Biomedical subjects

Holger Jeske

Publications and source records attributed to Holger Jeske.

14 recordsLinked to original sources

PubMed2006

Deletion and recombination events between the DNA-A and DNA-B components of Indian cassava-infecting geminiviruses generate defective molecules in Nicotiana benthamiana.

Cloned DNA-B components, belonging to the bipartite begomoviruses Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV), family Geminiviridae, when co-inoculated along with previously cloned DNA-A components of the respective viruses onto the experimental host Nicotiana benthamiana, generated defective DNAs (def-DNA) ranging in size from 549 to 1555 nucleotides. All the cloned def-DNAs contained the common region (CR) as well as portions of either DNA-A or DNA-B and, in a few cases, both DNA-A and DNA-B, representing recombinant products, the junction points of which correspond to repeats of 2-11 bases found in the parental molecules. The DNA-B-derived def-DNAs were, in some cases, associated with a decrease in levels of DNA-B, with a concomitant change in the symptoms from downward leaf curling in the older leaves to upward leaf-rolling in newly emerging leaves, more typical of monopartite begomoviruses.

Base Sequence↗

PubMed2006

Tissue and cell tropism of Indian cassava mosaic virus (ICMV) and its AV2 (precoat) gene product.

In order to establish defined viruses for challenging plants in resistance breeding programmes, Indian cassava mosaic virus (ICMV; family Geminiviridae) DNA clones were modified to monitor viral spread in plants by replacing the coat protein gene with the green fluorescent protein (GFP) reporter gene. Comparative in situ hybridization experiments showed that ICMV was restricted to the phloem in cassava and tobacco. GFP-tagged virus spread similarly, resulting in homogeneous fluorescence within nuclei and cytoplasm of infected cells. To analyze viral intercellular transport in further detail, GFP was fused to AV2, a protein that has been implicated in viral movement. Expressed from replicating viruses or from plasmids, AV2:GFP became associated with the cell periphery in punctate spots, formed cytoplasmic as well as nuclear inclusion bodies, the latter as conspicuous paired globules. Upon particle bombardment of expression plasmids, AV2:GFP was transported into neighboring cells of epidermal tissues showing that the intercellular transport of the AV2 protein is not restricted to the phloem. The results are consistent with a redundant function of ICMV AV2 acting as a movement protein, presumably as an evolutionary relic of a monopartite geminivirus that may still increase virus fitness but is no longer necessary in a bipartite genome. The fusion of ICMV ORF AV2 to the GFP gene is the first example of a reporter construct that follows the whole track of viral DNA from inside the nucleus to the cell periphery and to the next cell.

Capsid Proteins↗

PubMed2006

Rolling circle amplification revolutionizes diagnosis and genomics of geminiviruses.

Better, easier and cheaper than polymerase chain reaction (PCR) or antibody detection, rolling circle amplification (RCA) using the bacteriophage varphi 29 DNA polymerase allows for a reliable diagnosis of geminiviruses and presumably all viruses with small single-stranded circular DNA genomes. The results show the efficiency of this technique in characterizing viral DNA components of several geminiviruses from experimental and natural host plant sources. The advantages are: (a) that no expensive devices are necessary, (b) simple handling, (c) detection of all infecting circular DNA components without any knowledge of sequence information in a single step, and (d) low costs per reaction. In addition, RCA-amplified viral DNA can be characterized by restriction fragment length polymorphism analysis and directly sequenced up to 900 bases in a single run circumventing cloning and plasmid purification. This shortcut will considerably accelerate genomics of at least gemini-, nano- and circoviruses in the future.

DNA Restriction Enzymes↗

PubMed2006

Atomic layer deposition on biological macromolecules: metal oxide coating of tobacco mosaic virus and ferritin.

Decoration of nanoparticles, in particular biomolecules, gathered high attention in recent years.(1-7) Of special interest is the potential use of biomolecules as templates for the fabrication of semiconducting or metallic nanostructures.(1-7,26) In this work we show the application of atomic layer deposition, a gas-phase thin film deposition process, to biological macromolecules, which are frequently used as templates in nanoscale science, and the possibility to fabricate metal oxide nanotubes and thin films with embedded biomolecules.(1-13).

Adsorption↗

PubMed2006

A versatile transreplication-based system to identify cellular proteins involved in geminivirus replication.

A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing.

DNA, Viral↗

PubMed2005

Poinsettia latent virus is not a cryptic virus, but a natural polerovirus-sobemovirus hybrid.

The biochemical and genetic features of Poinsettia latent virus (PnLV, formerly named Poinsettia cryptic virus), which is spread worldwide in commercial cultivars of Euphorbia pulcherrima without inducing symptoms, have been determined using virus-purification, immunological techniques, electron microscopy, cloning, and sequencing. PnLV was found to be a chimeric virus with one 4652 bases, plus strand RNA showing a close relationship to poleroviruses within the first three quarters of its genome but to sobemoviruses in the last quarter. Thus, we propose to classify this virus as "polemovirus". Similarities of protein and nucleic acid sequences at the 5' and extreme 3' end of its RNA suggest a replication mode like that of poleroviruses, whereas the coat protein sequence is closely related to that of sobemoviruses. Consistent with these results, PnLV forms stable icosahedra of 34 nm in diameter. The consequences for the taxonomy of PnLV and for gardeners' practice are discussed.

Base Sequence↗

PubMed2005

Replicative intermediates of Tomato leaf curl virus and its satellite DNAs.

Several plant geminiviruses have been shown recently to utilize both rolling-circle replication (RCR) and recombination-dependent replication (RDR) strategies. A highly specific binding of the viral replication-associated protein (Rep) to its cognate DNA is essential for initiation of viral DNA replication and for the recognition of DNA components of the bipartite geminiviruses of the Begomovirus genus. We have extended the replication analysis to the monopartite Australian Tomato leaf curl virus (ToLCV), its Rep binding deficient mutants, and the satellite DNAs it supports. Analyses of viral DNA by two-dimensional agarose gel electrophoresis after fractionation by single-stranded (ss) DNA-selective cellulose chromatography revealed that DNA intermediates of ToLCV and its mutant were identical. Both RCR and RDR intermediates were identified. New ToLCV DNA forms were observed and characterized as subgenomic topoisomers, heterogeneous open circular double-stranded (ds) DNA, and degradation products. A 1350-nt DNA beta satellite associated with the unrelated Cotton leaf curl Multan virus (CLCuMV) was supported by ToLCV and produced intermediates of both RCR and RDR, suggesting that replication strategies of satellites are determined by the helper virus. Replicative intermediates of the 682 nt ToLCV satellite DNA could not be resolved; however, concatemers of up to octamer were detected, together with a field of hybridizing material suggestive of complementary strand replication on heterogeneous circular ssDNA templates.

DNA Replication↗

PubMed2004

Short deletions in nuclear targeting sequences of African cassava mosaic virus coat protein prevent geminivirus twinned particle formation.

Coat proteins (CPs) of geminiviruses are multifunctional proteins. Using transient expression experiments, we have recently identified putative sequence motifs of African cassava mosaic virus (ACMV) CP involved in nuclear import (NLS) and export (NES) (Virology 286 (2001) 373). Here, we report on the effect of corresponding deletion mutants in the context of infecting viruses. Since NLS and NES may overlap with DNA binding and multimerisation domains, we have investigated their effect on viral infection, particularly, on particle formation. All deletion mutants were infectious in Nicotiana benthamiana when co-inoculated with DNA B, but poorly sap-transmissible. Some of the mutants showed reduced levels of viral single-stranded DNA (ssDNA), whereas the amount of double-stranded DNA (dsDNA) was not greatly affected. None of these CP mutants was able to produce stable virus particles. In contrast, viruses with CP fused to Flag epitopes at the N- or C-terminus (CP:Flag or Flag:CP) were readily sap-transmissible and formed amorphous nucleoprotein particles but only few geminate structures. The relevance of the identified sequences in replicating viruses with reference to nuclear import and export as well as to particle stability and DNA binding is discussed.

Amino Acid Sequence↗

PubMed2004

Geminate structures of African cassava mosaic virus.

Two types of geminate structures were purified from African cassava mosaic geminivirus (ACMV)-infected Nicotiana benthamiana plants and analyzed by electron cryomicroscopy and image reconstruction. After cesium sulfate density gradient centrifugation, they were separated into lighter top (T) and heavier bottom (B) components. T particles comigrated with host proteins, whereas B particles were concentrated in a cesium density typical for complete virions. Both particles were composed of two incomplete icosahedra of 11 capsomers each, but T particles were slightly larger (diameter, 22.5 nm) and less dense in the interior than B particles (diameter, 21.5 nm). T particles were frequently associated with small globules of approximately 14 nm diameter of unknown origin. The overall structure of ACMV, a begomovirus transmitted by whiteflies, was similar to that of Maize streak virus (MSV), a mastrevirus transmitted by leafhoppers, although the vertices of the icosahedra were less pronounced. Models of ACMV coat proteins based on Satellite tobacco necrosis virus support the exposure of parts of the molecule essential for transmission specificity by whiteflies and provide possible structural explanations for the smaller protrusion of the ACMV capsid relative to MSV. The differences of ACMV and MSV virion shapes are discussed with reference to their different animal vectors.

Amino Acid Sequence↗

PubMed2004

Interaction of DNA with the movement proteins of geminiviruses revisited.

Geminiviruses manage the transport of their DNA within plants with the help of three proteins, the coat protein (CP), the nuclear shuttle protein (NSP), and the movement protein (MP). The DNA-binding capabilities of CP, NSP, and MP of Abutilon mosaic virus (AbMV; family Geminiviridae; genus Begomovirus) were scrutinized using gel mobility shift assays and electron microscopy. CP and NSP revealed a sequence-independent affinity for both double-stranded and single-stranded DNA, as has been previously reported for other begomoviruses. MP interacted selectively with dimeric supercoiled plasmid DNA in the electrophoretic assay. Further apparent size- and form-selective binding capacities of MP have been previously reported for another geminivirus (Bean dwarf mosaic virus), but in the case of AbMV, they have been identified as the result of electrophoretic interference rather than of complex formation. Without these complications, electron microscopy confirmed the assembly of double-stranded supercoiled DNA with NSP and MP into conspicuous structures and provided the first direct evidence for cooperative interaction of MP, NSP, and DNA. Based on these results and previous ones, a transport model of geminiviruses is discussed in which NSP packages DNA and MP anchors this complex to the protoplasmic leaflets of plasma membranes and microsomes for cell-to-cell movement.

Biological Transport↗

PubMed2004

Tête à tête of tomato yellow leaf curl virus and tomato yellow leaf curl sardinia virus in single nuclei.

Since 1997 two distinct geminivirus species, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), have caused a similar yellow leaf curl disease in tomato, coexisted in the fields of southern Spain, and very frequently doubly infected single plants. Tomatoes as well as experimental test plants (e.g., Nicotiana benthamiana) showed enhanced symptoms upon mixed infections under greenhouse conditions. Viral DNA accumulated to a similar extent in singly and doubly infected plants. In situ tissue hybridization showed TYLCSV and TYLCV DNAs to be confined to the phloem in both hosts, irrespective of whether they were inoculated individually or in combination. The number of infected nuclei in singly or doubly infected plants was determined by in situ hybridization of purified nuclei. The percentage of nuclei containing viral DNA (i.e., 1.4% in tomato or 6% in N. benthamiana) was the same in plants infected with either TYLCSV, TYLCV, or both. In situ hybridization of doubly infected plants, with probes that discriminate between both DNAs, revealed that at least one-fifth of infected nuclei harbored DNAs from both virus species. Such a high number of coinfected nuclei may explain why recombination between different geminivirus DNAs occurs frequently. The impact of these findings for epidemiology and for resistance breeding concerning tomato yellow leaf curl diseases is discussed.

Cell Nucleus↗

PubMed2003

Mapping of abutilon mosaic geminivirus minichromosomes.

The single-stranded circular DNA of Abutilon mosaic geminivirions is complemented to double-stranded DNA by host proteins after infecting cells. This double-stranded DNA serves as a template for replication as well as transcription and is assembled into host nucleosomes, yielding circular viral minichromosomes. Their chromatin structure was analyzed by use of isolated nuclei combining nuclease sensitivity assays with ligation-mediated PCR, evaluating nucleosomal ladders and topoisomer distributions in one- and two-dimensional gels by blot hybridization. Viral minichromosomes were found to exist in at least two defined structures covered with 11 or 12 nucleosomes, leaving open gaps accessible for interactions with other host factors. Nucleosome-free gaps were colocalized with promoter structures and the origin of replication in both components of genomic DNA (DNA A and DNA B). Nucleosomes were positioned over the entire viral DNA in at least two alternative phases with different periodicities. The distribution of topoisomers of monomeric viral circular double-stranded DNA confirmed the presence of variable chromatin structures revealing maximum frequencies of molecules with either 11, 12, or 13 superhelical turns (corresponding to respective numbers of nucleosomes) at maximal frequency at different stages during leaf development of infected plants. The role of variable chromatin structures for gene regulation of geminiviruses is discussed.

Base Sequence↗

PubMed2003

Multitasking in replication is common among geminiviruses.

Geminiviruses package single-stranded circular DNA and replicate via double-stranded DNA intermediates. During the past decade, increasing evidence has led to the general acceptance that their replication follows a rolling-circle replication mechanism like bacteriophages with single-stranded DNA. In a recent study, we showed that this is also true for Abutilon mosaic geminivirus (AbMV), but that this particular virus may also use a recombination-dependent replication (RDR) route in analogy to T4 phages. Because AbMV is a special case, since it has been propagated on ornamental plants for more than a hundred years, it was interesting to determine whether RDR is common among other geminiviruses. We analyzed geminiviruses from different genera and geographic origins by using BND cellulose chromatography in combination with an improved high resolution two-dimensional gel electrophoresis, and we conclude that multitasking in replication is widespread, at least for African cassava mosaic, Beet curly top, Tomato golden mosaic, and Tomato yellow leaf curl virus.

DNA↗

PubMed2001

The distinct disease phenotypes of the common and yellow vein strains of Tomato golden mosaic virus are determined by nucleotide differences in the 3'-terminal region of the gene encoding the movement protein.

In Nicotiana benthamiana, the common strain of the bipartite geminivirus Tomato golden mosaic virus (csTGMV) induces extensive chlorosis whereas the yellow vein strain (yvTGMV) produces veinal chlorosis on systemically infected leaves. In Datura stramonium, csTGMV produces leaf distortion and a severe chlorotic mosaic whereas yvTGMV produces only small chlorotic lesions on systemically infected leaves. Genetic recombination and site-directed mutagenesis studies using infectious clones of csTGMV and yvTGMV have identified a role in symptom production for the gene encoding the movement protein (MP). The MP amino acid at position 272, either valine (csTGMV) or isoleucine (yvTGMV), influenced symptoms in both hosts by inducing an intermediate phenotype when exchanged between the two strains. Exchange of an additional strain-specific MP amino acid at position 288, either glutamine (csTGMV) or lysine (yvTGMV), resulted in the change of symptom phenotype to that of the other strain. In situ hybridization analysis in N. benthamiana demonstrated that there was no qualitative difference in the tissue distribution of the two strains although csTGMV accumulated in higher amounts, suggesting that the efficiency of virus movement rather than distinct differences in tissue specificity of the strains is responsible for the symptom phenotypes.

3' Untranslated Regions↗