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Juan Antonio Garcia - One of the best experts on this subject based on the ideXlab platform.
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o glcnac modification of the coat protein of the potyvirus plum pox virus enhances viral infection
Virology, 2013Co-Authors: Jose De Jesus Perez, Namrata D Udeshi, Jeffrey Shabanowitz, Sergio Ciordia, Silvia Juarez, Cheryl L Scott, Neil E Olszewski, Donald F Hunt, Juan Antonio GarciaAbstract:Abstract O-GlcNAcylation is a dynamic protein modification which has been studied mainly in metazoans. We reported previously that an Arabidopsis thaliana O-GlcNAc transferase modifies at least two threonine residues of the Plum pox virus (PPV) capsid protein (CP). Now, six additional residues were shown to be involved in O-GlcNAc modification of PPV CP. CP O-GlcNAcylation was abolished in the PPV CP7-T/A mutant, in which seven threonines were mutated. PPV CP7-T/A infected Nicotiana clevelandii, Nicotiana benthamiana, and Prunus persica without noticeable defects. However, defects in infection of A. thaliana were readily apparent. In mixed infections of wild-type arabidopsis, the CP7-T/A mutant was outcompeted by wild-type virus. These results indicate that CP O-GlcNAcylation has a major role in the infection process. O-GlcNAc modification may have a role in virion assembly and/or stability as the CP of PPV CP7-T/A was more sensitive to protease digestion than that of the wild-type virus.
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Preservation of 5'-end integrity of a potyvirus genomic RNA is not dependent on template specificity.
Virology, 2000Co-Authors: Laureano Simón-buela, Juan Antonio Garcia, Lourdes Osaba, Juan José López-moyaAbstract:Abstract Full-length in vitro transcripts of plum pox potyvirus (PPV) genomic RNA with mutations altering the number of 5′-terminal adenosine residues were able to infect Nicotiana clevelandii plants, whereas a mutant with a substitution of adenosine in position 2 by guanosine failed to infect. The genomic 5′ end was template-independently repaired during in vivo RNA synthesis producing wild-type viral progeny. Putative models of replication initiation are discussed.
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Development of an antigen presentation system based on plum pox potyvirus
FEBS letters, 1998Co-Authors: M. Rosario Fernández-fernández, Jorge L. Martínez-torrecuadrada, J. Ignacio Casal, Juan Antonio GarciaAbstract:The development of an antigen presentation system based on the plum pox potyvirus (PPV) is here described. The amino-terminal part of PPV capsid protein was chosen as the site for expression of foreign antigenic peptides. Modifications in this site were engineered to avoid the capability of natural transmission by aphids of this PPV vector. As a first practical attempt, different forms of an antigenic peptide (single and tandem repetition) from the VP2 capsid protein of canine parvovirus (CPV) were expressed. Both chimeras are able to infect Nicotiana clevelandii plants with similar characteristics to wild-type virus and remain genetically stable after several plant passages. The antigenicity of purified chimeric virions was demonstrated, proving the suitability of this system for diagnostic purposes. Moreover, mice and rabbits immunized with chimeric virions developed CPV-specific antibodies, which showed neutralizing activity.
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Long Sequences in the 5′ Noncoding Region of Plum Pox Virus Are Not Necessary for Viral Infectivity but Contribute to Viral Competitiveness and Pathogenesis
Virology, 1997Co-Authors: Laureano Simón-buela, Juan Antonio GarciaAbstract:Abstract The 5′-terminal 31 nucleotides of the 146-nucleotides-long 5′ noncoding region of plum pox potyvirus (PPV) are highly conserved in all the members of the Potyvirus genus. To map the sequences of the 5′ noncoding region that are necessary in vivo for infectivity, we have constructed a nested set of substitution and deletion mutants. While we were not able to infect Nicotiana clevelandii plants with full-length PPV transcripts bearing mutations in the 5′-terminal 35 nucleotides of the viral genome, the deletion of long sequences located between nucleotides 39 and 145 did not alter either the rate of infection or viral accumulation. Nevertheless, these mutants were not able to compete with the wild-type strain in coinoculation experiments. Plants infected with a PPV mutant that lacked nucleotides 127 to 145 showed a very mild symptomathology; the wild-type symptom severity was recovered after spontaneous second-site mutations.
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Plum pox potyvirus RNA replication in a crude membrane fraction from infected Nicotiana clevelandii leaves
Journal of General Virology, 1991Co-Authors: María Teresa Martín, Juan Antonio GarciaAbstract:In vitro synthesis of plum pox potyvirus (PPV)-specific nucleic acid has been measured in a crude fraction prepared from leaves of PPV-infected Nicotiana clevelandii plants. Using alkali and DNase treatments, the synthesized nucleic acid was shown to be RNA. The electrophoretic mobility and the differing sensitivity to RNase at high and low salt concentrations allowed the identification of in vitro products probably corresponding to replicative form and replicative intermediate RNA, as well as to single-stranded RNA. Most of the PPV-specific RNA synthesized was shown to be of positive polarity. The in vitro RNA synthesis, performed in the presence of actinomycin D, required all four ribonucleoside triphosphates and Mg2+ ions. This enzyme extract contained about 6% of the leaf protein and most of the identified virus-encoded proteins. Altogether, the results presented in this paper suggest that in vitro RNA synthesis was carried out by the PPV replicase complex.
Ervin Balázs - One of the best experts on this subject based on the ideXlab platform.
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Identification a coat protein region of cucumber mosaic virus (CMV) essential for long-distance movement in cucumber
Archives of Virology, 2011Co-Authors: Katalin Salanki, Ákos Gellért, László Kiss, Ervin BalázsAbstract:To characterise the long-distance movement determinant of cucumoviral coat proteins (CPs), five mutants were engineered into the CMV CP bearing the corresponding tomato aspermy virus (TAV) loops exposed on the surface of the virion. Both viruses can move long-distance in Nicotiana clevelandii , but only CMV can move long-distance in cucumber. Investigation of the CMV chimeras identified three amino acids of the βB-βC loop that were essential for the CMV long-distance movement in cucumber. Introducing these mutations into the TAV CP was not sufficient for long-distance movement, indicating that this is not the sole region causing long-distance movement deficiency.
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Identification a coat protein region of cucumber mosaic virus (CMV) essential for long-distance movement in cucumber
Archives of Virology, 2011Co-Authors: Katalin Salanki, Ákos Gellért, László Kiss, Ervin BalázsAbstract:Springer-Verlag 2011 Abstract To characterise the long-distance movement determinant of cucumoviral coat proteins (CPs), five mutants were engineered into the CMV CP bearing the corresponding tomato aspermy virus (TAV) loops exposed on the surface of the virion. Both viruses can move long- distance in Nicotiana clevelandii, but only CMV can move long-distance in cucumber. Investigation of the CMV chimeras identified three amino acids of the bB-bC loop that were essential for the CMV long-distance movement in cucumber. Introducing these mutations into the TAV CP was not sufficient for long-distance movement, indicating that this is not the sole region causing long-distance movement deficiency.
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Consequences of gene transfer between distantly related tombusviruses
Gene, 1993Co-Authors: József Burgyán, Tamas Dalmay, Mario Tavazza, Alessandra Lucioli, Ervin BalázsAbstract:Hybrid cDNA clones were constructed by fusing the coat protein-encoding gene and/or the 3'-terminal region (including the 22- and 19-kDa protein-encoding genes) derived from a clone of artichoke mottled crinkle tombusvirus to the 5'-terminal region of a full-length clone of cymbidium ringspot tombusvirus. In vitro transcripts from recombinant clones were infectious when inoculated into Nicotiana clevelandii plants. Inoculated plants showed symptoms different from those induced by parent viruses. In particular, systemic invasion depended very much, although not exclusively, on the type of protein that coated progeny viral RNA, suggesting a role of the capsid protein in the long-distance movement of tombusvirus infections.
K. W. Buck - One of the best experts on this subject based on the ideXlab platform.
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Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus.
Journal of General Virology, 1995Co-Authors: Helen Bates, T. A. M. Osman, M Farjah, K. W. BuckAbstract:A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl β-d-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with several minor proteins possibly of host origin. After removal of endogenous RNA with micrococcal nuclease, the poly-merase became template-dependent. It was also template-specific, being able to utilize as templates RNA of two strains of RCNMV, but not RNAs of three viruses in different taxonomic groups, namely cucumber mosaic cucumovirus, tomato bushy stunt tombusvirus and tomato mosaic tobamovirus. The products of RNA polymerase reactions were double-stranded RNAs corresponding to RCNMV RNAs 1 and 2. The ability of the template-dependent RNA polymerase to synthesize RNA was completely inhibited by antibodies to a peptide containing the GDD motif, whereas the activity of the template-bound enzyme was unaffected by these antibodies.
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Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus.
The Journal of general virology, 1995Co-Authors: H J Bates, M Farjah, T A Osman, K. W. BuckAbstract:A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl beta-D-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with several minor proteins possibly of host origin. After removal of endogenous RNA with micrococcal nuclease, the polymerase became template-dependent. It was also template-specific, being able to utilize as templates RNA of two strains of RCNMV, but not RNAs of three viruses in different taxonomic groups, namely cucumber mosaic cucumovirus, tomato bushy stunt tombusvirus and tomato mosaic tobamovirus. The products of RNA polymerase reactions were double-stranded RNAs corresponding to RCNMV RNAs 1 and 2. The ability of the template-dependent RNA polymerase to synthesize RNA was completely inhibited by antibodies to a peptide containing the GDD motif, whereas the activity of the template-bound enzyme was unaffected by these antibodies.
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Detection of the 3a Protein of Cucumber Mosaic Virus in a Cell Wall Fraction from Infected Nicotiana clevelandii Plants
Journal of Phytopathology, 1994Co-Authors: A. J. Burman, T. A. M. Osman, K. W. BuckAbstract:The 3a protein of cucumber mosaic virus was expressed in Escherichia coli and, after purification, used to produce an antiserum. The 3a protein was detected in a cell wall fraction obtained from infected Nicotiana clevelandii leaf tissue by immunoblotting using the 3a antiserum. The 3a protein reached a maximum level 4 days after inoculation and remained at this level for a further 8 days before slowly declining. In contrast, the virus capsid protein, detected in an 80 000 g pellet by immunoblotting using a virus particle antiserum, reached a maximum 5 days after inoculation and remained at this level for at least a further 16 days. Zusammenfassung Nachweis des 3a Proteins des cucumber mosaic Virus in einer Zellwandfraktion von infizierten Nicotiana clevelandii Pflanzen Das 3a Protein des cucumber mosaic Virus wurde in Escherichia coli zur Expression gebracht und nach Reinigung fur die Antiserumproduktion verwendet. Das 3a Protein wurde in einer Zellwandfraktion, die von infiziertem Nicotiana clevelandii-Blattgewebe gewonnen worden war, durch Immunoblotting mit dem 3a Antiserum nachgewiesen. Das 3a Protein erreichte die hochsten Werte 4 Tage nach der Inokulation, diese Werte wurden fur weitere 8 Tage beibehalten bis eine langsame Verringerung eintrat. Im Gegensatz dazu konnte mit dem Viruscapsid-protein, welches in einem 80 000 g Sediment durch Immunoblotting mit einem Viruspartikelantiserum detektiert wurde, Hochstwerte 5 Tage nach der Inokulation erreicht werden, Werte die mindestens 16 Tage anhielten.
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Detection of the movement protein of red clover necrotic mosaic virus in a cell wall fraction from infected Nicotiana clevelandii plants.
Journal of General Virology, 1991Co-Authors: T. A. M. Osman, K. W. BuckAbstract:The movement protein of red clover necrotic mosaic virus (RCNMV) was expressed in Escherichia coli as a fusion with a maltose-binding protein using the vector pMAL-cRI and used to produce an antiserum. The RCNMV movement protein was detected in a cell wall fraction obtained from infected Nicotiana clevelandii leaf tissue by immunoblotting using the movement protein antiserum. The movement protein could be detected 6 h after inoculation and reached a maximum after 24 h. In contrast, the virus capsid protein, detected in a soluble fraction by immunoblotting using a capsid antiserum, continued to increase for 72 h after inoculation.
D S Teakle - One of the best experts on this subject based on the ideXlab platform.
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studies on the mechanism of transmission of pollen associated tobacco streak ilarvirus virus by thrips tabaci
Plant Pathology, 1993Co-Authors: R Sdoodee, D S TeakleAbstract:Mixed instars of Thrips tabaci were allowed to feed on test seedlings of cucumber, the thrips were removed and wounds caused by thrips feeding dusted with tobacco streak virus (TSV) carried in tomato pollen. Transmission of TSV occurred in three out of the four experiments conducted. When Chenopodium amaranticolor test seedlings were dusted with infective tomato pollen, and thrips introduced for varying periods before being killed with insecticide, TSV transmission occurred after exposure to thrips for 1 h, 5 h, 1 day or 2 days, but not 5 min. Pollen‐washing experiments indicated that TSV was carried both internally and externally in pollen of tomato and several weed hosts. T. tabaci was able to transmit TSV from pollen of Nicotiana clevelandii and Nicandra physalodes, and TSV from tomato pollen was transmitted to several weed species. It is concluded that T. tabaci transmits TSV associated with tomato pollen by a mechanical mechanism whereby virus carried externally, or released from inside the pollen, infects feeding wounds.
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High incidence of tobacco streak virus in tobacco and its transmission by Microcephalothrips abdominalis and pollen from Ageratum houstonianum.
Plant Disease, 1991Co-Authors: R. S. Greber, D S Teakle, M. J. Klose, J. R. MilneAbstract:Incidence of tobacco streak virus (TSV) in an Australian tobacco crop at flowering was shown by enzyme-linked immunosorbent assay (ELISA) to be 58−59% in outside rows near weeds infected with TSV, decreasing to 10−18% in the 12th row inside the crop. The most prevalent dicotyledonous weed, Ageratum houstonianum, had a 50% incidence of infection and its flower heads were commonly infested with Microcephalotrips abdominalis. In transmission tests with either five or 10 adult thrips and pollen taken from the nearby A. houstonianum, TSV infection occurred in 32 of 45 tobacco, 11 of 12 cucumber, two of 12 Nicotiana clevelandii, and 18 of 22 Chenopodium amaranticolor test seedlings (.)
Bryan D. Harrison - One of the best experts on this subject based on the ideXlab platform.
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Nuclear location of the 16K non-structural protein of tobacco rattle virus.
Journal of General Virology, 1991Co-Authors: D. H. Liu, David J. Robinson, G. H. Duncan, Bryan D. HarrisonAbstract:An antiserum, elicited by a synthetic peptide coupled to bovine serum albumin, reacted specifically with the non-structural 16K protein of tobacco rattle virus. The protein was detected in extracts of systemically infected Nicotiana clevelandii leaves, but only in those made with the aid of SDS, urea and 2-mercaptoethanol. Immunogold labelling of ultrathin sections showed that the protein was mainly associated with nuclei, but was also present in the cytoplasm. These observations suggest that the 16K protein binds to macromolecular components of infected cells, especially in nuclei, but do not clarify its function.