Characterization of Cucumber Mosaic Virus

Cucumber mosaic virus (CMV), generated from biologically active cDNA clones of Fny-CMV RNA 1 plus 2 and Sny-CMV RNA 3, derived from the Fny-and Sny-strains of CMV, was able to infect tobacco but not squash plants systemically. In squash, viral RNA, movement protein, and coat protein all accumulated in the inoculated cotyledons. The lack of systemic infection was associated with a reduced rate of cell-to-cell movement within the cotyledons. The restricted movement mapped to two sequence changes in the codons of amino acids 51 and 240 of the Sny-CMV 3a gene. These same sequence changes previously were shown to be associated with high levels of 3a protein accumulation and chronic vs acute, cyclic infection typical of Sny-CMV vs Fny-CMV [Gal-Onet al.(1996).Virology226, 354–361]. Fny-CMV, mutated in the codons of 3a gene amino acids 51 and 240, was still able to infect several solanaceous hosts (tobacco, tomato, and pepper) systemically, but did not elicit a typical CMV systemic infection on any of several cucurbit hosts (cucumber, melon, or squash). The significance of the location of amino acid positions 51 and 240 in the 3a movement protein is discussed.     

Phosphorylation of the Movement Protein of Cucumber Mosaic Virus in Transgenic Tobacco Plants

The 3a protein of Cucumber mosaic virus is essential for the cell-to-cell movement of the viral RNA through plasmodesmata. We have introduced an epitope peptide before the stop codon of the 3a protein and cloned the tagged ORF into a binary vector for Agrobacterium-mediated transformation. The established transgenic tobacco lines produced the 3a protein, which was specifically detected with anti-3a and anti-epitope antisera. Metabolic labeling and subsequent immunoprecipitation revealed that [³²P]-orthophosphate was incorporated into the 3a protein. The phosphoamino acid analysis indicated that the 3a protein contained phosphoserine but not phosphothreonine or phosphotyrosine. This is the first demonstration of the 3a protein phosphorylation in planta.     

Transgenically Expressed Cucumber Mosaic Virus RNA 1 Simultaneously Complements Replication of Cucumber Mosaic Virus RNAs 2 and 3 and Confers Resistance to Systemic Infection

Tobacco plants transformed with a cDNA copy of RNA 1 of the Fny strain of cucumber mosaic virus (CMV) promoted the asymptomatic accumulation of inoculated viral RNAs 2 and 3, which could be detected in noninoculated leaves, suggesting that the transgene also permitted viral long-distance movement. Typical symptoms of infection appeared later and correlated with the appearance of viral RNA 1 regenerated from the transgenic mRNA. Although all R₀-generation plants were susceptible to Fny-CMV, one line displaying variable susceptibility to the virus in R₁-and R₂-generations led to selected R₃-generation lines with systemic resistance to Fny-CMV. In the inoculated leaves of resistant plants, a dramatic decrease in the accumulation of viral RNA 1 was observed, relative to susceptible plants. No viral RNAs were detected in noninoculated leaves of the resistant plants, but such leaves were susceptible to infection. Furthermore, these leaves could sustain replication of inoculated CMV RNAs 2 and 3, indicating that a complete transgene-silencing had not been induced. Although a transgene-mediated, CMV RNA 1-suppression occurred in the inoculated leaf of resistant plants, the absence of a complete systemically acquired silencing suggests the existence of additional interferences with viral infection that prevented systemic infection by viral RNAs 2 and 3.     

Serological Behaviour of Tobacco Mosaic Virus and of its Protein Fragments

The protein obtained when tobacco mosaic virus is disrupted with alkali is antigenically heterogeneous, whereas the intact virus is homogeneous. Aggregating the protein by acidification makes it again antigenically homogeneous. In comparable conditions, antibody/antigen ratio is considerably higher in precipitates formed by the protein than in those formed by the virus. Aggregating the protein restores the ratio to that given by the original virus.

Electron-microscopic examination shows that in extreme antibody excess each virus particle is covered with a layer of antibody molecules. In these conditions, therefore, individual antibody molecules seem not to bridge directly between different virus particles, but they may do so in the zone of equivalence.

     

Rheo-optical characterization (flow-birefringence and flow-dichroism) of the Tobacco Mosaic Virus

Rheo-optical techniques of determining flow-birefringence Δn′ and flow-dichroism Δn″ were employed to investigate a Tobacco Mosaic Virus (TMV) solution under steady-state and relaxation conditions to obtain further information about molecular dynamics in terms of local and global orientation and flexibility. These effects were investigated in a concentration range of 0,5 g/L ? c ≦ 2,89 g/L, i.e. below and above the critical overlap concentration c* = 2,45 g/L. Although some aggregation was unavoidable it was determined that in the region of c* the dynamic behaviour was not influenced by varying the concentration. Furthermore it was shown that these aggregates naturally appearing in TMV are sensitive to shear force and dissolve at shear rates above 1 s^?1. Beyond the critical shear rate this rodlike molecule should show a good agreement of the orientation angles determined through dichroism and birefringence and a constant ratio Δn″/Δn′ which in fact was found. This reveals experimentally that in spite of the sensitivity to different length scales dichroism and birefringence exhibit similar responses to shear forces for rigid and monodisperse macromolecules.     

Complete Nucleotide Sequence of the Genomic RNA of Tobacco Mosaic Virus Strain Cg

Tobacco mosaic virus (TMV)-Cg is a crucifer-infecting tobamovirus that was isolated from field-grown garlic. We determined the complete nucleotide sequence of the genomic RNA of TMV-Cg. The genomic RNA of TMV-Cg consists of 6303 nucleotides and encodes four large open reading frames, organized basically in the same way as that of other tobamoviruses. The nucleotide and deduced amino acid sequences are very similar to those of the other crucifer-infecting tobamoviruses that have been sequenced so far. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of two strains of Tobacco Mosaic Virus by biological,biochemical, immunological and electron microscopical techniques

Two strains of Tobacco Mosaic Virus (TMV) isolated from tomato plants were characterized by biological, biochemical, immunological and electron microscopical techniques including immuno-electron microscopy. The biological tests indicated, on the basis of symptoms observed on differential hosts, differences between the two strains. However, detailed studies with the other techniques mentioned above did not reveal significant differences.     

Antigenic Relationships Among Newcastle Disease Virus Mutants Obtained from Laboratory Strains and from Recent California Isolates

The antigenic relationship between pairs of plaque mutants of Newcastle disease virus (NDV) derived from laboratory strains and from isolates from the 1971-72 California epizootic were examined by kinetic neutralization test. Comparing four sets of mutants from laboratory strains by both kinetic neutralization and hemagglutination inhibition tests, a similarity was found in the antigenic relationship expressed as an r value with both tests. However, kinetic neutralization was the more precise as well as sensitive assay. Antigenic diversity was greatest between pairs of mutants from different strains, but distinctions could also be made between mutants from the same strains such as Herts-L and Herts-S with an r value of 36%. Examination of mutants from the California epizootic isolated from separate locations and at different times showed antigenic divergence which was greatest between two red-plaque mutants with an r value of 39%. Antigenic distinctions were found between a red- and clear-plaque mutant obtained from isolates taken from brain and tracheal swabs of one infected chicken. In addition to antigenic divergence found between pairs of some mutants, two of the clear-plaque mutants reacted more avidly with antibody than did the corresponding red-plaque partner. Thus, both differences in antigenicity and avidity can be found among these NDV mutants. The antigenic variation found among these mutants is similar to that found within a serotype. This would imply that at the present NDV is a single serological type.     

The Synthesis of Liquid Crystalline Copolymers with Block Copolymer Grafts

The synthesis of novel copolymers consisting of a side‐group liquid crystalline backbone and poly(tetrahydrofuran)‐poly(methyl methacrylate) block copolymer grafts was realized by using cationic‐to‐free‐radical transformation reactions. Firstly, photoactive poly(tetrahydrofuran) macroinimers were prepared by cationic polymerization of tetrahydrofuran and subsequent termination with 2‐picoline N‐oxide. Secondly, the macroinimers and acrylate monomers containing different spaced cyanobiphenyl mesogenic groups were copolymerized to yield the respective graft copolymers. Eventually, these were used for indirect photochemical polymerization of methyl methacrylate by UV irradiation in the presence of anthracene as a photosensitizer leading to the final copolymers with block copolymer grafts. The liquid crystalline, semicrystalline, and amorphous blocks were micro‐phase separated in the graft copolymers.     

Packing and Uniaxial Alignment of Liquid Crystalline Oligofluorenes

Summary: Liquid crystalline oligomers of 9,9‐bis(2‐ethylhexyl)fluorene of defined degree of polymerization 4, 5, 6, and 7 were investigated by X‐ray diffraction in the non‐oriented and in the aligned state. The diffraction data give evidence for a smectic B type phase for all of the oligomers. Quenching below the glass transition does not change the structure of the liquid crystalline phase. This allows to align spin‐coated films of these oligomers on rubbed polyimide substrates to give monodomain films. These are stable against thermal disordering below T_g, e.g. at room temperature. The degree of alignment is characterized by the dichroic spectra and polarized fluorescence spectra. Dichroic ratios and polarization ratios increase substantially with the chain length and values as high as D = 23 and P = 41 are obtained for the heptamer. The type of packing of the oligomers in the LC phase is discussed based on the X‐ray single crystal structure of models. In one such model the packing of the 2‐ethylhexyl side chains could be fully resolved, while the other model reveals the torsional angle between adjacent fluorene units in the same molecule as 144.2° which corroborates earlier work based on fiber diffraction of corresponding polyfluorenes.