Final Report Summary - PEP-REP-2 (In vitro replication of Pepino mosaic virusand characterization of replication essential host (tomato) factors)
In Pep-Rep-2, the main objective was the purification of soluble, template-dependent RNA-dependent RNA polymerase (RdRp) from Pepino mosaic virus (PepMV)-infected tomato plants in order to facilitate analysis of viral synthesis and secondarily, to characterise two interactions between tomato and PepMV-encoded proteins.
Initially, a laboratory area was designated for this quarantine plant virus and pilot transcription assays from various plant extracts were performed. More specifically, homogenised extracts from PepMV-infected leaves were subjected to differential centrifugation and separation on sucrose gradients and the resultant fractions, containing membrane-bound RdRp, were tested for RNA transcriptional activity. Endogenous templates, namely incomplete, partially synthesized RNAs present in the replication complexes (RCs) of infected plants were used to test for RNA transcriptional activity. The resuspended P30 pellet (membrane-bound RdRp) transcribed mainly four endogenous RNA species corresponding to products of the genomic and sgRNAs (TGB1, TGB2-3, CP). All four RNAs were shown to be double-stranded (ds) in nature. We further characterised the relative amounts of the genomic and sgRNAs produced by membrane-bound PepMV RdRp. Genomic dsRNA predominantly hybridised to negative-sense PepMV 3'- terminal RNA transcripts suggesting that they represent mainly positive-sense RNAs. All three subgenomic dsRNAs hybridised with similar intensities to identical amounts 5 ug of the negative-sense 5'- terminal and positive-sense strand 3'- terminal RNAs suggesting that they are comprised of similar amounts of both RNA strands.
An important breakthrough was the initiation of specific plus-and minus-strand RNA synthesis by template-bound RdRp. Here, the endogenous PepMV RNA present in the RdRp-enriched fraction was digested with micrococcal nuclease and the resultant template-dependent RdRp was then incubated with either a full-length PepMV RNA transcript or PepMV virion RNA. Both templates successfully synthesized full-length genomic RNA products indicating the specificity of the purified RdRp system. The template-dependent PepMV RdRp was then programmed with several different PepMV RNA templates, including positive-and negative-sense RNA strands. These experiments revealed specific recognition of the 3'- termini of both positive-and negative-sense PepMV RNA transcripts. Additionally, a polyclonal antiserum was raised against a peptide epitope representing an immunogenic stretch of the PepMV RdRp. The purified IgGs detected the PepMV RdRp in the membrane-bound as well as the template-dependent fractions and in subsequent experiments, and significantly reduced the activity of the template-dependent RdRp in a concentration-dependent manner.
Finally RNA synthesis from the 3'- terminus of the PepMV genome was examined in order to permit analysis of replication-essential cis-acting RNA elements. Identical amounts of PepMV RNA transcripts, representing the positive-stranded 3'- terminus together with various lengths of the poly
(a) tail were introduced into the established template-dependent RdRp system. It was demonstrated that a poly
(a) tail of at least 20 adenosine residues was necessary for minus-strand synthesis. Further analysis of the cis-acting 3'- UTR elements for minus-strand RNA synthesis were undertaken and a prediction for the PepMV 3'- UTR structure was experimentally proven. Pin-point mutations were used to introduce deletions, substitutions and restorations of specific base pairs suggesting that stem loop 1 (SL1) containing the polyadenylation signal is replication redundant while, SL2 and SL3 and the predicted pseudoknot are replication essential.
Objective 2 was corroborated with research carried out in parallel at doctoral level. Here, two host factors (Hsc70 and PROTEIN-C) were shown to interact with the PepMV proteins, CP and p25. Specifically for Hsc70, the amino acid sequences of the identified interactors from the tomato cDNA library were not 100 % identical and later experiments (yeast two hybrid and BiFC assay) confirmed the interaction for one of the three complete tomato Hsc70s namely, Hsc70. 3. The PepMV CP-Hsc70. 3 interaction complex was localised in the cytoplasm and nucleus. Immunogold labeling, transmission electron microscopy, immunoblot analysis on purified PepMV virions and time course experiments (mRNA, proteins) suggested that elevated levels of Hsc70 accumulate and interact with virions/CP in PepMV-infected leaves. Of particular importance are the findings of the relative PROTEIN-C mRNA and protein levels in infected tissue in relation to PepMV p25. These data will permit future experimentation in order to confirm the actual role of the interaction in virus infection.
Overall, Pep-Rep-2 capitalised on a previous bilateral (Greece-Spain) Project results and ran in parallel with a PhD investigation (2009-2012) to produce a number of significant scientific advances in the field of RNA virus replication. Furthermore, Pep-Rep-2 fosters future collaborations and will have an impact on phytosanitary policy at a European level. More analytically, Pep-Rep-2 has had the following scientific and socioeconomic impact:
-The identification of plant proteins and viral cis-acting RNA elements involved in virus replication represent important scientific millestones
-In terms of methodology, the delicate nature of the research and the transfer of technology achieved suggest that a comparative advantage (competitiveness) in the field
-The collaboration with a) Dr. R. Olshoorn's lab (Leiden Institute of Chemistry, The Netherlands) and b) Dr. M. Aranda (CEBAS, Murcia, Spain) has proven of paramount importance during the project and sustainability has been foreseen. The former, is an expert on RNA structure analysis for both animal and plant RNA viruses and facilitated rapid progress and worldwide competitiveness. A new Pep-Rep-3 proposal has already been submitted to the Greek Research and Technology Secretariat (GSRT) under the call announcement, ARISTEIA (06/2012). In the submitted Pep-Rep-3, a joint PhD is foreseen. The collaboration with, and the use of novel innovative genetic tools developed by Dr. Aranda's laboratory was essential to many phases of Pep-Rep-2. Future collaboration is currently planned.
-EU trade policy. In the Barcelona Declaration and the Agadir Agreement signed by the Euro-Mediterranean Partners and Morocco, Tunisia, Egypt and Jordan respectively, the establishment of a Euro-Mediterranean Free Trade Area (EMFTA) by the year 2010 was agreed. Pep-Rep-2 results concern the replication of a quarantine plant virus and awareness has been raised during scientific conferences and student tuition within the Mediterranean Agronomic Institute. Dr. I. Livieratos has informed in detail the coordinator of the PEPEIRA European Union grant devoted on PepMV seed transmission, Dr. R. van der Vlugt, about Pep-Rep-2, during the AAB Conference (2012) in Dublin (Ireland).
-Publication of an article in Virus Research and the presentation of the results in two conferences (2010, 2012) of the Hellenic Phytopathology Society. At least one more publication is anticipated within the next 12 months to contain the analysis of the 3'- UTR and identified interactions with/or the PROTEIN-C.
-Guidance of MSc and PhD level students (transfer of technology) of the Laboratory of Plant Virology of the Mediterranean Agronomic Institute by the researcher was daily implemented during the whole lifetime of Pep-Rep-2.
Initially, a laboratory area was designated for this quarantine plant virus and pilot transcription assays from various plant extracts were performed. More specifically, homogenised extracts from PepMV-infected leaves were subjected to differential centrifugation and separation on sucrose gradients and the resultant fractions, containing membrane-bound RdRp, were tested for RNA transcriptional activity. Endogenous templates, namely incomplete, partially synthesized RNAs present in the replication complexes (RCs) of infected plants were used to test for RNA transcriptional activity. The resuspended P30 pellet (membrane-bound RdRp) transcribed mainly four endogenous RNA species corresponding to products of the genomic and sgRNAs (TGB1, TGB2-3, CP). All four RNAs were shown to be double-stranded (ds) in nature. We further characterised the relative amounts of the genomic and sgRNAs produced by membrane-bound PepMV RdRp. Genomic dsRNA predominantly hybridised to negative-sense PepMV 3'- terminal RNA transcripts suggesting that they represent mainly positive-sense RNAs. All three subgenomic dsRNAs hybridised with similar intensities to identical amounts 5 ug of the negative-sense 5'- terminal and positive-sense strand 3'- terminal RNAs suggesting that they are comprised of similar amounts of both RNA strands.
An important breakthrough was the initiation of specific plus-and minus-strand RNA synthesis by template-bound RdRp. Here, the endogenous PepMV RNA present in the RdRp-enriched fraction was digested with micrococcal nuclease and the resultant template-dependent RdRp was then incubated with either a full-length PepMV RNA transcript or PepMV virion RNA. Both templates successfully synthesized full-length genomic RNA products indicating the specificity of the purified RdRp system. The template-dependent PepMV RdRp was then programmed with several different PepMV RNA templates, including positive-and negative-sense RNA strands. These experiments revealed specific recognition of the 3'- termini of both positive-and negative-sense PepMV RNA transcripts. Additionally, a polyclonal antiserum was raised against a peptide epitope representing an immunogenic stretch of the PepMV RdRp. The purified IgGs detected the PepMV RdRp in the membrane-bound as well as the template-dependent fractions and in subsequent experiments, and significantly reduced the activity of the template-dependent RdRp in a concentration-dependent manner.
Finally RNA synthesis from the 3'- terminus of the PepMV genome was examined in order to permit analysis of replication-essential cis-acting RNA elements. Identical amounts of PepMV RNA transcripts, representing the positive-stranded 3'- terminus together with various lengths of the poly
(a) tail were introduced into the established template-dependent RdRp system. It was demonstrated that a poly
(a) tail of at least 20 adenosine residues was necessary for minus-strand synthesis. Further analysis of the cis-acting 3'- UTR elements for minus-strand RNA synthesis were undertaken and a prediction for the PepMV 3'- UTR structure was experimentally proven. Pin-point mutations were used to introduce deletions, substitutions and restorations of specific base pairs suggesting that stem loop 1 (SL1) containing the polyadenylation signal is replication redundant while, SL2 and SL3 and the predicted pseudoknot are replication essential.
Objective 2 was corroborated with research carried out in parallel at doctoral level. Here, two host factors (Hsc70 and PROTEIN-C) were shown to interact with the PepMV proteins, CP and p25. Specifically for Hsc70, the amino acid sequences of the identified interactors from the tomato cDNA library were not 100 % identical and later experiments (yeast two hybrid and BiFC assay) confirmed the interaction for one of the three complete tomato Hsc70s namely, Hsc70. 3. The PepMV CP-Hsc70. 3 interaction complex was localised in the cytoplasm and nucleus. Immunogold labeling, transmission electron microscopy, immunoblot analysis on purified PepMV virions and time course experiments (mRNA, proteins) suggested that elevated levels of Hsc70 accumulate and interact with virions/CP in PepMV-infected leaves. Of particular importance are the findings of the relative PROTEIN-C mRNA and protein levels in infected tissue in relation to PepMV p25. These data will permit future experimentation in order to confirm the actual role of the interaction in virus infection.
Overall, Pep-Rep-2 capitalised on a previous bilateral (Greece-Spain) Project results and ran in parallel with a PhD investigation (2009-2012) to produce a number of significant scientific advances in the field of RNA virus replication. Furthermore, Pep-Rep-2 fosters future collaborations and will have an impact on phytosanitary policy at a European level. More analytically, Pep-Rep-2 has had the following scientific and socioeconomic impact:
-The identification of plant proteins and viral cis-acting RNA elements involved in virus replication represent important scientific millestones
-In terms of methodology, the delicate nature of the research and the transfer of technology achieved suggest that a comparative advantage (competitiveness) in the field
-The collaboration with a) Dr. R. Olshoorn's lab (Leiden Institute of Chemistry, The Netherlands) and b) Dr. M. Aranda (CEBAS, Murcia, Spain) has proven of paramount importance during the project and sustainability has been foreseen. The former, is an expert on RNA structure analysis for both animal and plant RNA viruses and facilitated rapid progress and worldwide competitiveness. A new Pep-Rep-3 proposal has already been submitted to the Greek Research and Technology Secretariat (GSRT) under the call announcement, ARISTEIA (06/2012). In the submitted Pep-Rep-3, a joint PhD is foreseen. The collaboration with, and the use of novel innovative genetic tools developed by Dr. Aranda's laboratory was essential to many phases of Pep-Rep-2. Future collaboration is currently planned.
-EU trade policy. In the Barcelona Declaration and the Agadir Agreement signed by the Euro-Mediterranean Partners and Morocco, Tunisia, Egypt and Jordan respectively, the establishment of a Euro-Mediterranean Free Trade Area (EMFTA) by the year 2010 was agreed. Pep-Rep-2 results concern the replication of a quarantine plant virus and awareness has been raised during scientific conferences and student tuition within the Mediterranean Agronomic Institute. Dr. I. Livieratos has informed in detail the coordinator of the PEPEIRA European Union grant devoted on PepMV seed transmission, Dr. R. van der Vlugt, about Pep-Rep-2, during the AAB Conference (2012) in Dublin (Ireland).
-Publication of an article in Virus Research and the presentation of the results in two conferences (2010, 2012) of the Hellenic Phytopathology Society. At least one more publication is anticipated within the next 12 months to contain the analysis of the 3'- UTR and identified interactions with/or the PROTEIN-C.
-Guidance of MSc and PhD level students (transfer of technology) of the Laboratory of Plant Virology of the Mediterranean Agronomic Institute by the researcher was daily implemented during the whole lifetime of Pep-Rep-2.