-9258.
  • Panavas, T., and Nagy, P. D. 2003. The RNA replication enhancer element of tombusviruses contains two interchangeable hairpins that are functional during plus-strand synthesis. Journal of Virology, 77, 258-269.
  • Panaviene, Z., Baker, J. M., and Nagy, P. D. 2003. The overlapping RNA-binding domains of p33 and p92 replicase proteins are essential for tombusvirus replication. Virology, 308, 191-205.
  • Panavas, T., Panaviene, Z., Pogany J., and Nagy, P. D. 2003. Generation of a novel cis-acting replication element by promoter duplication in tombusviruses. Virology, 310, 118-129.


Progress 01/01/02 to 12/31/02

Outputs
The PI and his associates have developed biochemical methods to study the replication process of Tombusviruses, which are small plant viruses, in the test tube. This is based on purification of the viral replicase from infected plants, which is the enzyme complex responsible for multiplying the viral genome. The PI has characterized minimal promoter elements present within the RNA genomes of Tombusviruses that are necessary for virus replication. These promoter elements are 11-19 nucleotides in length and they are located at the 3' ends of both the plus-stranded and the minus-stranded RNAs. The promoters allow for precise initiation of complementary RNA synthesis initiated from the 3' ends, resulting in full-length RNA products. Internal initiation by the viral replicase was also detected, which might be important for the generation of small parasitic RNAs, called defective interfering RNAs. The PI has also discovered that Tombusviruses contain novel replication regulatory elements, such as RNA replication enhancers and replication silencers. These elements modulate virus replication, fitness, competitiveness and evolution. New RNA-protein and protein-protein interactions are currently being defined among the viral proteins and RNAs that are part of the replicase complex. In addition, a powerful genetic method to study Tombusvirus replication is under development based on yeast, the favorite host in virus-host interactions.

Impacts
RNA viruses cause major losses for agriculture and represent significant risk to human health. The PI studies replication, which is the central part of virus pathogenesis, of RNA viruses in infected hosts. The use of powerful model viral systems, such as Tombusviruses, small plant viruses, greatly contribute to our understanding of the replication process and the results obtained are expected to help in devising new strategies to control viral diseases.

Publications

  • Panavas, T., Pogany, J., and Nagy, P. D. (2002). Analysis of minimal promoter sequences for plus-strand synthesis by the Cucumber necrosis virus RNA-dependent RNA polymerase. Virology, 296: 263-274.
  • Panavas, T., Pogany, J., and Nagy, P. D. (2002). Internal initiation by the Cucumber necrosis virus RNA-dependent RNA polymerase is facilitated by promoter-like sequences. Virology, 296: 275-287.
  • Rajendran, K. S., Pogany, J., and Nagy, P. D. (2002). Comparison of Turnip crinkle virus RNA-dependent RNA polymerase preparations expressed in E. coli or derived from infected plants. Journal of Virology, 76:1707-1717.
  • Cheng, C.-P., Pogany, J., and Nagy, P. D. (2002). Mechanism of DI RNA formation in Tombusviruses: Dissecting the requirement for primer extension by the Tombusvirus RNA-dependent RNA polymerase in vitro. Virology, 304: 460-473.


Progress 01/01/01 to 12/31/01

Outputs
We have recently developed two new template-dependent in vitro RNA-dependent RNA polymerase (RdRp) systems for Tombusviruses. These RdRp preparations were purified from Cucumber necrosis virus (CNV)- and Tomato bushy stunt virus (TBSV)-infected plants. The Tombusvirus RdRp preparations initiated RNA synthesis from RNA templates without added primers (de novo initiation) in the presence of either the plus- or minus-strand initiation promoters of a TBSV defective interfering (DI) RNA. In addition, we have developed two new in vitro RdRp systems, based on the Turnip crinkle virus (TCV) RdRp proteins expressed in E. coli. These RdRp preparations can use the Tombusvirus-associated DI RNA and the TCV-associated sat-RNA efficiently as templates, while they cannot transcribe the phage Qbeta-associated satellite RNA or tRNA. Using the in vitro RdRp system, we demonstrated that the 3'-terminal 19 nucleotides (nt) of the plus-strand RNA contains the minimal minus-strand initiation promoter (termed genomic promoter, gPR). In contrast, the minimal plus-strand initiation promoter (complementary promoter, or cPR) includes the 3'-terminal 11 nt of the minus-strand RNA. These promoters are capable of directing de novo initiation from their 3'-terminal cytidylates. In vitro analysis of template activities of a series of deletion mutants of plus and minus-stranded DI RNAs revealed that a minus-strand region, termed region III, contains a replication enhancer. The region III(-) sequence enhanced RNA synthesis from the upstream minimal cPR promoter by ten-fold in the in vitro RdRp assay. RNA binding studies with the E. coli expressed and purified tombusvirus RdRp revealed that the RdRp could bind directly and efficiently to region III (-) while binding to other control RNAs were much weaker. This suggests that the role of the region III(-) is to recruit the RdRp during replication. A 17 nt-long sequence neighboring the minimal cPR promoter was found to stimulate RNA synthesis by 3-fold. We term this sequence the promoter proximal enhancer (PPE). The sequence of the PPE is similar to cPR, suggesting that sequence duplication of the promoter region was the likely mechanism of generation of PPE. This was supported by the 2.5-fold higher template activity of an artificial construct carrying directly duplicated cPR when compared to a construct with one copy of cPR. This suggests that RNA recombination is important in the evolution of cis-acting replication enhancer elements.

Impacts
Significan progress was made towards generating novel parasitic RNAs (termed defective interfering or DI RNAs) that are more competitive than wild type DI RNA isolates. These DI RNAs have potential for grealy reducing virus symptoms and crop loss caused by some plant viruses.

Publications

  • Peter D. Nagy, Judit Pogany and Anne E. Simon (2001). In vivo and in vitro characterization of an RNA replication enhancer in a satellite RNA associated with Turnip crinkle virus. Virology, 288, 315-324.


Progress 10/01/00 to 12/31/00

Outputs
Plus-strand RNA viruses, including the Tombusviruses, replicate in their hosts in a two-step process. The infecting plus-strand RNA is first copied to complementary minus-strand RNA, which then serves as a template for plus-strand RNA synthesis. Each RNA strand contains one initiation sequence (promoter), which is absolutely necessary for virus replication. Using our in vitro system, based on the purified viral replicase complex, we have found that the promoter region for plus-strand synthesis is located at the 3' end of the minus-stranded RNA within the terminal 28 nucleotides (nt). Further deletion studies localized the 'core' or minimal promoter to just 11 terminal nts. Mutations within this core region have had various debilitating effects on the promoter activity, with the terminal two cytidylates absolutely required for RNA initiation. Dissecting the minimal promoter for minus strand synthesis is currently underway.

Impacts
Plus-strand RNA viruses are the most abundant and significant virus pathogens of plants and animals. Virus replication is one of the most significant steps in the life cycle, infectivity and pathogenicity of viruses. Promoter sequences are crucial in these processes. Therefore, these studies are providing fundamental information on virus replication and will help develop future tools to interfere with viral infection processes in both plants and animals.

Publications

  • Nagy, P. D and Pogany, J. (2000) Partial purification and characterization of Cucumber necrosis virus and Tomato bushy stunt virus RNA-dependent RNA polymerases: Similarities and differences in template usage between tombusvirus and carmovirus RNA-dependent RNA polymerases. Virology 276; 279-288.