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At least 127 records · Page 7Linked to original sources

Hen egg yolk as a source of antiviral antibodies in the enzyme-linked immunosorbent assay (ELISA): a comparison of two plant viruses.

A method of raising antibodies against plant viruses in hen egg yolk is described. Laying hens were immunized with citrus tristeza virus (CTV) or tobacco mosaic virus-avocado isolate (TMV-A). Anti-viral antibodies in the yolks of sequentially laid eggs as well as in the serum were titrated by the (heterologous) antiglobulin double antibody sandwich form of the enzyme-linked immunosorbent assay (HADAS-ELISA). Antibodies first appeared in yolk 7 days after injection and peak levels were attained on day 9-11; these levels persisted for about 6-12 days. Non-specific yolk antibodies were removed by absorption with an extract of uninfected plant tissue. Using the HADAS-ELISA technique we found that yolk titres were equal to, or higher than those in serum. The benefits of using laying hens over conventional laboratory animals as a source of antiviral antibody are discussed.

Animals↗

Maculavirus, a new genus of plant viruses.

Maculavirus is a new genus of plant viruses typified by Grapevine fleck virus (GFkV). A possible second member is Grapevine redglobe virus (GRGV). Maculaviruses are phloem-limited non-mechanically transmissible viruses with isometric particles c. 30 nm in diameter that have a rounded contour and prominent surface structure. Vectors, if any, are unknown. GFkV preparations contain two centrifugal components, T made up of empty protein shells and B, which contains 35% RNA. The coat protein (CP) has a molecular mass of 24 kDa. The genome is a single-stranded RNA that has c. 50% cytosine residues. It is 7564 nt in size, excluding the poly(A) tail and contains four putative open reading frames (ORF) that encode a 215.4 kDa polypeptide with the conserved motifs of replication-associated proteins of positive-strand RNA viruses (ORF1), the CP (ORF2), and one (GRGV) or two (GFkV) proline-rich polyproteins of 31.4 kDa (ORF3) and 15.9 kDa (ORF4), respectively, with unknown function. Replication-associated proteins and CP are phylogenetically related to those of members of the genera Tymovirus and Marafivirus. GFkV-infected grapevine cells contain vesiculated mitochondria, the possible site of RNA replication. In the natural host, GFkV particles accumulate in great quantity, sometimes in crystalline arrays in phloem cells.

Open Reading Frames↗

[Investigation of helical plant virus ribonucleoprotein structures with the help of tritium planigraphy and theoretical modeling].

The results of the studies of helical plant virus structures by tritium planigraphy (TP) method are discussed. TP method is based on bombardment of macromolecular objects with a stream of tritium atoms, followed by analysis of tritium label distribution along the macromolecule. By combining the TP data with the results of theoretical predictions of the protein structure, it turned out to be possible to propose a model of the coat protein structure in the virions of potato virus X (the type member of potexvirus group) and potato virus A (one of the members of potyvirus group). With the help of TP it also managed to find subtle differences in the coat protein structure between wildtype tobacco mosaic virus (strain U1) and its mutant with two amino acid substitutions in the coat protein and alter host specificity.

Capsid Proteins↗

Plant virus expression systems for transient production of recombinant allergens in Nicotiana benthamiana.

In recent years, several studies have demonstrated the use of autonomously replicating plant viruses as vehicles to express a variety of therapeutic molecules of pharmaceutical interest. Plant virus vectors for expression of heterologous proteins in plants represent an attractive biotechnological tool to complement the conventional production of recombinant proteins in bacterial, fungal, or mammalian cells. Virus vectors are advantageous when high levels of gene expression are desired within a short time, although the instability of the foreign genes in the viral genome may present problems. Similar levels of foreign protein production in transgenic plants often are unattainable, in some cases because of the toxicity of the foreign protein. Now virus-based vectors are for the first time investigated as a means of producing recombinant allergens in plants. Several plant virus vectors have been developed for the expression of foreign proteins. Here, we describe the utilization of tobacco mosaic virus- and potato virus X-based vectors for the transient expression of plant allergens in Nicotiana benthamiana plants. One approach involves the inoculation of tobacco plants with infectious RNA transcribed in vitro from a cDNA copy of the recombinant viral genome. Another approach utilizes the transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA copies of recombinant plus-sense RNA viruses.

Allergens↗

Analysis of six DNA components of the faba bean necrotic yellows virus genome and their structural affinity to related plant virus genomes.

Faba bean necrotic yellows virus (FBNYV) has a multicomponent circular ssDNA genome. In addition to a previously described genome component (C1) coding for a replicase-associated protein (Rep), five further components (C2 to C6) have now been identified. Each of the six components is about 1 kb in size, contains one major open reading frame (ORF) in the virion sense with a TATA box and polyadenylation signal, and has a noncoding region containing a highly conserved sequence possibly forming a stem-loop structure. Similar to C1, C2 encodes another putative Rep of 33.1 kDa, which is closely related to the Rep of banana bunchy top virus (BBTV). Based on bacterial expression and immunoblot analysis, the ORF of C5 encodes the capsid protein (CP) with a deduced molecular mass of 19 kDa. The FBNYV CP shares the highest amino acid (aa) identity (56.2%) with that of subterranean clover stunt virus (SCSV). The ORF of C4 potentially codes for a hydrophobic protein which appears to be structurally and functionally similar to the BBTV-C4 and SCSV-C1 proteins. No protein sequence similarities were found in databases for the C3 and C6 ORFs of FBNYV. FBNYV is clearly distinct from any known virus but is taxonomically related to BBTV and SCSV.

Amino Acid Sequence↗

Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system.

Acetylajmalan esterase (AAE) plays an essential role in the late stage of ajmaline biosynthesis. Based on the partial peptide sequences of AAE isolated and purified from Rauvolfia cell suspensions, a full-length AAE cDNA clone was isolated. The amino acid sequence of AAE has the highest level of identity of 40% to putative lipases known from the Arabidopsis thaliana genome project. Based on the primary structure AAE is a new member of the GDSL lipase superfamily. The expression in Escherichia coli failed although a wide range of conditions were tested. With a novel virus-based plant expression system, it was possible to express AAE functionally in leaves of Nicotiana benthamiana Domin. An extraordinarily high enzyme activity was detected in the Nicotiana tissue, which exceeded that in Rauvolfia serpentina (L.) Benth. ex Kurz cell suspension cultures about 20-fold. This expression allowed molecular analysis of AAE for the first time and increased the number of functionally expressed alkaloid genes from Rauvolfia now to eight, and the number of ajmaline pathway-specific cDNAs to a total of six.

Ajmaline↗

The use of serological differentiation indices for the phylogenetic analysis of plant virus relationships.

The potential use of serological differentiation indices (SDIs) in the phylogenetic analysis of plant virus coat protein relationships is presented. Relationship dendrograms were constructed from SDI data for viruses in the tymovirus and Subgroup I geminivirus taxonomic groups, using distance or phenetic methods embodied in the computer programmes FITCH and NJTREE. Relationship dendrograms for geminiviruses agreed well with those constructed by others from sequence or other data; the dendrogram for tymoviruses was not in agreement with one calculated from amino acid composition data, or from other properties, but appears consistent with recently-shown sequence similarities between tymoviruses. Implications of these results for the classification and taxonomy of plant viruses are discussed.

Data Interpretation, Statistical↗

Enzyme-assisted immune detection of plant virus proteins electroblotted onto nitrocellulose paper.

A technique for the detection of plant virus coat proteins in plant sap is described. The method entails the electroblotting of sodium dodecyl sulphate-polyacrylamide gel electrophoresis-fractionated plant extracts onto nitrocellulose paper, probing the paper with virus-specific rabbit antisera, and indirect detection of virus proteins with horseradish peroxidase-conjugated goat anti-rabbit globulins. The sensitivity and specificity of the technique were tested using brome mosaic and barley stripe mosaic viruses. As little as 1 ng per track of virus protein was detectable, either as pure virus or when mixed with plant sap. Distant serological relationships were detected amongst tobamoviruses, and amongst the bromoviruses, with single antisera. The uses of the technique in probing capsid configuration in a presumed aphid picornavirus, and in routine diagnostic practice, are described.

Antigens, Viral↗

Cell-to-cell movement of plant viruses. Insights from amino acid sequence comparisons of movement proteins and from analogies with cellular transport systems.

Cell-to-cell movement is a crucial step in plant virus infection. In many viruses, the movement function is secured by specific virus-encoded proteins. Amino acid sequence comparisons of these proteins revealed a vast superfamily containing a conserved sequence motif that may comprise a hydrophobic interaction domain. This superfamily combines proteins of viruses belonging to all principal groups of positive-strand RNA viruses, as well as single-stranded DNA containing geminiviruses, double-stranded DNA-containing pararetroviruses (caulimoviruses and badnaviruses), and tospoviruses that have negative-strand RNA genomes with two ambisense segments. In several groups of positive-strand RNA viruses, the movement function is provided by the proteins encoded by the so-called triple gene block including two putative small membrane-associated proteins and a putative RNA helicase. A distinct type of movement proteins with very high content of proline is found in tymoviruses. It is concluded that classification of movement proteins based on comparison of their amino acid sequences does not correlate with the type of genome nucleic acid or with grouping of viruses based on phylogenetic analysis of replicative proteins or with the virus host range. Recombination between unrelated or distantly related viruses could have played a major role in the evolution of the movement function. Limited sequence similarities were observed between i) movement proteins of dianthoviruses and the MIP family of cellular integral membrane proteins, and ii) between movement proteins of bromoviruses and cucumoviruses and M1 protein of influenza viruses which is involved in nuclear export of viral ribonucleoproteins. It is hypothesized that all movement proteins of plant viruses may mediate hydrophobic interactions between viral and cellular macromolecules.

Amino Acid Sequence↗

Phylogeny of capsid proteins of small icosahedral RNA plant viruses.

Statistically significant alignment was generated between the amino acid sequences of the (putative) shell (S) domains of the capsid proteins of small RNA plant viruses with icosahedral capsids in the tombusvirus, carmovirus, dianthovirus, sobemovirus and luteovirus groups. Inspection of the alignment showed good correspondence between the experimentally defined beta-strands and alpha-helices of the capsid proteins of tomato bushy stunt, southern bean mosaic and turnip crinkle viruses, allowing prediction of the secondary structure elements in proteins with unresolved tertiary structure. It is concluded that this set of viral capsid proteins forms a tight evolutionary cluster. Comparison of the alignment of the proteins of this family with the sequences of other capsid proteins of icosahedral RNA viruses revealed more distant similarities to the satellites of tobacco necrosis, panicum mosaic, tobacco mosaic and maize white line mosaic viruses, as well as to nepo- and comoviruses. The tentative phylogenetic tree derived from the capsid protein alignment separated into three main lineages: (I) carmo-, tombus- and dianthoviruses, (II) southern bean mosaic, tobacco necrosis and maize chlorotic mottle viruses, and (III) luteoviruses. Comparison of this tree topology with the tentative evolutionary schemes for the respective virus RNA-dependent RNA polymerases suggested that gene shuffling is the universal trend in the evolution of small RNA plant virus genomes.

Amino Acid Sequence↗

RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block.

Cell-to-cell and long-distance transport of some plant viruses requires coordinated action of three movement proteins encoded by triple gene block (TGB). The largest of TGB proteins, TGBp1, is a member of the superfamily I of DNA/RNA helicases and possesses a set of conserved helicase sequence motifs necessary for virus movement. A recombinant His-tagged form of TGBp1 of two hordeiviruses and potato virus X, a potexvirus, produced in Escherichia coli had unwinding activity on a partially duplexed RNA, but not DNA substrate. The helicase activity of these proteins was dependent on Mg2+ and ATP. The isolated C-terminal half of the PSLV TGBp1 retaining all helicase motifs was also able to unwind RNA duplex.

Adenosine Triphosphatases↗

Foveavirus, a new plant virus genus.

Foveavirus is a novel genus of plant viruses with helically constructed filamentous particles ca. 800 nm long, typified by apple stem pitting virus (ASPV). Virions do not contain lipids or carbohydrates, have a positive sense, single-stranded, polyadenylated RNA genome 8.4 to 9.3 kb in size, and a single type of coat protein with a size of 28 to 44 kDa. The genome of definitive viral species is made up of five ORFs encoding respectively, the replication-related proteins (ORF 1), the putative movement proteins (ORF 2 to 4, constituting the triple block gene), and the coat protein (ORF 5). Virions accumulate in the cytoplasm, where replication is likely to occur with a strategy comparable to that of potexviruses, based on direct expression of the 5'-proximal ORF, and expression of downstream ORFs through subgenomic RNAs. No vector is known. Virus transmission is by grafting, and dispersal is through infected propagating material. The genome structure and organization (i.e., number and order of genes) closely resembles that of the genera Potexvirus, Carlavirus and Allexvirus, but ORF 1 and the coat protein cistron (ASPV only) are significantly larger.

Open Reading Frames↗

Use of ELISA for measuring the extent of serological cross-reactivity between plant viruses.

The degree of antigenic relatedness between two plant viruses is commonly expressed by a serological differentiation index (SDI) which corresponds to the average number of two-fold dilution steps separating homologous from heterologous precipitin titers. Results obtained with several tobamo- and tombusviruses indicated that the indirect form of the enzyme-linked immunosorbent assay (ELISA) can also be used for calculating SDI values. This was achieved by comparing the antiserum dilutions that lead to the same absorbance measurements (for instance 1.0) when homologous and heterologous viruses are assayed by ELISA. SDI values calculated from ELISA were similar to those obtained from precipitin tests. Because of its greater sensitivity, ELISA is able to quantify weak cross-reactions that are not detectable by precipitin tests.

Antigens, Viral↗